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Hepatitis B Virus X Protein Stimulates Gene Expression Selectively From Extrachromosomal DNA Templates
Abstract
Unlabelled:
Chronic hepatitis B virus (HBV) infection is a major risk factor for liver cancer development. HBV encodes the hepatitis B virus X (HBx) protein that promotes transcription of the viral episomal DNA genome by the host cell RNA polymerase II. Here we provide evidence that HBx accomplishes this task by a conserved and unusual mechanism. Thus, HBx strongly stimulates expression of transiently transfected reporter constructs, regardless of the enhancer and promoter sequences. This activity invariably requires HBx binding to the cellular UV-damaged DDB1 E3 ubiquitin ligase, suggesting a common mechanism. Unexpectedly, none of the reporters tested is stimulated by HBx when integrated into the chromosome, despite remaining responsive to their cognate activators. Likewise, HBx promotes gene expression from the natural HBV episomal template but not from a chromosomally integrated HBV construct. The same was observed with the HBx protein of woodchuck HBV. HBx does not affect nuclear plasmid copy number and functions independently of CpG dinucleotide methylation.
Conclusion:
We propose that HBx supports HBV gene expression by a conserved mechanism that acts specifically on episomal DNA templates independently of the nature of the cis-regulatory sequences. Because of its uncommon property and key role in viral transcription, HBx represents an attractive target for new antiviral therapies.
... In additional to its potential role in hepatocarcinogenesis, HBx also promotes HBV replication. HBx has long been known to activate the transcription of episomal DNA regardless of promoter or enhancer sequence (Colgrove et al., 1989;Seto et al., 1988;Spandau and Lee, 1988;Tang et al., 2005;Twu and Schloemer, 1987;van Breugel et al., 2012). It was found later that this transactivation activity relies on its interaction with the cellular DNA damage-binding protein 1 (DDB1) (Hodgson et al., 2012;Leupin et al., 2005;Li et al., 2010;van Breugel et al., 2012). ...
... HBx has long been known to activate the transcription of episomal DNA regardless of promoter or enhancer sequence (Colgrove et al., 1989;Seto et al., 1988;Spandau and Lee, 1988;Tang et al., 2005;Twu and Schloemer, 1987;van Breugel et al., 2012). It was found later that this transactivation activity relies on its interaction with the cellular DNA damage-binding protein 1 (DDB1) (Hodgson et al., 2012;Leupin et al., 2005;Li et al., 2010;van Breugel et al., 2012). Recently, two groups independently reported that HBx hijacks the host DDB1-containing E3 ubiquitin ligase to target the restriction factor, the structural maintenance of chromosomes 5/6 (Smc5/6), for degradation (Decorsiere et al., 2016;Murphy et al., 2016). ...
... One of the most important roles of HBx in HBV viral cycle is to promote the transcription of viral DNA. Actually, HBx has long been known to activate the transcription of episomal DNA regardless of promoter or enhancer sequence (Colgrove et al., 1989;Seto et al., 1988;Spandau and Lee, 1988;Tang et al., 2005;Twu and Schloemer, 1987;van Breugel et al., 2012). HBx activates transcription by degrading the restriction factor of HBV, Smc5/6 (Livingston et al., 2017;Murphy et al., 2016). ...
The host structural maintenance of chromosomes 5/6 complex (Smc5/6) is a restriction factor of hepatitis B virus (HBV) that inhibits the transcription of viral ccDNA. HBV antagonizes this restriction by expressing the regulatory X protein (HBx) which targets Smc5/6 for degradation via DNA damage-binding protein 1 (DDB1) E3 ubiquitin ligase. However, the molecular insights into how Smc5/6 interacts with HBx remain elusive. In this study, we systematically investigated the interaction between Smc5/6 and HBx. Smc5/6 interacts with HBx through multiple sites in the absence of DDB1 in the pull-down assay. HBx C-terminal is sufficient for the interaction. Most importantly, residue Phe132, which is strictly conserved in all HBV subtypes, is critical for interaction with Smc5/6 both in vitro and in vivo. Mutation of this site (F132A) results in defect in Smc5/6 interaction, extrachromosomal reporter transcription, and HBV production both in cells and in mouse model. Collectively, our data identifies a key residue on HBx for Smc5/6 interaction and viral production. These results provide valuable information for both basic research and therapeutic drugs targeting HBx.
... Kostyushev and colleagues (2019) first reported that methylated HBV cccDNA may be resistant to the degradation effect of CRISPR/Cas9-based anti-HBV therapeutics [82]. van Breugel et al. (2012) argued that the gene expression activation of HBx functions may only work for extrachromosomal DNA, rather than integrants [83]. They demonstrated its consistent activation effect on transiently transfected constructs independent of regulatory elements within them, and this effect disappeared once these constructs were incorporated into the host genome. ...
... Kostyushev and colleagues (2019) first reported that methylated HBV cccDNA may be resistant to the degradation effect of CRISPR/Cas9-based anti-HBV therapeutics [82]. van Breugel et al. (2012) argued that the gene expression activation of HBx functions may only work for extrachromosomal DNA, rather than integrants [83]. They demonstrated its consistent activation effect on transiently transfected constructs independent of regulatory elements within them, and this effect disappeared once these constructs were incorporated into the host genome. ...
... They demonstrated its consistent activation effect on transiently transfected constructs independent of regulatory elements within them, and this effect disappeared once these constructs were incorporated into the host genome. They showed that the activity required HBx binding to the cellular UV-damaged DDB1 E3 ubiquitin ligase [83]. ...
Hepatitis B virus (HBV), the well-studied oncovirus that contributes to the majority of hepatocellular carcinomas (HCC) worldwide, can cause a severe inflammatory microenvironment leading to genetic and epigenetic changes in hepatocyte clones. HBV replication contributes to the regulation of DNA methyltransferase gene expression, particularly by X protein (HBx), and subsequent methylation changes may lead to abnormal transcription activation of adjacent genes and genomic instability. Undoubtedly, the altered expression of these genes has been known to cause diverse aspects of infected hepatocytes, including apoptosis, proliferation, reactive oxygen species (ROS) accumulation, and immune responses. Additionally, pollutant-induced DNA methylation changes and aberrant methylation of imprinted genes in hepatocytes also complicate the process of tumorigenesis. Meanwhile, hepatocytes also contribute to epigenetic modification of the viral genome to affect HBV replication or viral protein production. Meanwhile, methylation levels of HBV integrants and surrounding host regions also play crucial roles in their ability to produce viral proteins in affected hepatocytes. Both host and viral changes can provide novel insights into tumorigenesis, individualized responses to therapeutic intervention, disease progress, and early diagnosis. As such, DNA methylation-mediated epigenetic silencing of cancer-related genes and viral replication is a compelling therapeutic goal to reduce morbidity and mortality from liver cancer caused by chronic HBV infection. In this review, we summarize the most recent research on aberrant DNA methylation associated with HBV infection, which is involved in HCC development, and provide an outlook on the future direction of the research.
... The SMC5/6 complex was previously proposed as a restriction factor for hepatitis B virus (HBV), which persists as an extrachromosomal covalently closed circular DNA (cccDNA) episomal viral genome, in the nuclei of infected cells (Decorsiè re et al., 2016;Murphy et al., 2016). Gene expression from HBV cccDNA, like unintegrated HIV-1, is restricted by the host cell yet is enhanced by the HBx viral protein (van Breugel et al., 2012), which degrades SMC5 and SMC6 (Decorsiè re et al., 2016;Murphy et al., 2016). We therefore tested whether ectopic expression of HBV HBx could substitute for HIV-1 Vpr and rescue gene expression from unintegrated HIV-1 reporters. ...
... SMC family proteins are ATP-dependent molecular motors that play central roles in regulating chromatin structure and genome stability. The SMC5/6 complex is best recognized for its role in DNA repair but is also essential for the maintenance of cellular DNA repeat regions (Peng et al., 2018;Torres-Rosell et al., 2005). In S. cerevisiae, the SMC5/6 complex functions in chromatin silencing of non-coding telomeric and ribosomal DNA repeat sequences, independent of its role in homologous recombination, and depletion of SMC5/6 leads to silencing defects and reduced repeat stability (Moradi-Fard et al., 2016. ...
... Aberrant recombination of repetitive DNA regions is a major source of cellular ecDNA (Cohen and Segal, 2009). The SMC5/6 complex limits recombination at these regions (Torres-Rosell et al., 2005) and, in the light of our results, may also act to compact and silence any ecDNA released as a result of inappropriate recombination. Given the high prevalence of additional viral and cellular extrachromosomal DNAs, we predict that the impact of this silencing mechanism is likely to extend beyond the confines of HIV biology. ...
Silencing of nuclear DNA is an essential feature of innate immune responses to invading pathogens. Early in infection, unintegrated lentiviral cDNA accumulates in the nucleus yet remains poorly expressed. In HIV-1-like lentiviruses, the Vpr accessory protein enhances unintegrated viral DNA expression, suggesting Vpr antagonizes cellular restriction. We previously showed how Vpr remodels the host proteome, identifying multiple cellular targets. We now screen these using a targeted CRISPR-Cas9 library and identify SMC5-SMC6 complex localization factor 2 (SLF2) as the Vpr target responsible for silencing unintegrated HIV-1. SLF2 recruits the SMC5/6 complex to unintegrated lentiviruses, and depletion of SLF2, or the SMC5/6 complex, increases viral expression. ATAC-seq demonstrates that Vpr-mediated SLF2 depletion increases chromatin accessibility of unintegrated virus, suggesting that the SMC5/6 complex compacts viral chromatin to silence gene expression. This work implicates the SMC5/6 complex in nuclear immunosurveillance of extrachromosomal DNA and defines its targeting by Vpr as an evolutionarily conserved antagonism.
... Through its interaction with other adaptor proteins, DDB1 recruits substrates to the complex for ubiquitination and eventual proteasomal degradation 22,23 . HBx-DDB1 interaction has been shown to subvert this process in the virus's favor, leading to enhanced cccDNA transcription and viral replication [24][25][26] . The fact that HBx requires interaction with cellular adaptor proteins to be functional suggest the possibility of targeting such interactions for treating liver diseases associated with HBV or HBx. ...
... K91, on the other hand, can tolerate K91T variation without losing 2A7 reactivity, but K91A mutation Anti-HBx 2A7 epitope coincides with DDB1 binding site allowing 2A7 to block HBx-DDB1 interaction. Previous studies of HBx functions have identified DDB1 as a cellular protein that binds HBx, and DDB1-HBx interactions have been shown to subvert normal functions of CUL4-DDB1 ubiquitination machinery to favor optimal HBV replication 24,25 . HBx interacts with DDB1 through a short segment encompassing a.a. ...
... Although interaction between HBx and DDB1 has long been known 19 , it was not until recent years that its functional significance in viral life cycle has been demonstrated [24][25][26] . Hijacking of the cellular CUL4-DDB1 E3 ligase by HBx caused abnormal ubiquitination and degradation of proteins such as Smc5/6 25,26 , which in turn resulted in enhanced transcription from episomal HBV cccDNA and viral replication 7,10 . ...
Hepatitis B virus (HBV) X protein (HBx) plays diverse roles in both viral life cycle and HBV-related carcinogenesis. Its interaction with DNA damage-binding protein 1 (DDB1) was shown to be essential for engendering cellular conditions favorable for optimal viral transcription and replication. Previously, we described a mouse monoclonal antibody against HBx (anti-HBx 2A7) recognizing HBx encoded by representative strains from 7 of 8 known HBV genotypes. In this work, we further characterized 2A7 in order to explore its potential usefulness in HBx-targeting applications. We demonstrated that 2A7 recognizes a linear epitope mapped to L89PKVLHKR96 on HBx, a segment that is highly conserved across genotypes and coincidentally overlaps with the DDB1-interacting segment. HBx-DDB1 binding could be inhibited by 2A7 in vitro, suggesting therapeutic potential. Nucleic acid and amino acid sequences of 2A7 were then obtained, which allowed construction of recombinant antibody and single chain variable fragments (scFv). 2A7-derived recombinant antibody and scFv recapitulate 2A7’s HBx-binding capacity and epitope specificity. We also reported preliminary results using cell-penetrating peptide for delivering 2A7 antibody across cell membrane to target intracellular HBx. Anti-HBx 2A7 and 2A7-derived scFv characterized here may give rise to novel HBx-targeting diagnostics and therapeutics for HBV- and HBx-related pathologies.
... It was therefore hypothesized that HBx binding to DDB1 could lead to proteasomal degradation of a specific cellular restriction factor[20,21]. In addition to binding DDB1, HBx has long been known to activate the transcription of a wide variety of genes encoded by episomal templates (i.e., closed-circular DNA molecules independent of cellular chromosomes), including cccDNA[22][23][24][25][26][27][28]. It was determined that HBx does so regardless of promoter or enhancer sequence, and thus acts as a non-specific transcriptional activator (transactivator) of episomal DNA. ...
... It was determined that HBx does so regardless of promoter or enhancer sequence, and thus acts as a non-specific transcriptional activator (transactivator) of episomal DNA. In contrast, HBx does not transactivate chromosomal genes[20,28]. Moreover, the transactivation of episomal DNA by HBx was shown to require an interaction of HBx with the DDB1?Cul4 ubiquitin ligase machinery[28]. ...
... In contrast, HBx does not transactivate chromosomal genes[20,28]. Moreover, the transactivation of episomal DNA by HBx was shown to require an interaction of HBx with the DDB1?Cul4 ubiquitin ligase machinery[28]. Taken together, these observations suggest that HBx transactivation activity is dependent upon DDB1-mediated degradation of a cellular restriction factor. ...
Hepatitis B X protein (HBx) plays an essential role in the hepatitis B virus (HBV) replication cycle, but the function of HBx has been elusive until recently. It was recently shown that transcription from the HBV genome (covalently-closed circular DNA, cccDNA) is inhibited by the structural maintenance of chromosome 5/6 complex (Smc5/6), and that a key function of HBx is to redirect the DNA-damage binding protein 1 (DDB1) E3 ubiquitin ligase to target this complex for degradation. By doing so, HBx alleviates transcriptional repression by Smc5/6 and stimulates HBV gene expression. In this review, we discuss in detail how the interplay between HBx and Smc5/6 was identified and characterized. We also discuss what is known regarding the repression of cccDNA transcription by Smc5/6, the timing of HBx expression, and the potential role of HBx in promoting hepatocellular carcinoma (HCC).
... HBx selectively transactivates HBV transcription from episomal DNA templates including the authentic cccDNA and the transfected HBV plasmid [50,[56][57][58]. Previous studies have reported that several TRIMs (5γ, 21, 31) inhibit HBV transcription through interacting with HBx and promoting its degradation [27][28][29]. ...
... In search of potential substrate(s) targeted by TRIM65 to inhibit HBV transcription, our initial protein of interest was HBx, a key regulatory viral protein that stimulates and maintains HBV transcription from episomal templates, including the authentic cccDNA and its surrogates [50,[56][57][58]. However, through comparative analyses of the antiviral effect of TRIM65 on the wild type and the HBx-null pHBV1.3 ...
Tripartite motif (TRIM) proteins, comprising a family of over 100 members with conserved motifs, exhibit diverse biological functions. Several TRIM proteins inffuence viral infections through direct antiviral mechanisms or by regulating host antiviral innate immune responses. To identify TRIM proteins modulating hepatitis B virus (HBV) replication, we assessed 45 human TRIMs in HBV-transfected HepG2 cells. Our study revealed that ectopic expression of 12 TRIM proteins signiffcantly reduced HBV RNA and subsequent capsid-associated DNA levels. Notably, TRIM65
uniquely downregulated viral pregenomic (pg) RNA in an HBV-promoter-speciffc manner, suggesting a targeted antiviral effect. Mechanistically, TRIM65 inhibited HBV replication primarily at the transcriptional level via its E3 ubiquitin ligase activity and intact B-box domain. Though HNF4α emerged as a potential TRIM65 substrate, disrupting its binding site on the HBV genome did not completely abolish TRIM65’s antiviral effect. In addition, neither HBx expression nor cellular MAVS signaling was essential to TRIM65-mediated regulation of HBV transcription. Furthermore, CRISPR mediated knock-out of TRIM65 in the HepG2-NTCP cells boosted HBV infection, validating its endogenous role. These ffndings underscore TRIM proteins’ capacity to inhibit HBV transcription and highlight TRIM65’s pivotal role in this process.
... HBx selectively transactivates HBV transcription from episomal DNA templates including the authentic cccDNA and the transfected HBV plasmid [50,[56][57][58]. Previous studies have reported that several TRIMs (5γ, 21, 31) inhibit HBV transcription through interacting with HBx and promoting its degradation [27][28][29]. ...
... In search of potential substrate(s) targeted by TRIM65 to inhibit HBV transcription, our initial protein of interest was HBx, a key regulatory viral protein that stimulates and maintains HBV transcription from episomal templates, including the authentic cccDNA and its surrogates [50,[56][57][58]. However, through comparative analyses of the antiviral effect of TRIM65 on the wild type and the HBx-null pHBV1.3 ...
Citation: Shen, S.; Yan, R.; Xie, Z.; Yu, X.; Liang, H.; You, Q.; Zhang, H.; Hou, J.; Zhang, X.; Liu, Y.; et al. Tripartite Motif-Containing Protein 65 (TRIM65) Inhibits Hepatitis B Virus Transcription. Viruses 2024, 16, 890.
... The HBV regulatory protein X (HBx), a product of the X gene, has been reported to play an important role in HBV life cycle through interaction with many host proteins. The best characterized HBx binding partner is the damage-specific DNA-binding protein 1 (DDB1), an adapter protein for the Cul4A E3 ligase complex 9 . The HBx binds to DDB1 through an H-box motif, which redirects the DDB1contaning E3 ubiquitin ligase to degrade the structural maintenance of chromosomes (SMC) 5/6 proteins localized to Nuclear Domain 10 (ND10, a.k.a. ...
... As anticipated, Spindlin1 50-262 displayed the most pronounced stabilization effect on HBx 2-21 peptide by +12°C (Fig. 1c). We also measured a dissociation constant (K d ) of 1.1 μM (Fig. 1d) between Spindlin1 50-262 and HBx [2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21] , which is about 44-fold and 55-fold stronger than that to HBx [11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30] and HBx 61-80 peptides, respectively ( Supplementary Fig. 1a). Then, the ability of Spindlin1 to interact with HBx was confirmed in HEK 293 T cells by immunoprecipitation assay (Fig. 1e). ...
Molecular interplay between host epigenetic factors and viral proteins constitutes an intriguing mechanism for sustaining hepatitis B virus (HBV) life cycle and its chronic infection. HBV encodes a regulatory protein, HBx, which activates transcription and replication of HBV genome organized as covalently closed circular (ccc) DNA minichromosome. Here we illustrate how HBx accomplishes its task by hijacking Spindlin1, an epigenetic reader comprising three consecutive Tudor domains. Our biochemical and structural studies have revealed that the highly conserved N-terminal 2–21 segment of HBx (HBx2–21) associates intimately with Tudor 3 of Spindlin1, enhancing histone H3 “K4me3-K9me3” readout by Tudors 2 and 1. Functionally, Spindlin1-HBx engagement promotes gene expression from the chromatinized cccDNA, accompanied by an epigenetic switch from an H3K9me3-enriched repressive state to an H3K4me3-marked active state, as well as a conformational switch of HBx that may occur in coordination with other HBx-binding factors, such as DDB1. Despite a proposed transrepression activity of HBx2-21, our study reveals a key role of Spindlin1 in derepressing this conserved motif, thereby promoting HBV transcription from its chromatinized genome.
... HBV encodes a nonstructural protein, HBx, whose function was for many years enigmatic, though it is essential for viral gene expression and replication [2] as, in the absence of HBx function, HBV episomes are epigenetically silenced [3]. Moreover, nuclear HBx promiscuously activates transcription from extrachromosomal DNA templates, including not only HBV DNA but also transfected expression plasmids, while having no effect on transcription of the same DNA molecule if integrated into a chromosome [4]. Finally, it was known that HBx bound the DNA-damage binding protein 1 (DDB1) subunit of a DDB1-containing E3 ubiquitin ligase [5]. ...
... Finally, it is worth noting that the SMC5/6 complex can also inhibit transcription from transfected expression plasmids [21]. Expression of the HBV HBx protein, which degrades SMC5 and SMC6, or knocking out these proteins, activates gene expression from episomal transfected plasmids but fails to activate the same expression plasmids after chromosomal integration [4,21]. ...
... Restriction by Smc5/6 is conserved in mammals 23 and is not limited to the HBV genome. Expression of any reporter construct is silenced by Smc5/6 and thus stimulated by HBx expression or Smc5/6 depletion, regardless of the enhancer and promoter type 20,24 . However, this occurs only if the DNA remains extrachromosomal. ...
... In addition to its essential roles in DNA replication and repair, Smc5/6 functions as a restriction factor against HBV. Smc5/6 binds to and silences the episomal HBV genome and, more generally, any extrachromosomal reporter template 20,21,24 . In this study, we explored the requirements for Smc5/6 to perform its restriction function. ...
In addition to its role in chromosome maintenance, the six-membered Smc5/6 complex functions as a restriction factor that binds to and transcriptionally silences viral and other episomal DNA. However, the underlying mechanism is unknown. Here, we show that transcriptional silencing by the human Smc5/6 complex is a three-step process. The first step is entrapment of the episomal DNA by a mechanism dependent on Smc5/6 ATPase activity and a function of its Nse4a subunit for which the Nse4b paralog cannot substitute. The second step results in Smc5/6 recruitment to promyelocytic leukemia nuclear bodies by SLF2 (the human ortholog of Nse6). The third step promotes silencing through a mechanism requiring Nse2 but not its SUMO ligase activity. By contrast, the related cohesin and condensin complexes fail to bind to or silence episomal DNA, indicating a property unique to Smc5/6.
... Therefore, it raises the possibility of using cccDNA surrogates to develop novel detection methods (Zhou et al. 2006). HBx can recruit transcription factors to transcriptionally active domain of cccDNA minichromosome and promote transcription of viral episome as well as transiently transfected plasmid (Reeves et al. 1985;van Breugel et al. 2012). However, HBx has no regulatory impact on HBV genes integrated into host chromosome (van Breugel et al. 2012). ...
... HBx can recruit transcription factors to transcriptionally active domain of cccDNA minichromosome and promote transcription of viral episome as well as transiently transfected plasmid (Reeves et al. 1985;van Breugel et al. 2012). However, HBx has no regulatory impact on HBV genes integrated into host chromosome (van Breugel et al. 2012). It suggests that HBx may apply a special mechanism to specifically activate expression of episome. ...
Hepatitis B is caused by hepatitis B virus (HBV), and persistent HBV infection is a global public health problem, with 257 million people as HBV chronic carriers. Viral covalently closed circular DNA (cccDNA) is a key factor to establish persistent infection in infected hepatocytes. Current antiviral therapies have no direct impact on pre-existing cccDNA reservoir, which can be assembled into minichromosome by hijacking host factors. Understanding the mechanisms of epigenetic regulation in cccDNA minichromosome is crucial to develop new therapy on cccDNA, an attractive target for HBV cure. This review summarizes the current advances in epigenetic regulation of cccDNA minichromosome, which might provide clues to novel druggable targets to cure hepatitis B by either silencing or eliminating cccDNA reservoir.
... Targeting neddylation inhibits transcription from cccDNA in de novo infection but not from integrated DNA in the PLC/PRF5 cells (Figure 3.24.). This phenotype is consistent with the fact that HBx protein enhances gene transcription selectively from extrachromosomal DNA templates but shows no effect lentiviral mediated insertion DNA in the host genome (van Breugel et al., 2012). ...
... MLN4924 also showed profound inhibition of transcription from cccDNA but not integrants, because of the HBx selectivity only on cccDNA templates (Figure 3.24.) (van Breugel et al., 2012). The selectivity of both ARC-520 and MLN4924 on cccDNA limits their future applications. ...
Translation of abstract (English)
Hepatitis B Virus (HBV) leads to chronic infection of the liver and is a risk factor for the development of cirrhosis and hepatocellular carcinoma. Virus persistence requires the establishment and maintenance of covalently closed circular (ccc)DNA, serving as the episomal template for transcription of viral genes in the nucleus of infected hepatocytes. Viral cccDNA-dependent gene expression requires HBV X protein (HBx) and HBx-mediated degradation of the host restriction factors structural maintenance of chromosome 5/6 (SMC5/6). A prerequisite for SMC5/6 ubiquitination and degradation is that HBx binds DNA damage-binding protein 1 (DDB1) and further recruits Cullin 4A ring ligase complex, which requires neddylation for its activation.
After the establishment of a reliable quantitative PCR, the kinetics of cccDNA formation in four in vitro infection systems and the effect of drugs (interferon-a, nucleos(t)ide analogues, entry inhibitors, and capsid inhibitors) on cccDNA were analyzed. Compared to replicative relaxed circular (rc)DNA, copy numbers of cccDNA in infected hepatocytes are unexpectedly low (1~6 copies per infected cell). Entry inhibitor, Myrcludex B, efficiently blocked cccDNA formation. Interferon-a reduced cccDNA level at high dose, whereas nucleos(t)ide analogues did not reduce it. Treatment with capsid inhibitors during the infection phase but not afterward led to a decline of cccDNA levels.
Using HBx-minus virion for infection, we verified HBx as a key controller of cccDNA transcription. HBx expressed by its authentic promoter showed nuclear localization. Using lentiviral-based transcomplementation assay to restore the transcription of HBx-minus virus to the wild-type level, key fragments and residues of HBx were identified. The whole C-terminus of HBx (51~154 amino acids) was sufficient and indispensable to enhance transcription of HBx-minus virus, whereas N-terminal HBx (1~50 amino acids) displayed no transactivation. In addition, two shorter truncations (51~142 and 58~142 amino acids) showed residual function. Remarkably, the HBx(R96E) mutant with impaired binding activity to DDB1 totally abolished its transactivation activity, supporting that foundation of the HBx-DDB1 complex is required for maximal transcription from cccDNA.
We further investigated whether blockage of neddylation hampers transcription. MLN4924, a specific NEDD8-activating enzyme 1 inhibitor, was identified to notably possess antiviral potential. MLN4924 potently reduced HBV transcription and viral antigen expression at nanomolar doses. After the establishment of cccDNA, the drug profoundly suppressed transcription from cccDNA without affecting cccDNA levels. Withdrawal of MLN4924 did not lead to restoration of cccDNA transcription in HepaRGhNTCP cells, however fast HBV rebound was observed in infected HepG2hNTCP cells. Peculiarly, transcription from cccDNA of HBx-minus virus was not significantly affected, suggesting that MLN4924 effect on wild-type virus replication is HBx dependent. MLN4924 selectively reduced transcription from all HBV promoters on cccDNA but not integrants. MLN4924 prevented SMC6 from degradation and the restoration of SMC6, in turn, silenced transcription from cccDNA.
Taken together, nuclear HBx binds DDB1, induces SMC6 degradation and thereby promotes transcription from cccDNA. Targeting neddylation blocks transcription from cccDNA and restores SMC6 restriction on cccDNA. Therefore, MLN4924 treatment traps cccDNA in “transcriptional silence”. Small molecules that target the HBx-DDB1-Cullin complex might be foresight as the next possible therapeutic option combating HBV.
... HBx is a nuclear protein that is expressed very early and at low levels during infection [71]. Its role is to promote the degradation of the Smc5/6 complex, a restriction factor of any episomal DNA expression [72], including HBV cccDNA [56]. For this reason, the HBx protein plays a central role in cccDNA expression. ...
Hepatitis B virus (HBV) is the etiologic agent of chronic hepatitis B, which puts at least 300 million patients at risk of developing fibrosis, cirrhosis, and hepatocellular carcinoma. HBV is a partially double-stranded DNA virus of the Hepadnaviridae family. While HBV was discovered more than 50 years ago, many aspects of its replicative cycle remain incompletely understood. Central to HBV persistence is the formation of covalently closed circular DNA (cccDNA) from the incoming relaxed circular DNA (rcDNA) genome. cccDNA persists as a chromatinized minichromosome and is the major template for HBV gene transcription. Here, we review how cccDNA and the viral minichromosome are formed and how viral gene transcription is regulated and highlight open questions in this area of research.
... HBx has been shown to exert a promiscuous transcriptional effect on episomal but not chromosomal promoters (21). To assess ...
Hepatitis B virus (HBV) remains a major public health threat with nearly 300 million people chronically infected worldwide who are at a high risk of developing hepatocellular carcinoma. Current therapies are effective in suppressing HBV replication but rarely lead to cure. Current therapies do not affect the HBV covalently closed circular DNA (cccDNA), which serves as the template for viral transcription and replication and is highly stable in infected cells to ensure viral persistence. In this study, we aim to identify and elucidate the functional role of cccDNA-associated host factors using affinity purification and protein mass spectrometry in HBV-infected cells. Nucleolin was identified as a key cccDNA-binding protein and shown to play an important role in HBV cccDNA transcription, likely via epigenetic regulation. Targeting nucleolin to silence cccDNA transcription in infected hepatocytes may be a promising therapeutic strategy for a functional cure of HBV.
... HBV cccDNA transcription is regulated by host factors and the viral X protein (HBx) (11)(12)(13). HBx is essential for sustaining HBV cccDNA transcription activity (14,15). The interactions of HBx with host proteins have been widely investigated in studies seeking to determine their various roles in HBV infection. ...
Hepatitis B virus (HBV) chronically infects approximately 300 million people worldwide, and permanently repressing transcription of covalently closed circular DNA (cccDNA), the episomal viral DNA reservoir, is an attractive approach toward curing HBV. However, the mechanism underlying cccDNA transcription is only partially understood. In this study, by illuminating cccDNA of wild-type HBV (HBV-WT) and transcriptionally inactive HBV that bears a deficient HBV X gene (HBV-ΔX), we found that the HBV-ΔX cccDNA more frequently colocalizes with promyelocytic leukemia (PML) bodies than that of HBV-WT cccDNA. A small interfering RNA (siRNA) screen targeting 91 PML body-related proteins identified SMC5-SMC6 localization factor 2 (SLF2) as a host restriction factor of cccDNA transcription, and subsequent studies showed that SLF2 mediates HBV cccDNA entrapment in PML bodies by interacting with the SMC5/6 complex. We further showed that the region of SLF2 comprising residues 590 to 710 interacts with and recruits the SMC5/6 complex to PML bodies, and the C-terminal domain of SLF2 containing this region is necessary for repression of cccDNA transcription. Our findings shed new light on cellular mechanisms that inhibit HBV infection and lend further support for targeting the HBx pathway to repress HBV activity.
IMPORTANCE Chronic HBV infection remains a major public health problem worldwide. Current antiviral treatments rarely cure the infection, as they cannot clear the viral reservoir, cccDNA, in the nucleus. Therefore, permanently silencing HBV cccDNA transcription represents a promising approach for a cure of HBV infection. Our study provides new insights into the cellular mechanisms that restrict HBV infection, revealing the role of SLF2 in directing HBV cccDNA to PML bodies for transcriptional repression. These findings have important implications for the development of antiviral therapies against HBV.
... These findings indicate that the SMC5/6-mediated defense against hepadnaviruses is evolutionarily conserved [191]. In agreement, the expression of any extrachromosomal plasmid, regardless of the enhancer or promoter sequence, can be suppressed by SMC5/6 and stimulated by HBx expression or SMC5/6 depletion [192]. ...
Promyelocytic leukemia nuclear bodies (PM NBs), often referred to as membraneless organelles, are dynamic macromolecular protein complexes composed of a PML protein core and other transient or permanent components. PML NBs have been shown to play a role in a wide variety of cellular processes. This review describes in detail the diverse and complex interactions between small and medium size DNA viruses and PML NBs that have been described to date. The PML NB components that interact with small and medium size DNA viruses include PML protein isoforms, ATRX/Daxx, Sp100, Sp110, HP1, and p53, among others. Interaction between viruses and components of these NBs can result in different outcomes, such as influencing viral genome expression and/or replication or impacting IFN-mediated or apoptotic cell responses to viral infection. We discuss how PML NB components abrogate the ability of adenoviruses or Hepatitis B virus to transcribe and/or replicate their genomes and how papillomaviruses use PML NBs and their components to promote their propagation. Interactions between polyomaviruses and PML NBs that are poorly understood but nevertheless suggest that the NBs can serve as scaffolds for viral replication or assembly are also presented. Furthermore, complex interactions between the HBx protein of hepadnaviruses and several PML NBs-associated proteins are also described. Finally, current but scarce information regarding the interactions of VP3/apoptin of the avian anellovirus with PML NBs is provided. Despite the considerable number of studies that have investigated the functions of the PML NBs in the context of viral infection, gaps in our understanding of the fine interactions between viruses and the very dynamic PML NBs remain. The complexity of the bodies is undoubtedly a great challenge that needs to be further addressed.
... Previously, we also showed that HBx induced proliferation via miR-21 in hepatic cells (27). Thus, HBx is indispensable for successful HBV infection and promotes replication from extrachromosomal templates (28). Though other proteins of HBV might play a role in modulating BANF1 expression, in this manuscript the role of HBx in HBV pathogenesis was studied. ...
Hepatitis B virus (HBV) infection targets host restriction factors that inhibit its replication and survival. Previous studies have shown that barriers to autointegration factor1 (BANF1) inhibited the replication of herpes simplex virus and vaccinia virus by binding to phosphate backbone of dsDNA. To date, no reports are available for the interplay between BANF1 and HBV. In this study, we elucidated the mechanisms by which HBV inhibit BANF1. First, the effect of HBV on BANF1 was observed in Huh-7, Hep G2, and Hep G2.2.15 cells. Huh-7 cells were transfected with pHBV1.3 or HBx plasmids. The results showed that there was a decreased expression of BANF1 in Hep G2.2.15 cells (P ≤ 0.005) or in HBV/HBx expressing Huh-7 cells (P ≤ 0.005), whereas BANF1 overexpression decreased viral replication (P ≤ 0.05). To study whether phosphorylation/dephosphorylation of BANF1 was responsible for antiviral activity, mutants were created, and it was found that inhibition due to mutants was less significant compared to BANF1 wild type. Previous studies have shown that HBV, at least in part, could regulate the expression of host miRNAs via HBx. It was found that miR-203 expression was high in Hep G2.2.15 cells (P ≤ 0.005) compared to Hep G2 cells. Next, the effect of HBx on miR-203 expression was studied and result showed that HBx upregulated miR-203 expression (P ≤ 0.005). Overexpression of miR-203 downregulated BANF1 expression (P ≤ 0.05) and viral titer was upregulated (P ≤ 0.05), while inhibition of miR-203, reversed these changes. In conclusion, BANF1 downregulated HBV, whereas HBV inhibited BANF1, at least in part, via HBx-mediated miR-203 upregulation in hepatic cells. IMPORTANCE In this study, for the first time, we found that BANF1 inhibited HBV replication and restricted the viral load. However, as previously reported for other viruses, the results in this study showed that BAF1 phosphorylation/dephosphorylation is not involved in its antiviral activity against HBV. HBV infection inhibited the intracellular expression of BANF1, via HBx-mediated upregulation of miR-203 expression. Overexpression of miR-203 downregulated BANF1 and increased the viral titer, while inhibition of miR-203 reversed these changes. This study helped us to understand the molecular mechanisms by which HBV survives and replicates in the host cells.
... The broad distribution of TSS of HBV X may reflect various lengths of HBx transcripts, which were independently verified by 59 rapid amplification of cDNA ends (RACE) (Fig. 2C), and they were also identified by two recent studies using 59 RACE or CAGE (13,22). However, the exact function, if any, of these transcripts remains unclear, although it is well established that the full-length HBx could substantially enhance HBV transcription (14,26,27). ...
Hepatitis B virus (HBV) infection can be effectively suppressed but rarely cured by available drugs. Chronic HBV infection is based on persistence of covalently closed circular DNA (cccDNA) and continuous infection and reinfection with HBV in the liver.
... plasmids), the transcriptional activity of cccDNA is repressed by the structural maintenance of chromosomes 5/6 (SMC5/6) complex. 5 To counteract this cellular downregulation, the HBV X protein (HBx) hijacks DNA damage-binding protein 1 (DDB1) and subsequently recruits NEDD8-Cullin4-RING ubiquitin E3 ligases to induce ubiquitination and proteasomal degradation of the SMC5/6 complex. This mechanism enables high levels of transcription from only a few cccDNA molecules in an actively HBV-replicating hepatocyte. ...
Background & Aims
240 million people are chronically infected with hepatitis B virus (HBV) with very limited curative treatment option. HBV persistence requires maintenance of the covalently closed circular (ccc)DNA template. Furthermore, integration of HBV fragments driving HBV surface antigen (HBsAg) expression occurs frequently. While cccDNA transcription is regulated by Cullin4A-DDB1-HBx-mediated degradation of the structural maintenance of chromosome 5 and 6 (SMC5/6) complex, HBsAg expression from integrants is largely SMC5/6 independent. Inhibition of Nedd8-conjugation of Cullin-RING ubiquitin ligases impairs degradation of substrates, reinstating SMC5/6 levels. Here, we show that targeting neddylation pathway components by siRNAs or the drug MLN4924 (Pevonedistat) suppresses expression of HBV proteins from both cccDNA and integrants.
Methods
A siRNA screen targeting secretory pathway regulators and neddylation genes was performed. Activity of MLN4924 was assessed in infection and integration models. Trans-complementation assays were used to study HBx function in cccDNA and integrant-driven expression.
Results
SiRNA screening uncovered neddylation pathway components (Nedd8, Ube2m) promoting HBsAg production post transcriptionally. Likewise, MLN4924 inhibited production of HBsAg encoded by integrants and reduced intracellular HBsAg levels, independent of HBx. MLN4924 also profoundly inhibited cccDNA transcription in three infection models. Using the HBV inducible cell line HepAD38 as a model we verified the dual action of MLN4924 on both cccDNA and integrants with sustained suppression of HBV markers during 42-days treatment.
Conclusions
HBV persists in chronic infection by forming a cccDNA reservoir and frequent genome integration. Both pathways require neddylation. Therefore, blocking neddylation might offer an attractive approach towards functional cure of chronic hepatitis B.
Lay summary
Here we report that targeting neddylation pathway components has multiple HBV suppressive effects by lowering the amount of HBsAg from HBV integrants and RNAs from cccDNA. Neddylation pathway inhibitor (MLN4924/Pevonedistat) treatment demonstrated antiviral activity in both HBV infection and integration models. Neddylation inhibition might have a therapeutic benefit by lowering HBsAg and increasing the rate of functional cure.
... HBx promotes transcription of the extrachromosomal HBV genome by hijacking the DNA-damage binding protein 1 (DDB1)-containing E3 ubiquitin ligase to target the "structural maintenance of chromosomes" (Smc) Smc5/6 complex for degradation (9). This mechanism stimulates transcription of episomal DNA templates as well as the HBV genome, giving HBx the title of a transcriptional transactivator (10). HBx has also been shown to promote viral replication by recruiting Parvulin 14 and 17 to the HBV covalently closed circular DNA to enhance transcriptional activation (11). ...
The viral protein HBx is the key regulatory factor of the hepatitis B virus (HBV) and the main etiology for HBV-associated liver diseases, such as cirrhosis and hepatocellular carcinoma. Historically, HBx has defied biochemical and structural characterization, deterring efforts to understand its molecular mechanisms. Here we show that soluble HBx fused to solubility tags copurifies with either a [2Fe-2S] or a [4Fe-4S] cluster, a feature that is shared among five HBV genotypes. We show that the O2-stable [2Fe-2S] cluster form converts to an O2-sensitive [4Fe-4S] state when reacted with chemical reductants, a transformation that is best described by a reductive coupling mechanism reminiscent of Fe-S cluster scaffold proteins. In addition, the Fe-S cluster conversions are partially reversible in successive reduction-oxidation cycles, with cluster loss mainly occurring during (re)oxidation. The considerably negative reduction potential of the [4Fe-4S]2+/1+ couple (-520 mV) suggests that electron transfer may not be likely in the cell. Collectively, our findings identify HBx as an Fe-S protein with striking similarities to Fe-S scaffold proteins both in cluster type and reductive transformation. An Fe-S cluster in HBx offers new insights into its previously unknown molecular properties and sets the stage for deciphering the roles of HBx-associated iron (mis)regulation and reactive oxygen species in the context of liver tumorigenesis.
... It has been revealed to serve a role in homologous recombination and in resolving replication-induced DNA supercoiling (41,42). Besides chromosome maintenance, certain data suggest that Smc5/6 binds episomes (including cccDNA) and blocks episome transcription (43). In placental trophoblast cells transfected with HBx plasmid, Smc5/6 expression was significantly lower compared with in mock-transfected cells. ...
Hepatitis B virus (HBV) infection is a global epidemic. The main transmission route of chronic HBV infection is from mother to child, yet the mechanisms underlying HBV intrauterine infection remain unclear. In the present study, the effect and the mechanism underlying hepatitis B virus X antigen (HBxAg) on HBV replication and EGFR activation in trophoblasts was investigated. Serum samples from pregnant women with HBV infection were used to infect trophoblasts and HBxAg expression was detected using ELISA. HBV plasmids carrying either full length hepatitis B virus X (HBx) or HBx with a deletion mutation (ΔHBx) were transfected into trophoblasts and expression levels of HBV DNA, hepatitis B e-antigen and pregenomic (pg)RNA, and structural maintenance of chromosomes (Smc) 5/6 were assessed. The association between HBx and EGFR promoters was characterized using a luciferase reporter assay and EGFR/PI3K/phosphorylated (p)-AKT expression and apoptosis rate were also monitored. The results of the present study indicated that HBxAg expression increased with the increasing titre of HBV DNA (P<0.05). Compared with the wild-type group, the amount of HBV DNA in the supernatant and cells was significantly reduced (P<0.05) in the ΔHBx group and the intracellular HBeAg and pgRNA levels were also significantly decreased (P<0.05). In addition, Smc5/6 expression was also significantly decreased (P<0.05) when the intracellular HBx protein was expressed compared with mock-transfected cells. Co-transfection of HBx and EGFR promoter plasmids in JEG-3 and HTR-8 cells significantly elevated EGFR promoter driven luciferase expression relative to the control group (P<0.01). In EGFR overexpressing cells, the expression of PI3K/p-AKT was significantly increased, whereas the apoptosis rate was significantly decreased (P<0.05). These results were reversed in the EGFR-knockdown group. In conclusion, the present study demonstrated that HBx promotes HBV replication in trophoblasts via downregulation of Smc5/6, activates the EGFR promoter and inhibits trophoblast apoptosis via the PI3K/p-AKT downstream signalling pathway, thereby increasing the risk of HBV intrauterine infection.
... In addition, the ubiquitination assays showed that HBx resulted in the substantial increase of WDR77 ubiquitination in HepG2 cells ( Figure 6B; Figure S6C), suggesting that HBx promotes the ubiquitination of WDR77. Previous studies suggested that HBx stimulates transcription of HBV genome by binding the DDB1 subunit of the DDB1-containing E3 ubiquitin ligase to target unknown host factors for ubiquitin-mediated degradation [32][33][34]. Then, we examined whether HBx promoted the degradation of WDR77 by hijacking DDB1-containing E3 ubiquitin ligase. ...
Rationale: Hepatitis B x protein (HBx) is required to initiate and maintain the replication of hepatitis B virus (HBV). Protein arginine methyltransferases 5 (PRMT5) negatively regulates HBV transcription. WD repeat domain 77 protein (WDR77) greatly enhances the methyltransferase activity of PRMT5. However, the role of WDR77 in the modulation of cccDNA transcription and HBV replication is poorly understood. In this study, we investigated the mechanism by which HBx modulated HBV replication involving WDR77 in the liver.
Methods: A human liver-chimeric mouse model was established. Immunohistochemistry (IHC) staining, Western blot analysis, Southern blot analysis, Northern blot analysis, immunofluorescence assays, ELISA, RT-qPCR, CoIP assays, and ChIP assays were performed in human liver-chimeric mouse model, primary human hepatocytes (PHHs), HepG2-NTCP, dHepaRG and HepG2 cell lines.
Results: HBV infection and HBx expression remarkably reduced the protein levels of WDR77 in human liver-chimeric mice and HepG2-NTCP cells. WDR77 restricted cccDNA transcription and HBV replication in PHHs and HepG2-NTCP cells. Mechanically, WDR77 enhanced PRMT5-triggered symmetric dimethylation of arginine 3 on H4 (H4R3me2s) on the cccDNA minichromosome to control cccDNA transcription. HBx drove the cellular DDB1-containing E3 ubiquitin ligase to degrade WDR77 through recruiting WDR77, leading to the disability of methyltransferase activity of PRMT5. Thus, HBx promoted HBV replication by driving a positive feedback loop of HBx-DDB1/WDR77/PRMT5/H4R3me2s/cccDNA/HBV/HBx in the liver.
Conclusions: HBx attenuates the WDR77-mediated HBV repression by driving DDB1-induced WDR77 degradation in the liver. Our finding provides new insights into the mechanism by which HBx enhances HBV replication in the liver.
... However, how SMC5/6 blocks transcription from cccDNA remains unclear. SMC5/6 may recognize an HBV-specific sequence motif, but this seems unlikely, as HBx-mediated augmentation of gene transcription is limited to extrachromosomal DNA templates: this phenomenon is not observed for HBV genomes with identical sequences integrated into host chromosomes [100]. Another possibility is that SMC5/6 senses the supercoiled cccDNA at transcription initiation and topologically fixes cccDNA in the supercoiled state, at which point transcription elongation is not possible. ...
Approximately 240 million people are chronically infected with hepatitis B virus (HBV), despite four decades of effective HBV vaccination. During chronic infection, HBV forms two distinct templates responsible for viral transcription: (1) episomal covalently closed circular (ccc)DNA and (2) host genome-integrated viral templates. Multiple ubiquitous and liver-specific transcription factors are recruited onto these templates and modulate viral gene transcription. This review details the latest developments in antivirals that inhibit HBV gene transcription or destabilize viral transcripts. Notably, nuclear receptor agonists exhibit potent inhibition of viral gene transcription from cccDNA. Small molecule inhibitors repress HBV X protein-mediated transcription from cccDNA, while small interfering RNAs and single-stranded oligonucleotides result in transcript degradation from both cccDNA and integrated templates. These antivirals mediate their effects by reducing viral transcripts abundance, some leading to a loss of surface antigen expression, and they can potentially be added to the arsenal of drugs with demonstrable anti-HBV activity. Thus, these candidates deserve special attention for future repurposing or further development as anti-HBV therapeutics.
... However, how SMC5/6 blocks transcription from cccDNA remains unclear. SMC5/6 may recognize an HBV-specific sequence motif but this seems unlikely as HBx-mediated augmentation of gene transcription is limited to extrachromosomal DNA templates: this phenomenon is not observed for HBV genomes with identical sequences integrated into host chromosomes [88]. Another possibility is that SMC5/6 senses the supercoiled cccDNA at transcription initiation and topologically fixes cccDNA in the supercoiled state, at which point transcription elongation is not possible. ...
Approximately 240 million people are chronically infected with hepatitis B virus (HBV), despite four decades of an effective HBV vaccine. During chronic infection, HBV forms two distinct templates responsible for viral gene transcription: (1) episomal covalently closed circular (ccc)DNA and (2) host-genome integrated viral templates. Multiple ubiquitous and liver-specific transcription factors are recruited onto these templates and modulate viral gene transcription. This review details the latest developments in antivirals that inhibit HBV gene transcription, and their impact on the stability of viral transcripts. Notably, nuclear receptor agonists exhibit potent inhibition of viral gene transcription from cccDNA, small molecule inhibitors repress HBV X protein-mediated transcription from cccDNA and small interfering RNAs and single-stranded oligonucleotides result in transcript degradation from both cccDNA and integrant templates. These antivirals mediate their effects by reducing viral transcripts abundance, eventually leading to loss of surface antigen expression, and can potentially be added to the arsenal of drugs with demonstrable anti-HBV activity. Thus, these candidates deserve special attention for future repurposing or further development as anti-HBV therapeutics.
... To date, no direct evidence has been reported regarding the role of CRL4B in HBV replication. However, crystallographic and functional analyses have revealed an interaction of HBx with UV-damaged DNA binding protein 1 (DDB1), an evolutionarily conserved adaptor protein for CUL4-RING E3 ubiquitin ligases 20 that is required for HBx protein stability 21 and has biological roles in viral promoter activation and cell cycle dysregulation 22,23 . Moreover, CRL4 is recruited by HBx and functions in the ubiquitination and degradation of the structural maintenance of chromosomes (SMC) complex proteins SMC5/6, thus restricting HBV replication by inhibiting HBV gene expression 24 . ...
Objective:
Hepatitis B virus (HBV) infection is a major public health problem worldwide. However, the regulatory mechanisms underlying HBV replication remain unclear. Cullin 4B-RING ubiquitin E3 ligase (CRL4B) is involved in regulating diverse physiological and pathophysiological processes. In our study, we aimed to explain the role of CUL4B in HBV infection.
Methods:
Cul4b transgenic mice or conditional knockout mice, as well as liver cell lines with CUL4B overexpression or knockdown, were used to assess the role of CUL4B in HBV replication. Immunoprecipitation assays and immunofluorescence staining were performed to study the interaction between CUL4B and HBx. Cycloheximide chase assays and in vivo ubiquitination assays were performed to evaluate the half-life and the ubiquitination status of HBx.
Results:
The hydrodynamics-based hepatitis B model in Cul4b transgenic or conditional knockout mice indicated that CUL4B promoted HBV replication (P < 0.05). Moreover, the overexpression or knockdown system in human liver cell lines validated that CUL4B increased HBV replication in an HBx-dependent manner. Importantly, immunoprecipitation assays and immunofluorescence staining showed an interaction between CUL4B and HBx. Furthermore, CUL4B upregulated HBx protein levels by inhibiting HBx ubiquitination and proteasomal degradation (P < 0.05). Finally, a positive correlation between CUL4B expression and HBV pgRNA level was observed in liver tissues from HBV-positive patients and HBV transgenic mice.
Conclusions:
CUL4B enhances HBV replication by interacting with HBx and disrupting its ubiquitin-dependent proteasomal degradation. CUL4B may therefore be a potential target for anti-HBV therapy.
... The outbreak of coronavirus disease 2019 (COVID- 19) The primary etiology of COVID-19 is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) of subfamily Coronavirinae in family Coronaviridae in order Nidovirales. 3 The positive-sense single stranded RNA genome of SARS-CoV-2 shares 80% sequence homology with severe acute respiratory syndrome coronavirus (SARS-CoV) and 50% homology to middle east respiratory syndrome coronavirus (MERS-CoV). 1,4 Compared to other known human coronaviruses associated with only mild symptoms, SARS-CoV, MERS-CoV and SARS-CoV-2 cause severe disease and result in higher mortality. ...
Background & Aims
The outbreak of coronavirus disease 2019 (COVID‐19) has been declared a pandemic. Although COVID‐19 is caused by infection in the respiratory tract, extrapulmonary manifestations including dysregulation of the immune system and hepatic injury have been observed. Given the high prevalence of hepatitis B virus (HBV) infection in China, we sought to study the impact of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) and HBV coinfection in patients.
Methods
Blood samples of 50 SARS‐CoV‐2 and HBV coinfected patients, 56 SARS‐CoV‐2 mono‐infected patients, 57 HBeAg‐negative chronic HBV patient controls and 57 healthy controls admitted to Renmin Hospital of Wuhan University were collected in this study. Complete blood count and serum biochemistry panels including markers indicative of liver functions were performed. Cytokines including IFN‐γ, TNF‐α, IL‐2, IL‐4, IL‐6 and IL‐10 were evaluated. T cell, B cell and NK cell counts were measured using flow cytometry.
Results
SARS‐CoV‐2 and HBV coinfection did not significantly affect the outcome of the COVID‐19. However, at the onset of COVID‐19, SARS‐CoV‐2 and HBV coinfected patients showed more severe monocytopenia and thrombocytopenia as well as more disturbed hepatic function in albumin production and lipid metabolism. Most of the disarrangement could be reversed after recovery from COVID‐19.
Conclusions
While chronic HBV infection did not predispose COVID‐19 patients to more severe outcomes, our data suggests SARS‐CoV‐2 and HBV coinfection poses a higher extent of dysregulation of host functions at the onset of COVID‐19. Thus, caution needs to be taken with the management of SARS‐CoV‐2 and HBV coinfected patients.
... It is made CC-BY-NC-ND 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The lentivirus vectors pWPT expressing GFP, GFP-HBx, and GFP-WHx have been 379 previously described (Leupin, Bontron, Schaeffer, & Strubin, 2005;Lin-Marq, Bontron, 380 Leupin, & Strubin, 2001;van Breugel et al., 2012). The X coding regions (synthesized by 381 Genewiz) from hepadnaviruses infecting the New World wooly monkey (Lagothrix) 382 (WMHBx), three distant bat species including the roundleaf bat (Hipposideros cf. ...
Infection with Hepatitis B virus (HBV) is a major cause of liver disease and cancer in humans. HBVs (family Hepadnaviridae ) have been associated with mammals for millions of years. Recently, the Smc5/6 complex, known for its essential housekeeping functions in genome maintenance, was identified as an antiviral restriction factor of human HBV. The virus has however developed a counteraction mechanism by degrading the complex via its regulatory HBx protein. Whether the antiviral activity of the Smc5/6 complex against hepadnaviruses is an important and evolutionary-conserved function is unknown. Here, we used a combined evolutionary and functional approach to address this question. We first performed phylogenetic and positive selection analyses of the six Smc5/6 complex subunits and found that they have been highly conserved in primates and mammals. Yet, the Smc6 subunit showed marks of adaptive evolution, potentially reminiscent of virus-host “arms-race” We then functionally tested the HBx from six very divergent hepadnaviruses now naturally infecting primates, rodents, and bats. Despite little sequence homology, we demonstrate that these HBx efficiently degraded mammalian Smc5/6 complexes, independently of the host species and of the sites under positive selection. Importantly, all also rescued the replication of an HBx-deficient HBV in primary human hepatocytes. These findings point to an evolutionary-conserved requirement for Smc5/6 inactivation by HBx, showing that the Smc5/6 antiviral activity has been an important defense mechanism against hepadnaviruses in mammals. Interestingly, Smc5/6 may further be a restriction factor of other yet unidentified viruses that have driven some of its adaptation.
Importance
Infection with hepatitis B virus (HBV) led to 887000 human deaths in 2015. HBV has been co-evolving with mammals for millions of years. Recently, the Smc5/6 complex, known for its essential housekeeping functions, was identified as a restriction factor of human HBV antagonized by the regulatory HBx protein. Here, we address whether the antiviral activity of Smc5/6 is an important evolutionary-conserved function. We found that all six subunits of Smc5/6 have been conserved in primates with only Smc6 showing signatures of “evolutionary arms-race” Using evolutionary-guided functional assays that include infections of primary human hepatocytes, we demonstrate that HBx from very divergent mammalian HBVs could all efficiently antagonize Smc5/6, independently of the host species and sites under positive selection. These findings show that the Smc5/6 antiviral activity against HBV is an important function in mammals. It also raises the intriguing possibility that Smc5/6 restricts other, yet unidentified viruses.
... C'est une protéine multifonctionnelle produite à un niveau très bas, durant l'infection aiguë et chronique, mais essentielle à la réplication complète du virus chez les orthohepadnavirus(371- 373). Elle transactive les promoteurs de l'ADNccc mais pas de l'ADN viral intégré(341,374). Bien qu'HBx ne semble pas pouvoir se fixer directement à l'ADN, elle interagit avec des facteurs de transcription et régule l'expression de gènes cellulaires impliqués dans l'apoptose ou la réparation de l'ADN(375). ...
Plusieurs données expérimentales suggèrent que la protéine Core du virus de l’Hépatite B (HBV), en plus de ses fonctions structurales pour la formation des nucléocapsides dans le cytoplasme, pourrait avoir des fonctions régulatrices importantes dans le noyau des hépatocytes infectés. En effet, Core s’associe à l’ADNccc et aux promoteurs de certains gènes cellulaires dans le noyau des hépatocytes infectés et pourrait ainsi contrôler leur régulation transcriptionnelle. De plus, de par sa capacité à lier les ARN, elle pourrait également participer au métabolisme post-transcriptionnel de gènes viraux et/ou cellulaires. Pour caractériser ces fonctions, nous avons réalisé une analyse protéomique des facteurs cellulaires qui interagissent avec la protéine Core dans le noyau d’hépatocytes humains. Cet interactome a mis en évidence un grand nombre de protéines de liaison aux ARN (RBP), qui participent au métabolisme des ARN et en particulier aux mécanismes d’épissage. Deux interactants majeurs de Core ont été plus particulièrement étudiés, SRSF10 et RBMX, impliqués notamment dans l’épissage et la réparation de l’ADN. Une analyse fonctionnelle effectuée par une approche siRNA a montré que SRSF10 et RBMX affectent différemment le niveau des ARN viraux, vraisemblablement en agissant à des étapes différentes du cycle viral. De même, un composé ciblant l’activité de certaines RBP diminue fortement la réplication d’HBV en affectant l’accumulation des ARN viraux. Ainsi, ces résultats suggèrent que Core pourrait interagir avec certaines RBP pour contrôler le destin des ARN viraux et/ou cellulaires, une piste intéressante pour le développement de nouvelles stratégies antivirales ciblant l’hôte
... A third possibility is epigenetic regulation of cccDNA through histone acetylation and methylation by host cellular machinery [23][24][25]. Fourth, hepatitis B virus X protein (HBX) has been described as being responsible for upregulating HBV transcription, thus HBX may autoregulate transcription [26,27]. In addition, the hepatitis B core protein may contribute to transcriptional regulation by directing epigenetic modifications on cccDNA [24]. ...
Background
Hepatitis B virus (HBV) is a leading cause of liver failure and hepatocellular carcinoma. Approximately 10% of people with HIV also have HBV and are at higher risk of liver disease progression than in HBV monoinfection. Antivirals, common to HIV and HBV, suppress HBV DNA levels but do not eradicate virus because the transcriptional template, covalently closed circular DNA (cccDNA), is long lived in infected hepatocytes.
Methods
Using single-cell laser capture microdissection, we isolated >1100 hepatocytes from 5 HIV/HBV coinfected persons with increasing exposure to HBV antivirals (HB1–HB5; no exposure to >7 years exposure), quantifying cccDNA and pregenomic RNA (pgRNA) in each cell using droplet digital polymerase chain reaction.
Results
The proportion of infected hepatocytes decreased with antiviral exposure from 96.4% (HB1) to 29.8% (HB5). Upper cccDNA range and median pgRNA decreased from HB1 to HB5 (P < .05 for both). The amount of pgRNA transcribed per cccDNA also decreased from HB1 to HB5 (P < .05). Cells with inactive pgRNA transcription were enriched from 0% (HB1) to 14.3% (HB5) of infected hepatocytes.
Conclusions
cccDNA transcription is reduced in HIV/HBV coinfected people with longer antiviral duration. Understanding HBV transcriptional regulation may be critical to develop a functional cure.
... Regarding IFN-α, the susceptibility assays show that it could reduce secNLuc expression in HBV-NLuc-35 cells. Because IFN-α could inhibit HBV replication, transcription, and expression [38] , and cccDNA was an exclusive template for virus transcription, this process depended on the presence of HBx [37,39] , implying that secNLuc recombinant cccDNA may be present in HBV-NLuc-35 cells. Altogether, these data demonstrated that new cell lines could secrete secNLuc recombinant viruses and were sensitive to existing anti-HBV drugs. ...
Background:
Previously, we have successfully constructed replication-competent hepatitis B virus (HBV) vectors by uncoupling the P open reading frame (ORF) from the preC/C ORF to carefully design the transgene insertion site to overcome the compact organization of the HBV genome and maintain HBV replication competence. Consequently, the replication-competent HBV vectors carrying foreign genes, including pCH-BsdR, carrying blasticidin resistance gene (399 bp), and pCH-hrGFP, carrying humanized renilla green fluorescent protein gene (720 bp), were successfully obtained. However, the replication efficiency of the former is higher but it is tedious to use, while that of the latter is poor and cannot be quantified. Hence, we need to search for a new reporter gene that is convenient and quantifiable for further research.
Aim:
To establish a helpful tool for intracellular HBV replication and anti-viral drugs screening studies.
Methods:
We utilized the replication-competent HBV viral vectors constructed by our laboratory, combined with the secreted luciferase reporter gene, to construct replication-competent HBV vectors expressing the reporter gene secretory Nanoluc Luciferase (SecNluc). HepG2.TA2-7 cells were transfected with this vector to obtain cell lines with stably secreted HBV particles carrying secNluc reporter gene.
Results:
The replication-competent HBV vector carrying the SecNluc reporter gene pCH-sNLuc could produce all major viral RNAs and a full set of envelope proteins and achieve high-level secreted luciferase expression. HBV replication intermediates could be produced from this vector. Via transfection with pTRE-sNLuc and selection by hygromycin, we obtained isolated cell clones, named HBV-NLuc-35 cells, which could secrete secNLuc recombinant viruses, and were sensitive to existing anti-HBV drugs. Using differentiated HepaRG cells, it was verified that recombinant HBV possessed infectivity.
Conclusion:
Our research demonstrated that a replication-competent HBV vector carrying a secreted luciferase transgene possesses replication and expression ability, and the established HBV replication and expression cell lines could stably secrete viral particles carrying secNluc reporter gene. More importantly, the cell line and the secreted recombinant viral particles could be used to trace HBV replication or infection.
... This defect is dependent on Vpr binding to DCAF CRL4 as it is fully abrogated upon infection with Vpr mutants (Q65R or H71R) lacking DCAF CRL4 interactions. It should be noted that numerous viruses besides HIV-1, most notably Hepatitis B virus, can also manipulate the E3 ubiquitin ligase DCAF CRL4 to enhance transcription (71). While, the mechanism of HIV-1 Vpr-mediated transcriptional enhancement remains unclear, previous research has shown Vpr-mediated degradation of HDACs (38) and members of the NuRD chromatin remodeling complex (72) which may globally enhance transcription. ...
Vpr is a 14 kDa accessory protein conserved amongst extant primate lentiviruses that is required for virus replication in vivo. Although many functions have been attributed to Vpr, its primary role, and the function under selective pressure in vivo, remains elusive. The minimal importance of Vpr in infection of activated CD4+ T cells in vitro suggests that its major importance lies in overcoming restriction to virus replication in non-cycling myeloid cell populations, such as macrophages and dendritic cells. HIV-1 replication is attenuated in the absence of Vpr in myeloid cells such as monocyte-derived dendritic cells (MDDCs) and macrophages, and is correlated with the ability of Vpr to overcome a post-integration transcriptional defect in these cells. Intriguingly, recent identification of the human hub silencing (HUSH) complex as a target for DCAFCRL4-mediated degradation by numerous ancestral SIV Vpr alleles, and the Vpr paralog Vpx, signifies the potential function of HIV-1 Vpr to alter yet-to-be identified chromatin remodeling complexes and prevent host-mediated transcriptional repression of both invading viral genomes and pro-inflammatory responses. Myeloid cells constitute an important bridge between innate and adaptive immune responses to invading pathogens. Here, we seek to illustrate the numerous means by which Vpr manipulates the myeloid cellular environment and facilitates virus replication, myeloid cell-dependent HIV transmission, and systemic virus dissemination.
... Recent studies demonstrated that cccDNA transcription is inhibited by the structural maintenance of chromosomes 5/6 complex (Smc5/6) and that HBx redirects a cellular DNA damage-binding protein 1 (DDB1)-containing E3 ubiquitin ligase to target this complex for degradation (7,8). This newly defined mechanism also accounts for the ability of HBx to transactivate episomal DNA templates (7,9). ...
The structural maintenance of chromosomes 5/6 complex (Smc5/6) is a host restriction factor that suppresses HBV transcription. HBV counters this restriction by expressing HBV X protein (HBx), which redirects a host ubiquitin ligase to target Smc5/6 for degradation. Despite this recent advance in understanding HBx function, the key regions and residues of HBx required for Smc5/6 degradation have not been determined. In the present study, we performed biochemical, biophysical, and cell-based analyses of HBx. By doing so, we mapped the minimal functional region of HBx and identified a highly conserved CCCH motif in HBx that is likely responsible for coordinating zinc and is essential for HBx function. We also developed a method to produce soluble recombinant HBx protein that likely adopts a physiologically relevant conformation. Collectively, this study provides new insights into the HBx structure-function relationship and suggests a new approach for structural studies of this enigmatic viral regulatory protein.
... By this simple yet elegant mechanism, viruses exploit the cell intrinsic ubiquitin-proteasome system to defeat antiviral immunity. Well-known examples of viral exploitation of DDB1 and/or CRLs include the hepatitis B virus (HBV) protein HBx [84,[89][90][91][92], the HIV-1-and HIV-2-encoded protein Vpr [93][94][95][96][97][98][99][100][101][102], HIV-2 Vpx [103][104][105][106][107][108][109], parainfluenza virus (PIV) V proteins [78,[110][111][112], bovine herpesvirus 1 (BoHV-1) VP8 [113,114], murine gamma herpesvirus (MHV68) M2 [115], as well as the cytomegalovirus proteins pM27 [116][117][118], pUL35 [119], and pUL145 [120]. ...
Human cytomegalovirus (HCMV) is a ubiquitous betaherpesvirus that frequently causes morbidity and mortality in individuals with insufficient immunity, such as transplant recipients, AIDS patients, and congenitally infected newborns. Several antiviral drugs are approved to treat HCMV infections. However, resistant HCMV mutants can arise in patients receiving long-term therapy. Additionally, side effects and the risk to cause birth defects limit the use of currently approved antivirals against HCMV. Therefore, the identification of new drug targets is of clinical relevance. Recent work identified DNA-damage binding protein 1 (DDB1) and the family of the cellular cullin (Cul) RING ubiquitin (Ub) ligases (CRLs) as host-derived factors that are relevant for the replication of human and mouse cytomegaloviruses. The first-in-class CRL inhibitory compound Pevonedistat (also called MLN4924) is currently under investigation as an anti-tumor drug in several clinical trials. Cytomegaloviruses exploit CRLs to regulate the abundance of viral proteins, and to induce the proteasomal degradation of host restriction factors involved in innate and intrinsic immunity. Accordingly, pharmacological blockade of CRL activity diminishes viral replication in cell culture. In this review, we summarize the current knowledge concerning the relevance of DDB1 and CRLs during cytomegalovirus replication and discuss chances and drawbacks of CRL inhibitory drugs as potential antiviral treatment against HCMV.
... HIV-2 additionally expresses Vpx, which exploits DDB1 and CRLs, also via VprBP, to induce the degradation of SAMHD1 enabling replication in myeloid and dendritic cells (31)(32)(33)(34)(35)(36)(37). HBx and WHx, derived from HBV and the Woodchuck hepatitis virus (WHV), respectively, bind DDB1 and CRLs to induce the degradation of restriction factors such as SMC5/6 (38)(39)(40)(41)(42). ...
Viruses and hosts are situated in a molecular arms race. To avoid morbidity and mortality, hosts evolved antiviral restriction factors. These restriction factors exert selection pressure on the viruses and drive viral evolution toward increasingly efficient immune antagonists. Numerous viruses exploit cellular DNA damage-binding protein 1 (DDB1)-containing Cullin RocA ubiquitin ligases (CRLs) to induce the ubiquitination and subsequent proteasomal degradation of antiviral factors expressed by their hosts. To establish a comprehensive understanding of the underlying protein interaction networks, we performed immuno-affinity precipitations for a panel of DDB1-interacting proteins derived from viruses such as mouse cytomegalovirus (MCMV, Murid herpesvirus [MuHV] 1), rat cytomegalovirus Maastricht MuHV2, rat cytomegalovirus English MuHV8, human cytomegalovirus (HCMV), hepatitis B virus (HBV), and human immunodeficiency virus (HIV). Cellular interaction partners were identified and quantified by mass spectrometry (MS) and validated by classical biochemistry. The comparative approach enabled us to separate unspecific interactions from specific binding partners and revealed remarkable differences in the strength of interaction with DDB1. Our analysis confirmed several previously described interactions like the interaction of the MCMV-encoded interferon antagonist pM27 with STAT2. We extended known interactions to paralogous proteins like the interaction of the HBV-encoded HBx with different Spindlin proteins and documented interactions for the first time, which explain functional data like the interaction of the HIV-2-encoded Vpr with Bax. Additionally, several novel interactions were identified, such as the association of the HIV-2-encoded Vpx with the transcription factor RelA (also called p65). For the latter interaction, we documented a functional relevance in antagonizing NF-κB-driven gene expression. The mutation of the DDB1 binding interface of Vpx significantly impaired NF-κB inhibition, indicating that Vpx counteracts NF-κB signaling by a DDB1- and CRL-dependent mechanism. In summary, our findings improve the understanding of how viral pathogens hijack cellular DDB1 and CRLs to ensure efficient replication despite the expression of host restriction factors.
... HBV cccDNA is persistent in the hepatocyte nucleus, functioning as a minichromosome and as a transcriptional template for all HBV viral RNAs. 5 Transcription of the viral genome is promoted by HBV regulatory X (HBx) protein. 6,7 Previous studies have suggested that this process requires the binding of HBx with the host protein damage-specific DNA-binding protein 1 (DDB1), 8,9 but the underlying mechanisms of the interaction of HBx with DDB1 and the promotion of viral transcription have long remained unknown. Recently, HBx was found to assemble an HBx-DDB1-CUL4-ROC1 E3 ligase complex to target structural maintenance of chromosomes 5/6 (Smc5/6), a host restriction factor that blocks viral transcription, for ubiquitination and degradation, resulting in enhanced viral transcription from cccDNA. ...
Background & Aims
Hepatitis B virus (HBV) infection is a major health concern worldwide. Although currently used nucleos(t)ide analogs efficiently inhibit viral replication, viral proteins transcribed from the episomal viral covalently closed circular DNA (cccDNA) minichromosome continue to be expressed long-term. Because high viral RNA or antigen loads may play a biological role during this chronicity, the elimination of viral products is an ultimate goal of HBV treatment. HBV regulatory protein X (HBx) was recently found to promote transcription of cccDNA with degradation of Smc5/6 through the interaction of HBx with the host protein DDB1. Here, this protein–protein interaction was considered as a new molecular target of HBV treatment.
Methods
To identify candidate compounds that target the HBx–DDB1 interaction, a newly constructed split luciferase assay system was applied to comprehensive compound screening. The effects of the identified compounds on HBV transcription and cccDNA maintenance were determined using HBV minicircle DNA, which mimics HBV cccDNA, and the natural HBV infection model of human primary hepatocytes.
Results
We show that nitazoxanide (NTZ), a thiazolide anti-infective agent that has been approved by the FDA for protozoan enteritis, efficiently inhibits the HBx–DDB1 protein interaction. NTZ significantly restores Smc5 protein levels and suppresses viral transcription and viral protein production in the HBV minicircle system and in human primary hepatocytes naturally infected with HBV.
Conclusions
These results indicate that NTZ, which targets an HBV-related viral–host protein interaction, may be a promising new therapeutic agent and a step toward a functional HBV cure.
... As a transactivating factor, HBx can bind to a variety of factors related to transcription and gene regulation and widely activate the promoters of the virus and cells. Thus, HBx is involved in regulation of host gene expression [17][18][19][20][21]. ...
Background
Hepatitis B virus (HBV) infection causes acute and chronic liver diseases that can eventually develop into cirrhosis and hepatocellular carcinoma (HCC), but the carcinogenesis of HBV is not fully understood. Carboxyl-terminal-binding protein 2 (CtBP2) plays an important role in tumorigenesis and progression. The aim of this study was to investigate the effect of HBV on CtBP2 expression and to explore its mechanism.
Methods
Real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) and western blotting were used to evaluate the CtBP2 mRNA and protein expression levels in tissues and cells. The HBV infectious clone pHBV1.3 and plasmids expressing a single gene of the HBV genome were cotransfected with the CtBP2 gene promoter pGL3-CtBP2 into the human hepatoma cell line HepG2, and luciferase activity was determined using a luminometer.
Results
CtBP2 expression was higher in HBV-related HCC tissues than in paracancerous tissues. CtBP2 expression was higher in HepG2.2.15 cells integrated with the HBV genome than in HepG2 cells. pHBV1.3 upregulated CtBP2 mRNA and protein expression. The HBV X gene significantly activated CtBP2 gene promoter activity, and CtBP2 mRNA and protein expression were upregulated by the HBV X gene. This activation effect was enhanced by the increase in the dose of the X gene, showing metrological dependence.
Conclusion
HBV may be involved in the occurrence and development of HCC by upregulating CtBP2 expression.
... Intriguingly, none of these reporters was stimulated by HBx when integrated into the host genome. This suggests the existence of a yet unexplained selective transactivation mechanism, where HBx specifically targets episomal DNA templates, but not integrated DNA of the same sequence [61]. A valid explanation for this selective transactivation pattern, which apparently does not affect integrated sequences, would be its predominance in inactive chromatin compartments that have no access to active transcription compartments. ...
Background
In hepatocyte nuclei, hepatitis B virus (HBV) genomes occur episomally as covalently closed circular DNA (cccDNA). The HBV X protein (HBx) is required to initiate and maintain HBV replication. The functional nuclear localization of cccDNA and HBx remains unexplored.
Results
To identify virus–host genome interactions and the underlying nuclear landscape for the first time, we combined circular chromosome conformation capture (4C) with RNA-seq and ChIP-seq. Moreover, we studied HBx-binding to HBV episomes. In HBV-positive HepaRG hepatocytes, we observed preferential association of HBV episomes and HBx with actively transcribed nuclear domains on the host genome correlating in size with constrained topological units of chromatin. Interestingly, HBx alone occupied transcribed chromatin domains. Silencing of native HBx caused reduced episomal HBV stability.
Conclusions
As part of the HBV episome, HBx might stabilize HBV episomal nuclear localization. Our observations may contribute to the understanding of long-term episomal stability and the facilitation of viral persistence. The exact mechanism by which HBx contributes to HBV nuclear persistence warrants further investigations.
... Expression plasmids. The lentivirus vectors pWPT expressing GFP, GFP-HBx, and GFP-WHx have been previously described (60)(61)(62). The X coding regions (synthesized by Genewiz) from hepadnaviruses infecting the New World wooly monkey (Lagothrix) (WMHBx) and three distant bat species, including the roundleaf bat (Hipposideros cf. ...
Infection with hepatitis B virus (HBV) led to 887,000 human deaths in 2015. HBV has been coevolving with mammals for millions of years. Recently, the Smc5/6 complex, which has essential housekeeping functions, was identified as a restriction factor of human HBV antagonized by the regulatory HBx protein. Here we address whether the antiviral activity of Smc5/6 is an important evolutionarily conserved function. We found that all six subunits of Smc5/6 have been conserved in primates, with only Smc6 showing signatures of an “evolutionary arms race.” Using evolution-guided functional analyses that included infections of primary human hepatocytes, we demonstrated that HBx proteins from very divergent mammalian HBVs could all efficiently antagonize Smc5/6, independently of the host species and sites under positive selection. These findings show that Smc5/6 antiviral activity against HBV is an important function in mammals. They also raise the intriguing possibility that Smc5/6 may restrict other, yet-unidentified viruses.
... The mechanism by which Smc5/6 recognizes cccDNA is still unknown. It appears that the recognition is not sequence specific because the HBx transactivation activity, which is dependent on Smc5/6 degradation, is observed in a variety of cellular and viral promoter and regulatory elements only when they are present in an episomal context but not when the same elements are integrated in the host chromosomal DNA [53]. Recent data suggest that Smc5/6 localization in promyelocytic nuclear bodies (PML-NBs) might be required for cccDNA silencing [47]. ...
Hepatitis B virus (HBV) is a major etiologic agent of acute and chronic hepatitis, and end-stage liver disease. Establishment of HBV infection, progression to persistency and pathogenesis are determined by viral and cellular factors, some of which remain still undefined. Key steps of HBV life cycle e.g., transformation of genomic viral DNA into transcriptionally active episomal DNA (cccDNA) or transcription of viral mRNAs from cccDNA, take place in the nucleus of infected cells and strongly depend on enzymatic activities provided by cellular proteins. In this regard, DNA damage response (DDR) pathways and some DDR proteins are being recognized as important factors regulating the infection. On one hand, HBV highjacks specific DDR proteins to successfully complete some of the steps of its life cycle. On the other hand, HBV subverts DDR pathways to presumably create a cellular environment that favours its replication. Direct consequences of these interactions are: HBV DNA integration into host chromosomal DNA, and accumulation of mutations in host chromosomal DNA that could eventually trigger carcinogenic processes, which would explain in part the incidence of hepatocellular carcinoma in chronically infected patients. Unravelling the interactions that HBV establishes with DDR pathways might help identify new molecular targets for therapeutic intervention.
... HBx is a small regulatory protein that is essential for viral gene expression and has been implicated in the pathogenesis of HCC [36][37][38]. It also seems to play a role in a number of cellular processes influencing the HBV life cycle including epigenetic control of the cccDNA minichromosome. ...
Hepatitis B virus (HBV) infection is a global health threat with 240 million chronic carriers at high risk to develop hepatocellular carcinoma. Current antiviral treatment can efficiently control viral replication and reduce liver inflammation, but is still quite far from achieving a cure. Significant progress has been made in understanding the virus life cycle and virus–host interaction in the past few years. With identification of the HBV receptor, cell-culture infection systems have become available that allow drug screening and establishing a pipeline of potential antivirals targeting either viral or host factors. Most of the candidate antivirals summarized in this review are still in preclinical development, but some have already entered or are about to enter early clinical trials.
... Therapeutic targeting of the HBV X protein (HBx) is attractive because this viral protein is essential for HBV infection in vivo [4][5][6] and is required for the initiation and maintenance of viral replication after in vitro infection [7]. Recent work has indicated that HBx plays this key role in the viral lifecycle by maintaining the covalently-closed circular DNA (cccDNA) HBV genome in a transcriptionally active state [7][8][9]. Pharmaceutical targeting of HBx therefore has the potential to transcriptionally silence cccDNA. This would be an attractive therapeutic response since reducing viral antigen levels may restore effective antiviral immunity and drive patients towards functional cure [10]. ...
The structural maintenance of chromosome 5/6 complex (Smc5/6) is a restriction factor that represses hepatitis B virus (HBV) transcription. HBV counters this restriction by expressing HBV X protein (HBx), which targets Smc5/6 for degradation. However, the mechanism by which Smc5/6 suppresses HBV transcription and how HBx is initially expressed is not known. In this study we characterized viral kinetics and the host response during HBV infection of primary human hepatocytes (PHH) to address these unresolved questions. We determined that Smc5/6 localizes with Nuclear Domain 10 (ND10) in PHH. Co-localization has functional implications since depletion of ND10 structural components alters the nuclear distribution of Smc6 and induces HBV gene expression in the absence of HBx. We also found that HBV infection and replication does not induce a prominent global host transcriptional response in PHH, either shortly after infection when Smc5/6 is present, or at later times post-infection when Smc5/6 has been degraded. Notably, HBV and an HBx-negative virus establish high level infection in PHH without inducing expression of interferon-stimulated genes or production of interferons or other cytokines. Our study also revealed that Smc5/6 is degraded in the majority of infected PHH by the time cccDNA transcription could be detected and that HBx RNA is present in cell culture-derived virus preparations as well as HBV patient plasma. Collectively, these data indicate that Smc5/6 is an intrinsic antiviral restriction factor that suppresses HBV transcription when localized to ND10 without inducing a detectable innate immune response. Our data also suggest that HBx protein may be initially expressed by delivery of extracellular HBx RNA into HBV-infected cells.
Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA), serving as the viral persistence form and transcription template of HBV infection, hijacks host histone and non-histone proteins to form a minichromosome and utilizes posttranslational modifications (PTMs) "histone code" for its transcriptional regulation. HBV X protein (HBx) is known as a cccDNA transcription activator. In this study we established a dual system of the inducible reporter cell lines modelling infection with wildtype (wt) and HBx-null HBV, both secreting HA-tagged HBeAg as a semi-quantitative marker for cccDNA transcription. The cccDNA-bound histone PTM profiling of wt and HBx-null systems, using chromatin immunoprecipitation coupled with quantitative PCR (ChIP-qPCR), confirmed that HBx is essential for maintenance of cccDNA at transcriptionally active state, characterized by active histone PTM markers. Differential proteomics analysis of cccDNA minichromosome established in wt and HBx-null HBV cell lines revealed group-specific hits. One of the hits in HBx-deficient condition was a non-histone host DNA-binding protein high mobility group box 1 (HMGB1). Its elevated association to HBx-null cccDNA was validated by ChIP-qPCR assay in both the HBV stable cell lines and infection systems in vitro. Furthermore, experimental downregulation of HMGB1 in HBx-null HBV inducible and infection models resulted in transcriptional re-activation of the cccDNA minichromosome, accompanied by a switch of the cccDNA-associated histones to euchromatic state with activating histone PTMs landscape and subsequent upregulation of cccDNA transcription. Mechanistically, HBx interacts with HMGB1 and prevents its binding to cccDNA without affecting the steady state level of HMGB1. Taken together, our results suggest that HMGB1 is a novel host restriction factor of HBV cccDNA with epigenetic silencing mechanism, which can be counteracted by viral transcription activator HBx.
Hepatitis B virus infection can develop into chronic infection, cirrhosis, and hepatocellular carcinoma. Treatment of chronic hepatitis B requires novel approaches to directly target the viral minichromosome, which is responsible for the persistence of the disease.
The hepatitis B virus (HBV) infects 257 million people worldwide. HBV infection requires establishment and persistence of covalently closed circular (ccc) DNA, a viral episome, in nucleus. Here, we study cccDNA spatial localization in the 3D host genome by using chromosome conformation capture-based sequencing analysis and fluorescence in situ hybridization (FISH). We show that transcriptionally inactive cccDNA is not randomly distributed in host nucleus. Rather, it is preferentially accumulated at specialized areas, including regions close to chromosome 19 (chr.19). Activation of the cccDNA is apparently associated with its re-localization, from a pre-established heterochromatin hub formed by 5 regions of chr.19 to transcriptionally active regions formed by chr.19 and nearby chromosomes including chr.16, 17, 20, and 22. This active versus inactive positioning at discrete regions of the host genome is primarily controlled by the viral HBx protein and by host factors including the structural maintenance of chromosomes protein 5/6 (SMC5/6) complex.
Hepatitis B virus (HBV) is the leading cause of hepatocellular carcinoma (HCC) worldwide. The prolyl hydroxylase domain (PHD)-hypoxia inducible factor (HIF) pathway is a key mammalian oxygen sensing pathway and is frequently perturbed by pathological states including infection and inflammation. We discovered a significant upregulation of hypoxia regulated gene transcripts in patients with chronic hepatitis B (CHB) in the absence of liver cirrhosis. We used state-of-the-art in vitro and in vivo HBV infection models to evaluate a role for HBV infection and the viral regulatory protein HBx to drive HIF-signalling. HBx had no significant impact on HIF expression or associated transcriptional activity under normoxic or hypoxic conditions. Furthermore, we found no evidence of hypoxia gene expression in HBV de novo infection, HBV infected human liver chimeric mice or transgenic mice with integrated HBV genome. Collectively, our data show clear evidence of hypoxia gene induction in CHB that is not recapitulated in existing models for acute HBV infection, suggesting a role for inflammatory mediators in promoting hypoxia gene expression.
The host structural maintenance of chromosomes 5/6 complex (Smc5/6) suppresses hepatitis B virus (HBV) transcription. HBV counters this restriction by expressing the X protein (HBx), which redirects the cellular DDB1-containing E3 ubiquitin ligase to target Smc5/6 for degradation. However, the details of how HBx modulates the interaction between DDB1 and Smc5/6 remain to be determined. In this study, we performed biophysical analyses of recombinant HBx and functional analysis of HBx mutants in HBV-infected primary human hepatocytes (PHH) to identify key regions and residues that are required for HBx function. We determined that recombinant HBx is soluble and exhibits stoichiometric zinc binding when expressed in the presence of DDB1. Mass spectrometry-based hydrogen-deuterium exchange and cysteine-specific chemical footprinting of the HBx:DDB1 complex identified several HBx cysteine residues (located between amino acids 61-137) that are likely involved in zinc binding. These cysteine residues did not form disulphide bonds in HBx expressed in human cells. In line with the biophysical data, functional analysis demonstrated that HBx amino acids 45-140 are required for Smc6 degradation and HBV transcription in PHH. Furthermore, site-directed mutagenesis determined that C61, C69, C137 and H139 are necessary for HBx function, although are likely not essential for DDB1 binding. This CCCH motif is highly conserved in HBV as well as in the X protein from various mammalian hepadnaviruses. Collectively, our data indicate that the essential HBx cysteine and histidine residues form a zinc-binding motif that is required for HBx function.IMPORTANCE The structural maintenance of chromosomes 5/6 complex (Smc5/6) is a host restriction factor that suppresses HBV transcription. HBV counters this restriction by expressing HBV X protein (HBx), which redirects a host ubiquitin ligase to target Smc5/6 for degradation. Despite this recent advance in understanding HBx function, the key regions and residues of HBx required for Smc5/6 degradation have not been determined. In the current study, we performed biochemical, biophysical and cell-based analyses of HBx. By doing so, we mapped the minimal functional region of HBx and identified a highly conserved CCCH motif in HBx that is likely responsible for coordinating zinc and is essential for HBx function. We also developed a method to produce soluble recombinant HBx protein that likely adopts a physiologically relevant conformation. Collectively, this study provides new insights into the HBx structure-function relationship and suggests a new approach for structural studies of this enigmatic viral regulatory protein.
Hepatitis B virus (HBV) has infected one-third of world population and 240 million people are chronic carriers, to whom a curative therapy is still not available. Similar to other viruses, persistent HBV infection relies on the virus to exploit host cell functions to support its replication and efficiently evade host innate and adaptive antiviral immunity. Understanding HBV replication and concomitant host cell interactions is thus instrumental for development of therapeutics to disrupt the virus-host interactions critical for its persistence and cure chronic hepatitis B. Although the currently available cell culture systems of HBV infection are refractory to genome-wide high throughput screening of key host cellular factors essential for and/or regulating HBV replication, classic one-gene (or pathway)-at-a-time studies in last several decades have already revealed many aspects of HBV-host interactions. An overview of the landscape of HBV-hepatocyte interaction indicates that in addition to more tightly suppressing viral replication by directly targeting viral proteins, disruption of key viral-host cell interactions to eliminate or inactivate the covalently closed circular (ccc) DNA, the most stable HBV replication intermediate that exists as an episomal minichromosome in the nucleus of infected hepatocyte, is essential to achieve a functional cure of chronic hepatitis B. Moreover, therapeutic targeting of integrated HBV DNA and their transcripts may also be required to induce HBsAg seroclearance and prevent liver carcinogenesis.
Worldwide, chronic hepatitis B virus (HBV) infection is a major health problem and no cure exists. Importantly, hepatocyte intrusion by HBV particles results in a complex deregulation of both viral and host cellular genetic and epigenetic processes. Among the attempts to develop novel therapeutic approaches against HBV infection, several options targeting the epigenomic regulation of HBV replication are gaining attention. These include the experimental treatment with 'epidrugs'. Moreover, as a targeted approach, the principle of 'epigenetic editing' recently is being exploited to control viral replication. Silencing of HBV by specific rewriting of epigenetic marks might diminish viral replication, viremia and infectivity, eventually controlling the disease and its complications. Additionally, epigenetic editing can be used as an experimental tool to increase our limited understanding regarding the role of epigenetic modifications in viral infections. Aiming for permanent epigenetic reprogramming of the viral genome without unspecific side-effects, this breakthrough may pave the roads for an ambitious technological pursuit: to start designing a curative approach utilizing manipulative molecular therapies for viral infections in vivo. This article is protected by copyright. All rights reserved.
Lay summary:
Long-term treatment with antiviral drugs carries the risk of selecting mutations in the hepatitis B virus (HBV). We herein report two cases of patients with insufficient response to dual tenofovir and entecavir therapy. Molecular analyses identified a distinct mutation, rtS78T/sC69∗, that abolishes HBsAg detection, enhanced replication, sustained exosome-mediated virion secretion and decreased susceptibility to antivirals, thereby representing a potential high-risk mutation for HBV-infected individuals.
Macrophages and dendritic cells have key roles in viral infections, providing virus reservoirs that frequently resist antiviral therapies and linking innate virus detection to antiviral adaptive immune responses. Human immunodeficiency virus 1 (HIV-1) fails to transduce dendritic cells and has a reduced ability to transduce macrophages, due to an as yet uncharacterized mechanism that inhibits infection by interfering with efficient synthesis of viral complementary DNA. In contrast, HIV-2 and related simian immunodeficiency viruses (SIVsm/mac) transduce myeloid cells efficiently owing to their virion-associated Vpx accessory proteins, which counteract the restrictive mechanism. Here we show that the inhibition of HIV-1 infection in macrophages involves the cellular SAM domain HD domain-containing protein 1 (SAMHD1). Vpx relieves the inhibition of lentivirus infection in macrophages by loading SAMHD1 onto the CRL4(DCAF1) E3 ubiquitin ligase, leading to highly efficient proteasome-dependent degradation of the protein. Mutations in SAMHD1 cause Aicardi-Goutières syndrome, a disease that produces a phenotype that mimics the effects of a congenital viral infection. Failure to dispose of endogenous nucleic acid debris in Aicardi-Goutières syndrome results in inappropriate triggering of innate immune responses via cytosolic nucleic acids sensors. Thus, our findings show that macrophages are defended from HIV-1 infection by a mechanism that prevents an unwanted interferon response triggered by self nucleic acids, and uncover an intricate relationship between innate immune mechanisms that control response to self and to retroviral pathogens.
Polysomal messenger RNA (mRNA) populations change rapidly in response to alterations in the physiological status of the cell. For this reason, translational regulation, mediated principally at the level of initiation, plays a key role in the maintenance of cellular homeostasis. In an earlier translational profiling study, we followed the impact of rapamycin on polysome re-seeding. Despite the overall negative effect on transcript recruitment, we nonetheless observed that some mRNAs were significantly less affected. Consequently, their relative polysomal occupancy increased in the rapamycin-treated cells. The behaviour of one of these genes, mdm2, has been further analysed. Despite the absence of internal ribosome entry site activity we demonstrate, using a dual reporter assay, that both the reported mdm2 5'-UTRs confer resistance to rapamycin relative to the 5'-UTR of β-actin. This relative resistance is responsive to the downstream targets mTORC1 but did not respond to changes in the La protein, a reported factor acting positively on MDM2 translational expression. Furthermore, extended exposure to rapamycin in the presence of serum increased the steady-state level of the endogenous MDM2 protein. However, this response was effectively reversed when serum levels were reduced. Taken globally, these studies suggest that experimental conditions can dramatically modulate the expressional output during rapamycin exposure.
The cullin 4-DNA-damage-binding protein 1 (CUL4-DDB1) ubiquitin ligase machinery regulates diverse cellular functions and can be subverted by pathogenic viruses. Here we report the crystal structure of DDB1 in complex with a central fragment of hepatitis B virus X protein (HBx), whose DDB1-binding activity is important for viral infection. The structure reveals that HBx binds DDB1 through an alpha-helical motif, which is also found in the unrelated paramyxovirus SV5-V protein despite their sequence divergence. Our structure-based functional analysis suggests that, like SV5-V, HBx captures DDB1 to redirect the ubiquitin ligase activity of the CUL4-DDB1 E3 ligase. We also identify the alpha-helical motif shared by these viral proteins in the cellular substrate-recruiting subunits of the E3 complex, the DDB1-CUL4-associated factors (DCAFs) that are functionally mimicked by the viral hijackers. Together, our studies reveal a common yet promiscuous structural element that is important for the assembly of cellular and virally hijacked CUL4-DDB1 E3 complexes.
HBV cccDNA, the template for transcription of all viral mRNAs, accumulates in the nucleus of infected cells as a stable episome organized into minichromosomes by histones and non-histone viral and cellular proteins. Using a cccDNA-specific chromatin immunoprecipitation (ChIP)-based quantitative assay, we have previously shown that transcription of the HBV minichromosome is regulated by epigenetic changes of cccDNA-bound histones and that modulation of the acetylation status of cccDNA-bound H3/H4 histones impacts on HBV replication. We now show that the cellular histone acetyltransferases CBP, p300, and PCAF/GCN5, and the histone deacetylases HDAC1 and hSirt1 are all recruited in vivo onto the cccDNA. We also found that the HBx regulatory protein produced in HBV replicating cells is recruited onto the cccDNA minichromosome, and the kinetics of HBx recruitment on the cccDNA parallels the HBV replication. As expected, an HBV mutant that does not express HBx is impaired in its replication, and exogenously expressed HBx transcomplements the replication defects. p300 recruitment is severely impaired, and cccDNA-bound histones are rapidly hypoacetylated in cells replicating the HBx mutant, whereas the recruitment of the histone deacetylases hSirt1 and HDAC1 is increased and occurs at earlier times. Finally, HBx mutant cccDNA transcribes significantly less pgRNA. Altogether our results further support the existence of a complex network of epigenetic events that influence cccDNA function and HBV replication and identify an epigenetic mechanism (i.e., to prevent cccDNA deacetylation) by which HBx controls HBV replication.
The hepatitis B-X (HBx) protein is strongly associated with hepatocellular carcinoma. It is implicated not to directly cause cancer but to play a role in hepatocellular carcinoma as a co-factor. The oncogenic potential of HBx primarily lies in its interaction with transcriptional regulators resulting in aberrant gene expression and deregulated cellular pathways. Utilizing ultraviolet irradiation to simulate a tumor-initiating event, we integrated chip-based chromatin immunoprecipitation (ChIP-chip) with expression microarray profiling and identified 184 gene targets directly deregulated by HBx. One-hundred forty-four transcription factors interacting with HBx were computationally inferred. We experimentally validated that HBx interacts with some of the predicted transcription factors (pTF) as well as the promoters of the deregulated target genes of these pTFs. Significantly, we demonstrated that the pTF interacts with the promoters of the deregulated HBx target genes and that deregulation by HBx of these HBx target genes carrying the pTF consensus sequences can be reversed using pTF small interfering RNAs. The roles of these deregulated direct HBx target genes and their relevance in cancer was inferred via querying against biogroup/cancer-related microarray databases using web-based NextBio(TM) software. Six pathways, including the Jak-STAT pathway, were predicted to be significantly deregulated when HBx binds indirectly to direct target gene promoters. In conclusion, this study represents the first ever demonstration of the utilization of ChIP-chip to identify deregulated direct gene targets from indirect protein-DNA binding as well as transcriptional factors directly interacting with HBx. Increased knowledge of the gene/transcriptional factor targets of HBx will enhance our understanding of the role of HBx in hepatocellular carcinogenesis and facilitate the design of better strategies in combating hepatitis B virus-associated hepatocellular carcinoma.
The recently described hepatic cell line HepaRG is the sole hepatoma cell line susceptible to hepatitis B virus (HBV) infection. It provides a unique tool for investigating some unresolved issues of the virus' biology, particularly the formation of the viral mini-chromosome believed to be responsible for the persistence of infection. In this study, we characterized the main features of HBV infection: it is restricted to a subpopulation of differentiated hepatocyte-like cells that express albumin as a functional marker and represents around 10 % of all differentiated HepaRG cells. Infection may persist for more than 100 days in cells maintained at the differentiated state. Even though infected cells continued to produce infectious viral particles, very limited or no spreading of infection was observed. Low genetic variation was also observed in the viral DNA from viruses found in the supernatant of infected cells, although this cannot explain the lack of reinfection. HBV infection of HepaRG cells appears to be a very slow process: viral replication starts at around day 8 post-infection and reaches a maximum at day 13. Analysis of viral DNA showed slow and inefficient conversion of the input relaxed circular DNA into covalently closed circular (CCC) DNA, but no further amplification. Continuous lamivudine treatment inhibited viral replication, but neither prevented viral infection nor initial formation of CCC DNA. In conclusion, HBV infection in differentiated HepaRG cells is characterized by long-term persistence without a key feature of hepadnaviruses, the so-called 'CCC DNA amplification' described in the duck hepatitis B model.
The potential of the hepadnavirus X gene product to activate gene expression in trans was tested through a series of cotransfections of X expression vectors with a variety of potential targets for transactivation. The X gene products from human hepatitis B virus (HBV), woodchuck hepatitis virus, and ground squirrel hepatitis virus are all equally active in augmenting the expression of a wide array of target promoters in both permissive and nonpermissive cells. Using the HBV genome itself as the source of X protein, we demonstrate that transactivation of HBV and heterologous genes occurs when X protein is expressed in its native state during productive infection of permissive cells. Run-on transcription analysis indicates that this transactivation occurs at the level of primary transcription.
The induction of beta-interferon is markedly enhanced in some cell types by a pretreatment with type I interferon itself (priming). We show that induction in response to double-stranded RNA is completely dependent upon priming in HeLa cells. However, unprimed cells can be partially induced by Sendai virus. This indicates that Sendai virus can provide a signal that is different from, or additional to, that provided by double-stranded RNA. The requirement for priming cannot be localized to a specific sequence element in the beta-interferon promoter, suggesting that priming may induce an essential component for signal transduction in response to double-stranded RNA.
The X gene of the mammalian hepadnaviruses is believed to encode a protein of 17 kDa which has been shown to transactivate a wide range of viral and cellular promoters. The necessity for X gene expression during the viral life cycle in vivo has recently been suggested (H.-S. Chen, S. Kaneko, R. Girones, R. W. Anderson, W. E. Hornbuckle, B. C. Tennant, P. J. Cote, J. L. Gerin, R. H. Purcell, and R. H. Miller, J. Virol. 67:1218-1226, 1993). We have independently constructed two variants of woodchuck hepatitis virus (WHV) with mutations in the X coding region. Transient transfection of two different hepatoma cell lines showed that these WHV X gene mutants were competent for virus replication in vitro. To determine whether X expression was required for viral replication in vivo, we injected mutant and wild-type genomes into the livers of susceptible woodchucks. While the wild-type WHV genomes were infectious in all animals examined, the mutant genomes did not initiate a WHV infection in woodchucks. These results indicate that the X gene of the hepadnaviruses plays a major role in viral replication in vivo.
All mammalian hepadnaviruses possess a gene, termed X, that encodes a protein capable of transactivating virus gene expression. The X gene overlaps the polymerase and precore genes as well as two newly identified open reading frames (ORFs) termed ORF5 and ORF6. In this investigation, we examined whether ORF5, ORF6, and the X gene were important for the replication of woodchuck hepatitis virus (WHV) in susceptible woodchucks. First, we investigated whether proteins were produced from ORF5 and ORF6 by in vitro translation of appropriate viral transcripts, searched for antibodies against the putative proteins in the sera of animals infected with wild-type virus, and looked for an antisense WHV transcript, necessary for expression of a protein from ORF6, in the livers of acutely or chronically infected woodchucks. All such experiments yielded negative results. Next, we used oligonucleotide-directed mutagenesis to introduce termination codons into ORF5 and ORF6 at two locations within each ORF. Adult woodchucks in groups of three were transfected with one of the four mutant genomes. All of these woodchucks developed WHV infections that were indistinguishable from those of animals transfected with the wild-type WHV recombinant. Polymerase chain reaction amplification and direct DNA sequencing confirmed that reversion of the mutants to a wild-type genotype did not occur. Taken together, these data indicate that ORF5 and ORF6 are not essential for virus replication and are unlikely to represent authentic genes. Finally, we generated five WHV X-gene mutants that either removed the initiation codon for protein synthesis or truncated the carboxyl terminus of the protein by 3, 16, 31, or 52 amino acids. Groups of three adult woodchucks were transfected with one of the five X-gene mutants. Only the mutant that possessed an X gene lacking 3 amino acids from the carboxyl terminus was capable of replication within the 6-month time frame of the experiment. In contrast, all seven woodchucks transfected with wild-type WHV DNA developed markers consistent with viral infection. Thus, it is likely (P < 0.01) that the WHV X gene is important for virus replication in the natural host.
The study of mammalian gene expression through the use of transient transfection assays has greatly expanded our knowledge of transcriptional mechanisms. However, transfected promotor constructs do not always serve as appropriate 'stand-ins' for endogenous genes, particularly in cases where chromatin remodeling must take place. The examples described above indicate that a level of caution is advised when studying regulation of various promoters and transcription factor function with the use of transient transfection assays. When possible, function of the corresponding endogenous promoters should be tested to assess the validity of regulatory mechanisms defined on transiently transfected, non-replicating templates. Future studies using template comparison as a tool will undoubtedly identify which transcription factors are critical in initiating essential changes in chromatin structure at endogenous target genes and assist in the elucidation of mechanisms involved in chromatin transitions. As in the MyoD experiments (36), these studies will also allow identification of functional domains in these factors, which are necessary for chromatin remodeling. Functional differences between transiently transfected and stable replicating templates need not be considered artifactual but rather can be exploited to identify and characterize regulatory mechanisms that involve chromatin components. Full understanding of gene expression in vivo will not be achieved until these mechanisms are understood in detail.
Inefficient nuclear delivery of plasmid DNA is thought to be one of the daunting hurdles to gene transfer, utilizing a nonviral delivery system such as polycation-DNA complex. Following its internalization by endocytosis, plasmid DNA has to be released into the cytosol before its nuclear entry can occur. However, the stability of plasmid DNA in the cytoplasm, that may play a determinant role in the transfection efficiency, is not known. The turnover of plasmid DNA, delivered by microinjection into the cytosol, was determined by fluorescence in situ hybridization (FISH) and quantitative single-cell fluorescence video-image analysis. Both single- and double-stranded circular plasmid DNA disappeared with an apparent half-life of 50-90 min from the cytoplasm of HeLa and COS cells, while the amount of co-injected dextran (MW 70,000) remained unaltered. We propose that cytosolic nuclease(s) are responsible for the rapid-degradation of plasmid DNA, since (1) elimination of plasmid DNA cannot be attributed to cell division or to the activity of apoptotic and lysosomal nucleases; (2) disposal of microinjected plasmid DNA was inhibited in cytosol-depleted cells or following the encapsulation of DNA in phospholipid vesicles; (3) generation and subsequent elimination of free 3'-OH ends could be detected by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay (TUNEL), reflecting the fragmentation of the injected DNA; and finally (4) isolated cytosol, obtained by selective permeabilization of the plasma membrane, exhibits divalent cation-dependent, thermolabile nuclease activity, determined by Southern blotting and 32P-release from end-labeled DNA. Collectively, these findings suggest that the metabolic instability of plasmid DNA, caused by cytosolic nuclease, may constitute a previously unrecognized impediment for DNA translocation into the nucleus and a possible target to enhance the efficiency of gene delivery.
The hepatitis B virus X protein (HBx) is essential for viral infection and strongly interferes with cell growth and viability in culture. These activities involve interaction of HBx with the DDB1 subunit of UV-damaged DNA-binding factor UV-DDB. UV-DDB consists of DDB1 and a DDB2 subunit that mediates nuclear import and has recognized functions in DNA repair and E2F1-mediated transcription. Here we show that HBx retains DDB1-binding-dependent cytotoxic activities when engineered to accumulate in the nucleus but not when excluded from the nucleus. Nuclear localization of HBx does not require binding to DDB1 and remains unaffected by ectopically expressed UV-DDB subunits, indicating that HBx reaches the nuclear compartment independently of UV-DDB. Unexpectedly, HBx appears to largely exist in association with DDB1 and is in direct competition with DDB2 for binding to DDB1. Hence, HBx-mediated cell death can be relieved by increased levels of DDB2, an effect that is not observed with a naturally occurring mutant of DDB2 that lacks DDB1-binding activity. These findings indicate that HBx acts through a pathway that involves a DDB2-independent nuclear function of DDB1 and that this activity will depend on the relative concentration of DDB1 and DDB2 in cells.
The UV-damaged DNA-binding activity protein (UV-DDB) consists of two subunits, DDB1 and DDB2, and functions in DNA repair and cell cycle regulation. The DDB1 subunit is a target for the hepatitis B virus X protein (HBx). Binding of HBx to DDB1 interferes with cell growth and viability in culture and has been implicated in the establishment of viral infection. DDB1 also interacts with the V proteins encoded by several paramyxoviruses including simian virus 5 (SV5), which prevent interferon signaling by targeting either STAT1 or STAT2 proteins for proteolysis. The role of V binding to DDB1, however, remains unclear. Here we show that the V protein of SV5 (SV5-V) and HBx exhibit strikingly similar DDB1 binding properties. Thus, SV5-V and HBx bind to DDB1 in a mutually exclusive manner, and SV5-V shares with HBx the ability to enhance the steady-state levels of DDB1 and to inhibit its association with DDB2. Yet only HBx induces cell death, and SV5-V can prevent HBx from doing so by blocking its interaction with DDB1. Binding of SV5-V to DDB1 may serve another function, since SV5-V shows a decreased ability to induce STAT1 degradation in cells expressing reduced amounts of DDB1. These findings demonstrate that HBx performs a unique function through its association with DDB1 for which SV5-V cannot substitute and suggest that SV5-V and HBx have evolved to bind DDB1 to achieve distinct functions, both by a mechanism that does not involve DDB2.
With an estimate of 350 million people chronically infected with the hepatitis B virus (HBV) worldwide, it is critically important to understand how persistent HBV infection is maintained and linked to chronic hepatitis, cirrhosis, and de- velopment of liver cancer (hepatocellular carcinoma (HCC)) (39). This review will focus on the HBV nonstructural X pro- tein (HBx), a key regulatory protein of the virus that is at the intersection of HBV infection, replication, pathogenesis, and possibly carcinogenesis. The exact role of HBx in viral repli- cation has yet to be established, and its link to the progression of HCC remains controversial. Moreover, it is still unclear whether development of HCC associated with chronic infec- tion by HBV involves a viral protein, is solely the consequence of a continual inflammatory response to infection, or requires both. Understanding the role of HBx in HBV replication and its effect on hepatocyte biology may help resolve this issue. This review describes key studies and activities of HBx, often interpreted within the context of the viral life cycle. The reader is referred to several comprehensive reviews on the reported biological properties of HBx (3, 78, 137). HBV
The hepatitis B virus (HBV) X protein (HBx) is essential for virus infection and has been implicated in the development of
liver cancer associated with chronic infection. HBx can interact with a number of cellular proteins, and in cell culture,
it exhibits pleiotropic activities, among which is its ability to interfere with cell viability and stimulate HBV replication.
Previous work has demonstrated that HBx affects cell viability by a mechanism that requires its binding to DDB1, a highly
conserved protein implicated in DNA repair and cell cycle regulation. We now show that an interaction with DDB1 is also needed
for HBx to stimulate HBV genome replication. Thus, HBx point mutants defective for DDB1 binding fail to complement the low
level of replication of an HBx-deficient HBV genome when provided in trans, and one such mutant regains activity when directly fused to DDB1. Furthermore, DDB1 depletion by RNA interference specifically
compromises replication of wild-type HBV, indicating that HBx produced from the viral genome also functions in a DDB1-dependent
fashion. We also show that HBx in association with DDB1 acts in the nucleus and stimulates HBV replication mainly by enhancing
viral mRNA levels, regardless of whether the protein is expressed from the HBV genome itself or supplied in trans. Interestingly, whereas HBx induces cell death in both HepG2 and Huh-7 hepatoma cell lines, it enhances HBV replication only
in HepG2 cells, suggesting that the two activities involve distinct DDB1-dependent pathways.
The role and functional domain of hepatitis B virus (HBV) X protein (HBx) in regulating HBV transcription and replication were investigated with a transient transfection system in the human hepatoma cell line HepG2 using wild-type or HBx-minus HBV genome constructs and a series of deletion or mutation HBx expression plasmids. We show here that HBx has augmentation effects on HBV transcription and replication as a HBV mutant genome with defective X gene led to decreased levels of 3.5-kb HBV RNA and HBV replication intermediates and that these decreases can be restored by either transient ectopic expression of HBx or a stable HBx expression cell line. The C-terminal two-thirds (amino acids [aa] 51 to 154), which contain the transactivation domain, is required for this function of HBx; the N-terminal one-third (aa 1 to 50) is not required. Using the alanine scanning mutagenesis strategy, we demonstrated that the regions between aa 52 to 65 and 88 to 154 are important for the augmentation function of HBx in HBV replication. By the luciferase reporter gene analysis, we found that the transactivation and coactivation activities of HBx coincide well with its augmentation function in HBV transcription and replication. These results suggest that HBx has an important role in stimulating HBV transcription and replication and that the transcriptional transactivation function of HBx may be critical for its augmentation effect on HBV replication.
Precise control of the level of protein expression in cells can yield quantitative and temporal information on the role of a given gene in normal cellular physiology and on exposure to chemicals and drugs. This is particularly relevant to liver cells, in which the expression of many proteins, such as phase I and phase II drug-metabolizing enzymes, vary widely between species, among individual humans, and on exposure to xenobiotics. The most widely used gene regulatory system has been the tet-on/off approach. Although a second-generation tet-on transactivator was recently described, it has not been widely investigated for its potential as a tool for regulating genes in cells and particularly in cells previously recalcitrant to the first-generation tet-on approach, such as hepatocyte-derived cells. Here we demonstrate the development of two human (HepG2 and HuH7) and one mouse (Hepa1c1c7) hepatoma-derived cell lines incorporating a second-generation doxycycline-inducible gene expression system and the application of the human lines to control the expression of different transgenes. The two human cell lines were tested for transient or stable inducibility of five transgenes relevant to liver biology, namely phase I (cytochrome P-450 2E1; CYP2E1) and phase II (glutathione S-transferase P1; GSTP1) drug metabolism, and three transcription factors that respond to chemical stress [nuclear factor erythroid 2 p45-related factors (NRF)1 and 2 and NFKB1 subunit of NF-kappaB]. High levels of functional expression were obtained in a time- and dose-dependent manner. Importantly, doxycycline did not cause obvious changes in the cellular proteome. In conclusion, we have generated hepatocyte-derived cell lines in which expression of genes is fully controllable.
Nuclear spatial positioning plays an important role in the epigenetic regulation of eukaryotic gene expression. Here we show a role for nuclear spatial positioning in regulating episomal transgenes that are delivered by virus-like particles (VLPs). VLPs mediate the delivery of plasmid DNA (pDNA) to cell nuclei but lack viral factors involved in initiating and regulating transcription. By tracking single fluorescently labeled VLPs, coupled with luciferase reporter gene assays, we found that VLPs transported pDNA to cell nuclei efficiently but transgenes were immediately silenced by the cell. An investigation of the nuclear location of fluorescent VLPs revealed that the pDNAs were positioned next to centromeric heterochromatin. The activation of transcription by providing viral factors or inhibiting histone deacetylase activity resulted in the localization to euchromatin regions. Further, the activation of transcription induced the recruitment of PML nuclear bodies (PML-NBs) to the VLPs. This association did not play a role in regulating transgene expression, but PML protein was necessary for the inhibition of transgene expression with alpha interferon (IFN-alpha). These results support a model whereby cells can prevent foreign gene expression at two levels: by positioning transgenes next to centromeric heterochromatin or, if that is overcome, via the type I IFN response facilitated by PML-NB recruitment.
The hepatitis B virus infects more than 350 million people worldwide and is a leading cause of liver cancer. The virus encodes a multifunctional regulator, the hepatitis B virus X protein (HBx), that is essential for virus replication. HBx is involved in modulating signal transduction pathways and transcription mediated by various factors, notably CREB that requires the recruitment of the co-activators CREB-binding protein (CBP)/p300. Here we investigated the role of HBx and its potential interaction with CBP/p300 in regulating CREB transcriptional activity. We show that HBx and CBP/p300 synergistically enhanced CREB activity and that CREB phosphorylation by protein kinase A was a prerequisite for the cooperative action of HBx and CBP/p300. We further show that HBx interacted directly with CBP/p300 in vitro and in vivo. Using chromatin immunoprecipitation, we provide evidence that HBx physically occupied the CREB-binding domain of CREB-responsive promoters of endogenous cellular genes such as interleukin 8 and proliferating cell nuclear antigen. Moreover expression of HBx increased the recruitment of p300 to the interleukin 8 and proliferating cell nuclear antigen promoters in cells, and this is associated with increased gene expression. As recruitment of CBP/p300 is known to represent the limiting event for activating CREB target genes, HBx may disrupt this cellular regulation, thus predisposing cells to transformation.
The 3.2-kb hepatitis B virus (HBV) genome encodes a single regulatory protein termed HBx. While multiple functions have been identified for HBx in cell culture, its role in virus replication remains undefined. In the present study, we combined an HBV plasmid-based replication assay with the hydrodynamic tail vein injection model to investigate the function(s) of HBx in vivo. Using a greater-than-unit-length HBV plasmid DNA construct (payw1.2) and a similar construct with a stop codon at position 7 of the HBx open reading frame (payw1.2*7), we showed that HBV replication in transfected HepG2 cells was reduced 65% in the absence of HBx. These plasmids were next introduced into the livers of outbred ICR mice via hydrodynamic tail vein injection. At the peak of virus replication, at 4 days postinjection, intrahepatic markers of HBV replication were reduced 72% to 83% in mice injected with HBx-deficient payw1.2*7 compared to those measured in mice receiving wild-type payw1.2. A second plasmid encoding HBx was able to restore virus replication from payw1.2*7 to wild-type levels. Finally, viremia was monitored over the course of acute virus replication, and at 4 days postinjection, it was reduced by nearly 2 logs in the absence of HBx. These studies establish that the role for HBx in virus replication previously shown in transfected HepG2 cells is also apparent in the mouse liver within the context of acute hepatitis. Importantly, the function of HBx can now be studied in an in vivo setting that more closely approximates the cellular environment for HBV replication.
Methylation of CpG dinucleotides within proximal promoters is often associated with transcriptional silencing. Methylation-dependent repression is well established for hypermethylated CpG island promoters that are characterized by a high density of CpG residues. The effect of CpG DNA methylation on CpG-poor promoters is less well characterized, probably due to the lack of convenient assay systems to test promoter activities in vitro. In this report, we describe a novel luciferase reporter vector, pCpGL, which completely lacks CpG dinucleotides and can be used to study the effect of promoter DNA methylation in transfection assays. Whereas a traditional reporter vector that contains a large number of backbone CpG residues significantly represses a CpG-free promoter when methylated, our new reporter vector is only repressed due to the presence of functionally important, methylated CpG residues. The pCpGL vector provides a useful tool to study the effects of CpG methylation on CpG-rich and CpG-poor promoters.
The replicative intermediate of hepatitis B virus (HBV), the covalently closed, circular DNA, is organized into minichromosomes in the nucleus of the infected cell by histone and non-histone proteins. In this study we investigated the architecture of the HBV minichromosome in more detail. In contrast to cellular chromatin the nucleosomal spacing of the HBV minichromosome has been shown to be unusually reduced by approximately 10 %. A potential candidate responsible for an alteration in the chromatin structure of the HBV minichromosome is the HBV core protein. The HBV core protein has been implicated in the nuclear targeting process of the viral genome. The association of the HBV core protein with nuclear HBV replicative intermediates could strengthen this role. Our findings, confirmed by in vivo and in vitro experiments indicate that HBV core protein is a component of the HBV minichromosome, binds preferentially to HBV double-stranded DNA, and its binding results in a reduction of the nucleosomal spacing of the HBV nucleoprotein complexes by 10 %. From this model of the HBV minichromosome we propose that the HBV core protein may have an impact on the nuclear targeting of the HBV genome and be involved in viral transcription by regulating the nucleosomal arrangement of the HBV regulatory elements, probably in a positive manner.
Hepatitis B virus (HBV), a major causative agent of hepatocelluar carcinoma (HCC), encodes an oncogenic X-protein (HBx) which has been known as a transcriptional transactivator on multiple viral and celluar promoters. In the report, we verified that HBx transcriptionally repress insulin-like growth factor binding protein-3 (IGFBP-3) by promoting HBx/histone deacetylase 1 (HDAC1) complex formation. HBx recruited HDAC1 forms complex with Sp1 in a p53-independent manner) and deacetylates Sp1 which resulted in the diminished binding of Sp1 on targeted DNA during transcriptional repression. Deacetylation of Sp1 by HBx recruited HDAC1 likely to be a part of the mechanism that controls HBx induced IGFBP-3 repression and the modification of chromatin structure.
The hepatitis B virus and the mammalian hepadnavirus genomes encode for a short open reading frame called x. Expression of the protein product (HBx) appears necessary for establishment of natural infection. However, in vitro studies have suggested a multifunctional role for HBx as an indirect transcriptional transactivator of a variety of different viral and cellular promoters. Indeed, HBx has no known direct DNA binding properties but may interact with transcription factors as well as activate intracellular signaling pathways associated with cell growth. To further address the possible functional role of HBx in the life cycle of hepatitis B virus, we performed an analysis using the yeast two-hybrid system to screen a cDNA library derived from a hepatocellular carcinoma cell line with a HBx fusion bait in an attempt to identify cellular partners that may bind to and alter the biologic properties of HBx. A HBx-interacting protein that specifically complexes with the carboxy terminus of wild-type HBx was identified and designated XIP. This 9.6-kDa protein is capable of binding to HBx in vitro, and transient and stable expression in hepatocellular carcinoma cells abolishes the transactivation properties of HBx on luciferase constructs driven by AP-1 and endogenous hepatitis B virus enhancer/promoter elements. Investigation of the role of XIP in hepatitis B virus replication in differentiated hepatocellular carcinoma cells revealed that XIP expression reduces wild-type hepatitis B virus replication to levels observed following transfection with an HBx-minus virus. In contrast, the replication levels of the duck hepatitis B virus, a hepadnavirus that lacks the x open reading frame, were unchanged in the context of XIP expression. We propose that one of the physiologic functions of the cellular protein XIP is to negatively regulate HBx activity and thus to alter the replication life cycle of the virus.
The primate lentivirus auxiliary protein Vpx counteracts an unknown restriction factor that renders human dendritic and myeloid cells largely refractory to HIV-1 infection. Here we identify SAMHD1 as this restriction factor. SAMHD1 is a protein involved in Aicardi-Goutières syndrome, a genetic encephalopathy with symptoms mimicking congenital viral infection, that has been proposed to act as a negative regulator of the interferon response. We show that Vpx induces proteasomal degradation of SAMHD1. Silencing of SAMHD1 in non-permissive cell lines alleviates HIV-1 restriction and is associated with a significant accumulation of viral DNA in infected cells. Concurrently, overexpression of SAMHD1 in sensitive cells inhibits HIV-1 infection. The putative phosphohydrolase activity of SAMHD1 is probably required for HIV-1 restriction. Vpx-mediated relief of restriction is abolished in SAMHD1-negative cells. Finally, silencing of SAMHD1 markedly increases the susceptibility of monocytic-derived dendritic cells to infection. Our results demonstrate that SAMHD1 is an antiretroviral protein expressed in cells of the myeloid lineage that inhibits an early step of the viral life cycle.
The molecular biology of hepatitis B virus (HBV) has been extensively studied but the exact role of the hepatitis B X protein (HBx) in the context of natural HBV infections remains unknown.
Primary human hepatocytes and differentiated HepaRG cells allowing conditional trans complementation of HBx were infected with wild type (HBV(wt)) or HBx deficient (HBV(x-)) HBV particles and establishment of HBV replication was followed.
We observed that cells inoculated with HBx-deficient HBV particles (HBV(x-)) did not lead to productive HBV infection contrary to cells inoculated with wild type HBV particles (HBV(wt)). Although equal amounts of nuclear covalently closed circular HBV-DNA (cccDNA) demonstrated comparable uptake and nuclear import, active transcription was only observed from HBV(wt) genomes. Trans-complementation of HBx was able to rescue transcription from the HBV(x-) genome and led to antigen and virion secretion, even weeks after infection. Constant expression of HBx was necessary to maintain HBV antigen expression and replication. Finally, we demonstrated that HBx is not packaged into virions during assembly but is expressed after infection within the new host cell to allow epigenetic control of HBV transcription from cccDNA.
Our results demonstrate that HBx is required to initiate and maintain HBV replication and highlight HBx as the key regulator during the natural infection process.
The hepatitis B virus (HBV) is a widespread human pathogen and a major health problem in many countries. Molecular cloning and sequencing of the viral DNA genome has demonstrated a small and compact structure organized into four overlapping reading frames that encode the viral proteins. Besides structural proteins of the core and the envelope, HBV encodes a DNA polymerase with reverse transcriptase activity, a secreted antigen of unknown function, and a transcriptional activator that is essential for viral replication. Major steps of the viral life cycle have been unraveled, including transcription of all viral RNAs from nuclear covalently closed circular DNA (cccDNA), followed by encapsidation of pregenomic RNA, a more-than-genome length transcript, and reverse transcription of pregenomic RNA leading to asymmetric synthesis of the DNA strands. Although HBV has been recognized as a human tumor virus, no direct transforming activity could be evidenced in different cellular and animal models. However, the transcriptional regulatory protein HBx encoded by the X gene is endowed with weak oncogenic activity. HBx harbors pleiotropic activities and plays a major role in HBV pathogenesis and in liver carcinogenesis.
The non-structural X protein, HBx, of hepatitis B virus (HBV) is assumed to play an important role in HBV replication. Woodchuck hepatitis virus X protein is indispensable for virus replication, but the duck hepatitis B virus X protein is not. In this study, we investigated whether the HBx protein is indispensable for HBV replication in vivo using human hepatocyte chimeric mice. HBx-deficient (HBx-def) HBV was generated in HepG2 cells by transfection with an overlength HBV genome. Human hepatocyte chimeric mice were infected with HBx-def HBV with or without hepatic HBx expression by hydrodynamic injection of HBx expression plasmids. Serum virus levels and HBV sequences were determined with mice sera. The generated HBx-def HBV peaked in the sucrose density gradient at points equivalent to the generated HBV wild type and the virus in a patient's serum. HBx-def HBV-injected mice developed measurable viraemia only in continuously HBx-expressed liver. HBV DNA in the mouse serum increased up to 9 log(10) copies ml(-1) and the viraemia persisted for more than 2 months. Strikingly, all revertant viruses had nucleotide substitutions that enabled the virus to produce the HBx protein. It was concluded that the HBx protein is indispensable for HBV replication and could be a target for antiviral therapy.
Identifying the requirements for the regulatory HBx protein in hepatitis B virus (HBV) replication is an important goal. A plasmid-based HBV replication assay was used to evaluate whether HBx subcellular localization influences its ability to promote virus replication, as measured by real time PCR quantitation of viral capsid-associated DNA. HBx targeted to the nucleus by a nuclear localization signal (NLS-HBx) was able to restore HBx-deficient HBV replication, while HBx containing a nuclear export signal (NES-HBx) was not. Both NLS-HBx and NES-HBx were expressed at similar levels (by immunoprecipitation and Western blotting), and proper localization of the signal sequence-tagged proteins was confirmed by deconvolution microscopy using HBx, NLS-HBx, and NES-HBx proteins fused to GFP. Importantly, these findings were confirmed in vivo by hydrodynamic injection into mice. Our results demonstrate that in these HBV replication assays, at least one function of HBx requires its localization to the nucleus.
The hepatitis B virus X protein (HBx) has been implicated as a potential trigger of the epigenetic deregulation of some genes, but the underlying mechanisms remain unknown. The aim of this study was to identify underlying mechanisms involved in HBx-mediated epigenetic modification.
Interactions between HBx and DNA methyltransferase (DNMT) or histone deacetylase-1 (HDAC1) were assessed by co-immunoprecipitation. DNA methylation of gene promoters was detected by bisulfite sequencing, and HBx-mediated protein binding to gene regulatory elements was evaluated by chromatin immunoprecipitation. Target gene transcriptional activity was measured by real-time polymerase chain reaction.
HBx can interact directly with DNMT3A and HDAC1. HBx recruited DNMT3A to the regulatory promoters of interleukin-4 receptor and metallothionein-1F and subsequently silenced their transcription via de novo DNA methylation. By contrast, the transcription of CDH6 and IGFBP3 was triggered by HBx through the deprivation of DNMT3A from their promoters. Transcriptional levels of target genes in hepatocellular carcinoma (HCC) specimens were strongly correlated with the occurrence of HBx.
The interaction of HBx and DNMT3A facilitates cellular epigenetic modification (via regional hypermethylation or hypomethylation) at distinct genomic loci, providing an alternative mechanism within HBx-mediated transcriptional regulation, and a profound understanding of hepatitis and HCC pathogenesis.
We carried out a comparative analysis of several proposed host protein partners of the human hepatitis B virus X protein (HBx) using both the GAL4- and the LexA-based yeast two-hybrid system. We showed that the interaction of HBx with the UV-damaged DNA-binding protein (UVDDB) is positive in both yeast systems, detectable in cotransfected human cells, conserved by rodent hepadnavirus X proteins (known to transactivate in human cells), and tightly correlated with the transactivation proficiency of X-insertion mutants. Taken together, our results strongly suggest that UVDDB is involved in X-mediated transactivation.
Hepatitis B virus produces chronic infections of the liver leading to cirrhosis and hepatocellular carcinoma. The X protein of hepatitis B virus (HBx) is a multifunctional protein that can interact with p53 but can also influence a variety of signal transduction pathways within the cell. In most instances this small viral protein favors cell survival and probably initiates hepatocarcinogenesis. HBx upregulates the activity of a number of transcription factors including NF-kappa B, AP-1, CREB, and TBP. However, the majority of HBx is localized to the cytoplasm where it interacts with and stimulates protein kinases such as protein kinase C, Janus kinase/STAT, IKK, PI-3-K, stress-activated protein kinase/Jun N-terminal kinase, and protein kinase B/Akt. This small viral protein can localize to the mitochondrion. HBx may act as an adaptor or kinase activator to influence signal transduction pathways. This review will attempt to analyze the involvement of HBx in signal transduction pathways during hepatitis B viral infections and hepatocellular carcinoma development.
The hepatitis B virus X protein (HBx) is essential for establishing natural viral infection and has been implicated in the development of liver cancer associated with chronic infection. The basis for HBx function in either process is not understood. In cell culture, HBx exhibits pleiotropic activities affecting transcription, DNA repair, cell growth, and apoptotic cell death. Numerous cellular proteins including the p127-kDa subunit of UV-damaged DNA-binding activity have been reported to interact with HBx but the functional significance of these interactions remains unclear. Here we show that the binding of HBx to p127 interferes with cell viability. Mutational analysis reveals that HBx contacts p127 via a region to which no function has been assigned previously. An HBx variant bearing a single-charge reversal substitution within this region loses p127 binding and concomitant cytotoxicity. This mutant regains activity when directly fused to p127. These studies confirm that p127 is an important cellular target of HBx, and they indicate that HBx does not exert its effect by sequestering p127, and thereby preventing its normal function, but instead by conferring to p127 a deleterious activity.
Efficient and sustained transgene expression are desirable features for many envisioned gene therapy applications, yet synthetic vectors tested to date are rarely successful in achieving these properties. Substantial research efforts have focused on protection of plasmid DNA from nuclease attack as well as increasing nuclear transport of plasmids, resulting in significant but still limited gains. We show here that a further barrier to efficient and sustained expression exists for synthetic vectors: plasmid DNA methylation. We have investigated this barrier for transient expression of a green fluorescent protein (GFP) transgene delivered via Lipofectamine, by testing the effects of culturing C3A human hepatoblastoma cells with 5-Azacytidine (AzaC), an irreversible inhibitor of DNA methyltransferase. To control for loss of plasmids by dilution during mitosis, transfected cells were growth-arrested for 1 week and their subsequent GFP expression quantified by FACS. In the presence of AzaC, a significantly greater fraction of transfected cells remained GFP-positive and possessed higher levels of GFP production relative to AzaC-untreated cells. Additionally, we have applied a Methyl-Assisted PCR (MAP) assay to quantify a subset of methylated CpG sites in the GFP gene. When MAP was performed on plasmids isolated from transfected cells, the extent of methylation was found to be inversely related to the level of GFP expression.
Hepatitis B Virus (HBV) infection is one of the major causes of hepatocellular carcinoma (HCC). X protein (HBx) has been suspected to be oncogenic, although the precise role(s) remain uncertain. HBx is a multifunctional viral regulator that modulates transcription, cell responses to genotoxic stress, protein degradation, and signaling pathways. These modulations affect viral replication and viral proliferation, directly or indirectly. HBx also affects cell cycle checkpoints, cell death, and carcinogenesis. This article presents an overview of the progress in HBx research over the past several years.
Hepatitis B virus (HBV) infects more than 300 million people and is a leading cause of liver cancer and disease. The HBV HBx protein is essential for infection; HBx activation of Src is important for HBV DNA replication. In our study, HBx activated cytosolic calcium-dependent proline-rich tyrosine kinase-2 (Pyk2), a Src kinase activator. HBx activation of HBV DNA replication was blocked by inhibiting Pyk2 or calcium signaling mediated by mitochondrial calcium channels, which suggests that HBx targets mitochondrial calcium regulation. Reagents that increased cytosolic calcium substituted for HBx protein in HBV DNA replication. Thus, alteration of cytosolic calcium was a fundamental requirement for HBV replication and was mediated by HBx protein.
Gene transcription and ubiquitin-mediated proteolysis are two processes that have seemingly nothing in common: transcription is the first step in the life of any protein and proteolysis the last. Despite the disparate nature of these processes, a growing body of evidence indicates that ubiquitin and the proteasome are intimately involved in gene control. Here, we discuss the deep mechanistic connections between transcription and the ubiquitin-proteasome system, and highlight how the intersection of these processes tightly controls expression of the genetic information.
The natural history of hepatitis B virus (HBV) infection is complex and variable and is greatly influenced by the age at infection, the level of HBV replication, and host immune status. Chronic HBV infection generally consists of an early replicative phase with active liver disease (hepatitis B e antigen [HBeAg]-positive chronic hepatitis) and a late low or nonreplicative phase with HBeAg seroconversion and remission of liver disease (inactive carrier state). After HBeAg seroconversion, some patients may have active hepatitis due to HBV variants not expressing HBeAg (HBeAg-negative chronic hepatitis). Morbidity and mortality are linked to development of cirrhosis and hepatocellular carcinoma. Survival is reasonably good (about 85% probability at 5 years) in compensated cirrhosis but very poor in decompensated cirrhosis. Both cirrhotic and noncirrhotic patients with sustained reduction of HBV replication and normalization of aminotransferase after interferon alfa therapy have a reduced risk for liver-related complications.
Hepatitis B virus (HBV), the causative agent of B-type hepatitis in humans, is the type member of the Hepadnaviridae, hepatotropic DNA viruses that replicate via reverse transcription. Beyond long-established differences to retroviruses in gene expression and overall replication strategy newer work has uncovered additional distinctions in the mechanism of reverse transcription per se. These include protein-priming by the unique extra terminal protein domain of the reverse transcriptase (RT) utilizing an RNA hairpin for de novo initiation of first strand DNA synthesis, and the strict dependence of this process on cellular chaperones. Recent in vitro reconstitution systems enabled first biochemical insights into this multifactorial reaction, complemented by high resolution structural information on the RNA, though not yet the protein, level. Genetic approaches have revealed long-distance interactions in the nucleic acid templates as an important factor enabling the puzzling template switches required to produce the relaxed circular (RC) DNA found in infectious virions. Finally, the failure of even potent HBV RT inhibitors to eliminate nuclear covalently closed circular (ccc) DNA, the functional equivalent of integrated proviral DNA, has spurred a renewed interest in the mechanism of cccDNA generation. These new developments are in the focus of this review.
Toll-like receptors (TLRs) as well as the receptors for tumor necrosis factor (TNF-R) and interleukin-1 (IL-1R) play an important role in innate immunity by regulating the activity of distinct transcription factors such as nuclear factor-kappaB (NF-kappaB). TLR, IL-1R and TNF-R signaling to NF-kappaB converge on a common IkappaB kinase complex that phosphorylates the NF-kappaB inhibitory protein IkappaBalpha. However, upstream signaling components are in large part receptor-specific. Nevertheless, the principles of signaling are similar, involving the recruitment of specific adaptor proteins and the activation of kinase cascades in which protein-protein interactions are controlled by poly-ubiquitination. In this review, we will discuss our current knowledge of NF-kappaB signaling in response to TLR-4, TNF-R and IL-1R stimulation, with a special focus on the similarities and dissimilarities among these pathways.
- Van Breugel
- Et Al
HEPATOLOGY, Vol. 56, No. 6, 2012
VAN BREUGEL, ROBERT, MUELLER, ET AL. 2123
- Robert Van Breugel
- Et Al
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VAN BREUGEL, ROBERT, MUELLER, ET AL.
HEPATOLOGY, December 2012
Advances in Viral Oncology
- Schödel F
- Sprengel R
- Weimer T
- Fernholz D
- Schneider R
- Will H
How the ubiquitin-proteasome system controls transcription
- Muratani