[Show abstract][Hide abstract] ABSTRACT: Long INterspersed Element-1 (LINE-1 or L1) retrotransposition poses a mutagenic threat to human genomes. Human cells have therefore evolved strategies to regulate L1 retrotransposition. The APOBEC3 (A3) gene family consists of seven enzymes that catalyze deamination of cytidine nucleotides to uridine nucleotides (C-to-U) in single-strand DNA substrates. Among these enzymes, APOBEC3A (A3A) is the most potent inhibitor of L1 retrotransposition in cultured cell assays. However, previous characterization of L1 retrotransposition events generated in the presence of A3A did not yield evidence of deamination. Thus, the molecular mechanism by which A3A inhibits L1 retrotransposition has remained enigmatic. Here, we have used in vitro and in vivo assays to demonstrate that A3A can inhibit L1 retrotransposition by deaminating transiently exposed single-strand DNA that arises during the process of L1 integration. These data provide a mechanistic explanation of how the A3A cytidine deaminase protein can inhibit L1 retrotransposition.DOI: http://dx.doi.org/10.7554/eLife.02008.001.
[Show abstract][Hide abstract] ABSTRACT: Maintaining genome integrity and transmission of intact genomes is critical for cellular, organismal, and species survival. Cells can detect damaged DNA, activate checkpoints, and either enable DNA repair or trigger apoptosis to eliminate the damaged cell. Aberrations in these mechanisms lead to somatic mutations and genetic instability, which are hallmarks of cancer. Considering the long history of host-microbe coevolution, an impact of microbial infection on host genome integrity is not unexpected, and emerging links between microbial infections and oncogenesis further reinforce this idea. In this review, we compare strategies employed by viruses, bacteria, and parasites to alter, subvert, or otherwise manipulate host DNA damage and repair pathways. We highlight how microbes contribute to tumorigenesis by directly inducing DNA damage, inactivating checkpoint controls, or manipulating repair processes. We also discuss indirect effects resulting from inflammatory responses, changes in cellular metabolism, nuclear architecture, and epigenome integrity, and the associated evolutionary tradeoffs.
[Show abstract][Hide abstract] ABSTRACT: Identifying cellular and molecular differences between human and non-human primates (NHPs) is essential to the basic understanding of the evolution and diversity of our own species. Until now, preserved tissues have been the main source for most comparative studies between humans, chimpanzees (Pan troglodytes) and bonobos (Pan paniscus). However, these tissue samples do not fairly represent the distinctive traits of live cell behaviour and are not amenable to genetic manipulation. We propose that induced pluripotent stem (iPS) cells could be a unique biological resource to determine relevant phenotypical differences between human and NHPs, and that those differences could have potential adaptation and speciation value. Here we describe the generation and initial characterization of iPS cells from chimpanzees and bonobos as new tools to explore factors that may have contributed to great ape evolution. Comparative gene expression analysis of human and NHP iPS cells revealed differences in the regulation of long interspersed element-1 (L1, also known as LINE-1) transposons. A force of change in mammalian evolution, L1 elements are retrotransposons that have remained active during primate evolution. Decreased levels of L1-restricting factors APOBEC3B (also known as A3B) and PIWIL2 (ref. 7) in NHP iPS cells correlated with increased L1 mobility and endogenous L1 messenger RNA levels. Moreover, results from the manipulation of A3B and PIWIL2 levels in iPS cells supported a causal inverse relationship between levels of these proteins and L1 retrotransposition. Finally, we found increased copy numbers of species-specific L1 elements in the genome of chimpanzees compared to humans, supporting the idea that increased L1 mobility in NHPs is not limited to iPS cells in culture and may have also occurred in the germ line or embryonic cells developmentally upstream to germline specification during primate evolution. We propose that differences in L1 mobility may have differentially shaped the genomes of humans and NHPs and could have continuing adaptive significance.
[Show abstract][Hide abstract] ABSTRACT: Macrophages play important roles in host immune defense against virus infection. During infection by herpes simplex virus type 1 (HSV-1), macrophages acquire enhanced antiviral potential. Restriction of HSV-1 replication and progeny production is important to prevent viral spread but the cellular mechanisms that inhibit the DNA virus in macrophages are unknown. SAMHD1 was recently identified as a retrovirus restriction factor highly expressed in macrophages. The SAMHD1 protein is expressed in both undifferentiated monocytes and differentiated macrophages, but retroviral restriction is limited to differentiated cells by modulation of SAMHD1 phosphorylation. It is proposed to block reverse transcription of retroviral RNA into DNA by depleting cellular deoxynucleotide triphosphates (dNTPs). Viruses with DNA genomes do not employ reverse transcription during infection, but replication of their viral genomes is also dependent on intracellular dNTP concentrations. Here, we demonstrate that SAMHD1 restricts replication of the HSV-1 DNA genome in differentiated macrophage cell lines. Depleting SAMHD1 in THP-1 cells enhanced HSV-1 replication, while ectopic overexpression of SAMHD1 in U937 cells repressed HSV-1 replication. SAMHD1 did not impact viral gene expression from incoming HSV-1 viral genomes. HSV-1 restriction involved the dNTP triphosphohydrolase activity of SAMHD1, and was partially overcome by addition of exogenous deoxynucleosides. Unlike retroviruses, restriction of HSV-1 was not affected by SAMHD1 phosphorylation status. Our results suggest that SAMHD1 functions broadly to inhibit replication of DNA viruses in non-dividing macrophages.
Journal of Virology 09/2013; 87(23):12949-56. · 5.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The intracellular parasite Theileria is the only eukaryote known to transform its mammalian host cells. We investigated the host mechanisms involved in parasite-induced transformation phenotypes. Tumour progression is a multistep process, yet 'oncogene addiction' implies that cancer cell growth and survival can be impaired by inactivating a single gene, offering a rationale for targeted molecular therapies. Furthermore, feedback loops often act as key regulatory hubs in tumorigenesis. We searched for microRNAs involved in addiction to regulatory loops in leukocytes infected with Theileria parasites. We show that Theileria transformation involves induction of the host bovine oncomiR miR-155, via the c-Jun transcription factor and AP-1 activity. We identified a novel miR-155 target, DET1, an evolutionarily-conserved factor involved in c-Jun ubiquitination. We show that miR-155 expression led to repression of DET1 protein, causing stabilization of c-Jun and driving the promoter activity of the BIC transcript containing miR-155. This positive feedback loop is critical to maintain the growth and survival of Theileria-infected leukocytes; transformation is reversed by inhibiting AP-1 activity or miR-155 expression. This is the first demonstration that Theileria parasites induce the expression of host non-coding RNAs and highlights the importance of a novel feedback loop in maintaining the proliferative phenotypes induced upon parasite infection. Hence, parasite infection drives epigenetic rewiring of the regulatory circuitry of host leukocytes, placing miR-155 at the crossroads between infection, regulatory circuits and transformation.
[Show abstract][Hide abstract] ABSTRACT: In mammals, one of the most pronounced consequences of viral infection is the induction of type I interferons, cytokines with potent antiviral activity. Schlafen (Slfn) genes are a subset of interferon-stimulated early response genes (ISGs) that are also induced directly by pathogens via the interferon regulatory factor 3 (IRF3) pathway 1 . However, many ISGs are of unknown or incompletely understood function. Here we show that human SLFN11 potently and specifically abrogates the production of retroviruses such as human immunodeficiency virus 1 (HIV-1). Our study revealed that SLFN11 has no effect on the early steps of the retroviral infection cycle, including reverse transcription, integration and transcription. Rather, SLFN11 acts at the late stage of virus production by selectively inhibiting the expression of viral proteins in a codon-usage-dependent manner. We further find that SLFN11 binds transfer RNA, and counteracts changes in the tRNA pool elicited by the presence of HIV. Our studies identified a novel antiviral mechanism within the innate immune response, in which SLFN11 selectively inhibits viral protein synthesis in HIV-infected cells by means of codon-bias discrimination. SLFN genes encode a family of proteins limited to mammalian organisms. Nine murine and six human SLFN genes share a conserved NH2-terminus containing a putative AAA-domain, and long SLFN genes possess motifs resembling DNA/RNA helicase domains, a trait they share with the nucleic acid sensors RIG-I and MDA-5 2 . Beyond that, SLFN proteins harbour no sequence similarity to other proteins. In vivo, short and long murine SLFN proteins inhibit T-cell develop-ment 3–5 , and levels of murine SLFN proteins are elevated after infection with Brucella or Listeria 4 . Lipopolysaccharide, poly-inosine-cytosine (poly-IC) or interferon (IFN)-a/b treatment of macrophages results in induction of several murine Slfn genes (our unpublished results). Treatment of human foreskin fibroblasts with IFN-b, poly-IC or poly-dAdT revealed similar induction of SLFN genes (Supplemen-tary Fig. 1a), and human SLFN5 and SLFN11 were consistently the most prominent family members (Supplementary Fig. 1b). Notably, we observed a striking difference in SLFN levels between HEK293 (293) and HEK293T (293T) cells (Supplementary Fig. 1b), and exploited this differential expression to focus on SLFN11 for further studies. We further used SLFN11-targeted short hairpin RNA to generate stable 293 cells that specifically lack SLFN11 expression (293shRNA SLFN) (Supplementary Fig. 1c, d). To test whether lack of SLFN11 in 293shRNA SLFN or 293T cells alters their ability to subdue viral infections, we infected these cells with vesicular stomatitis virus (VSV)-G pseudotyped HIV (HIV VSV-G), or amphotropic murine stem cell virus (MSCV), adeno-associated virus (AAV), or herpes simplex virus (HSV). HIV VSV-G -infected cells expressed luciferase after integration of the viral complementary DNAs into the host genome. Regardless of SLFN11 expression, all cell lines had comparable luciferase levels after HIV VSV-G infection (Sup-plementary Fig. 2). A similar lack of influence of SLFN11 was observed when cells were infected with MSCV, AAV or HSV (not shown). HEK293T cells are used as packaging cells for production of retroviruses, and we therefore considered the possibility that virus production rather than the response towards them is afflicted by SLFN11. Indeed, 293T cells produced markedly higher HIV VSV-G (Fig. 1b) or MCSV (Supplementary Fig. 3a) titres than 293 cells from the viral vectors pNL4-3.Luc.R 1 E 2 or MSCV-IRES-GFP, respectively. Most importantly, this increase in viral titre was also clearly evident in 293shRNA SLFN cells, whereas 293 and 293shRNA Ctl cells produced the same low levels of virus (Fig. 1b and Supplementary Fig. 3a). Notably, the modulation of virus production is limited to particular viruses, as fabrication of retroviruses (Fig. 1b and Supplementary Fig. 3a), but not of AAV (Supplementary Fig. 3c), was affected by SLFN11. We also did not observe any modulation of ISGs such as ISG15, ISG54 or APOBEC3G (not shown) as a consequence of SLFN11 expression, supporting the notion that SLFN11 does not create a general virus-resistant phenotype. To corroborate that the observed differences are attributable to dissimilar SLFN11 expression, we expressed full-length SLFN11 (amino acids 1–901) in 293T cells and analysed their ability to produce HIV VSV-G . Indeed, SLFN11 strongly inhibited HIV VSV-G (Fig. 1a) or MSCV (Supplementary Fig. 3b) production from 293T cells, with the inhibitory activity residing in the AAA-domain-containing, amino-terminal region (SLFN11-N; amino acids 1–579). No effect of the isolated carboxy-terminal region (SLFN11-C; amino acids 523–901) harbouring the putative helicase sequence was observed (Fig. 1a and Supplementary Fig. 3b). Intriguingly, SLFN5 failed to inhibit retrovirus production but yielded slightly elevated viral titres, illustrating specificity among SLFN proteins in their antiviral activity. To discern whether SLFN11 reduced the number or the viability of released virus, we measured p24 capsid and viral RNA (vRNA) levels in supernatants of pNL4-3.Luc.R 1 E 2 -transfected, HIV VSV-G -producing cells. Extracellular p24 (Fig. 1c and d) or vRNA (Fig. 1e and f) concentration patterns closely matched the titre results from infection assays, demonstrating that SLFN11 diminishes the number of viral particles released from the cells. To assess a possible reduction of intracellular vRNA, we determined its levels in 293T cells expressing chloramphenicol acetyl transferase (CAT), SLFN5, SLFN11, SLFN11-N or SLFN11-C (Fig. 1g), as well as in 293, 293shRNA Ctl and 293shRNA SLFN cells (Fig. 1h). In contrast to the pronounced variations in extracellular vRNA, only insignificant differences in intracellular vRNA were evident among those cells. We also analysed vRNA in the cytoplasmic fraction specifically, as nuclear export of unspliced vRNA is a hallmark of retroviral RNA proces-sing 6–8 , but again found no significant alterations attributable to SLFN11 (Supplementary Fig. 4a). Unlike in BST2-expressing cells 9,10 , electron-microscopic analysis failed to document accumulation of virions inside or on the surface of virus-producing cells in the presence of SLFN11 (Supplementary Fig. 4b). As such, SLFN11 greatly diminishes the formation of viral particles inside the cell, despite the fact that vRNA is equally available.
[Show abstract][Hide abstract] ABSTRACT: Viral hijacking of cellular processes relies on the ability to mimic the structure or function of cellular proteins. Many viruses encode ubiquitin ligases to facilitate infection, although the mechanisms by which they select their substrates are often unknown. The Herpes Simplex Virus type-1-encoded E3 ubiquitin ligase, ICP0, promotes infection through degradation of cellular proteins, including the DNA damage response E3 ligases RNF8 and RNF168. Here we describe a mechanism by which this viral E3 hijacks a cellular phosphorylation-based targeting strategy to degrade RNF8. By mimicking a cellular phosphosite, ICP0 binds RNF8 via the RNF8 forkhead associated (FHA) domain. Phosphorylation of ICP0 T67 by CK1 recruits RNF8 for degradation and thereby promotes viral transcription, replication, and progeny production. We demonstrate that this mechanism may constitute a broader viral strategy to target other cellular factors, highlighting the importance of this region of the ICP0 protein in countering intrinsic antiviral defenses.
[Show abstract][Hide abstract] ABSTRACT: The human APOBEC3 family of cytidine deaminases constitutes a cellular intrinsic defense mechanism that is effective against a range of viruses and retro-elements. While it is well established that these enzymes are powerful mutators of viral DNA, the possibility that their activity could threaten the integrity of the host genome has only recently begun to be investigated. Here, we discuss the implications of new evidence suggesting that APOBEC3 proteins can mediate the deamination of cellular DNA. The maintenance of genomic integrity in the face of this potential off-target activity must require high fidelity DNA repair and strict regulation of APOBEC3 gene expression and enzyme activity. Conversely, the ability of specific members of the APOBEC3 family to activate DNA damage signaling pathways might also reflect another way that these proteins contribute to the host immune response.
[Show abstract][Hide abstract] ABSTRACT: Adeno-associated virus type 2 (AAV2) is a human parvovirus that relies on a helper virus for efficient replication. Herpes simplex virus 1 (HSV-1) supplies helper functions and changes the environment of the cell to promote AAV2 replication. In this study, we examined the accumulation of cellular replication and repair proteins at viral replication compartments (RCs) and the influence of replicating AAV2 on HSV-1-induced DNA damage responses (DDR). We observed that the ATM kinase was activated in cells coinfected with AAV2 and HSV-1. We also found that phosphorylated ATR kinase and its cofactor ATR-interacting protein were recruited into AAV2 RCs, but ATR signaling was not activated. DNA-PKcs, another main kinase in the DDR, was degraded during HSV-1 infection in an ICP0-dependent manner, and this degradation was markedly delayed during AAV2 coinfection. Furthermore, we detected phosphorylation of DNA-PKcs during AAV2 but not HSV-1 replication. The AAV2-mediated delay in DNA-PKcs degradation affected signaling through downstream substrates. Overall, our results demonstrate that coinfection with HSV-1 and AAV2 provokes a cellular DDR which is distinct from that induced by HSV-1 alone.
Journal of Virology 01/2012; 86(1):143-55. · 5.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cellular restriction factors responding to herpesvirus infection include the ND10 components PML, Sp100 and hDaxx. During the initial stages of HSV-1 infection, novel sub-nuclear structures containing these ND10 proteins form in association with incoming viral genomes. We report that several cellular DNA damage response proteins also relocate to sites associated with incoming viral genomes where they contribute to the cellular front line defense. We show that recruitment of DNA repair proteins to these sites is independent of ND10 components, and instead is coordinated by the cellular ubiquitin ligases RNF8 and RNF168. The viral protein ICP0 targets RNF8 and RNF168 for degradation, thereby preventing the deposition of repressive ubiquitin marks and counteracting this repair protein recruitment. This study highlights important parallels between recognition of cellular DNA damage and recognition of viral genomes, and adds RNF8 and RNF168 to the list of factors contributing to the intrinsic antiviral defense against herpesvirus infection.
[Show abstract][Hide abstract] ABSTRACT: Viruses often induce signaling through the same cellular cascades that are activated by damage to the cellular genome. Signaling triggered by viral proteins or exogenous DNA delivered by viruses can be beneficial or detrimental to viral infection. Viruses have therefore evolved to dissect the cellular DNA damage response pathway during infection, often marking key cellular regulators with ubiquitin to induce their degradation or change their function. Signaling controlled by ubiquitin or ubiquitin-like proteins has recently emerged as key regulator of the cellular DNA damage response. Situated at the interface between DNA damage signaling and the ubiquitin system, viruses can reveal key convergence points in this important cellular pathway. In this review, we examine how viruses harness the diversity of the cellular ubiquitin system to modulate the DNA damage signaling pathway. We discuss the implications of viral infiltration of this pathway for both the transcriptional program of the virus and for the cellular response to DNA damage.
[Show abstract][Hide abstract] ABSTRACT: Human apolipoprotein-B mRNA-editing catalytic polypeptide-like 3 (APOBEC3) proteins constitute a family of cytidine deaminases that mediate restriction of retroviruses, endogenous retro-elements and DNA viruses. It is well established that these enzymes are potent mutators of viral DNA, but it is unclear whether their editing activity is a threat to the integrity of the cellular genome. We show that expression of APOBEC3A can lead to induction of DNA breaks and activation of damage responses in a deaminase-dependent manner. Consistent with these observations, APOBEC3A expression induces cell-cycle arrest. These results indicate that cellular DNA is vulnerable to APOBEC3 activity and deregulated expression of APOBEC3A could threaten genomic integrity.
[Show abstract][Hide abstract] ABSTRACT: The adenovirus (Ad) E1b55K and E4orf6 gene products assemble an E3 ubiquitin ligase complex that promotes degradation of cellular proteins. Among the known substrates are p53 and the Mre11-Rad50-Nbs1 (MRN) complex. Since members of the RecQ helicase family function together with MRN in genome maintenance, we investigated whether adenovirus affects RecQ proteins. We show that Bloom helicase (BLM) is degraded during adenovirus type 5 (Ad5) infection. BLM degradation is mediated by E1b55K/E4orf6 but is independent of MRN. We detected BLM localized at discrete foci around viral replication centers. These studies identify BLM as a new substrate for degradation by the adenovirus E1b55K/E4orf6 complex.
Journal of Virology 02/2011; 85(4):1887-92. · 5.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Members of the human APOBEC3 family of editing enzymes can inhibit various mobile genetic elements. APOBEC3A (A3A) can block the retrotransposon LINE-1 and the parvovirus adeno-associated virus type 2 (AAV-2) but does not inhibit retroviruses. In contrast, APOBEC3G (A3G) can block retroviruses but has only limited effects on AAV-2 or LINE-1. What dictates this differential target specificity remains largely undefined. Here, we modeled the structure of A3A based on its homology with the C-terminal domain of A3G and further compared the sequence of human A3A to those of 11 nonhuman primate orthologues. We then used these data to perform a mutational analysis of A3A, examining its ability to restrict LINE-1, AAV-2, and foreign plasmid DNA and to edit a single-stranded DNA substrate. The results revealed an essential functional role for the predicted single-stranded DNA-docking groove located around the A3A catalytic site. Within this region, amino acid differences between A3A and A3G are predicted to affect the shape of the polynucleotide-binding groove. Correspondingly, transferring some of these A3A residues to A3G endows the latter protein with the ability to block LINE-1 and AAV-2. These results suggest that the target specificity of APOBEC3 family members is partly defined by structural features influencing their interaction with polynucleotide substrates.
Journal of Virology 02/2011; 85(4):1765-76. · 5.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Adeno-associated virus (AAV) was first discovered as a contaminant of adenovirus stocks in the 1960s. The development of recombinant AAV vectors (rAAV) was facilitated by early studies that generated infectious molecular clones, determined the sequence of the genome, and defined the genetic elements of the virus. The refinement of methods and protocols for the production and application of rAAV vectors has come from years of studies that explored the basic biology of this virus and its interaction with host cells. Interest in improving vector performance has in turn driven studies that have provided tremendous insights into the basic biology of the AAV lifecycle. In this chapter, we review the background on AAV biology and its exploitation for vectors and gene delivery.
[Show abstract][Hide abstract] ABSTRACT: Oncogenic viruses infect many cells but rarely lead to tumorigenesis. In this issue of Cell Host & Microbe, Nikitin et al. describe how a protective DNA damage response acts to suppress transformation in the majority of cells latently infected with Epstein-Barr virus (EBV).
[Show abstract][Hide abstract] ABSTRACT: Genomes are subject to constant threat by damaging agents that generate DNA double-strand breaks (DSBs). The ends of linear chromosomes need to be protected from DNA damage recognition and end-joining, and this is achieved through protein-DNA complexes known as telomeres. The Mre11-Rad50-Nbs1 (MRN) complex plays important roles in detection and signaling of DSBs, as well as the repair pathways of homologous recombination (HR) and non-homologous end-joining (NHEJ). In addition, MRN associates with telomeres and contributes to their maintenance. Here, we provide an overview of MRN functions at DSBs, and examine its roles in telomere maintenance and dysfunction.
[Show abstract][Hide abstract] ABSTRACT: The ICP0 protein of herpes simplex virus type 1 is an E3 ubiquitin ligase and transactivator required for the efficient switch between latent and lytic infection. As DNA damaging treatments are known to reactivate latent virus, we wished to explore whether ICP0 modulates the cellular response to DNA damage. We report that ICP0 prevents accumulation of repair factors at cellular damage sites, acting between recruitment of the mediator proteins Mdc1 and 53BP1. We identify RNF8 and RNF168, cellular histone ubiquitin ligases responsible for anchoring repair factors at sites of damage, as new targets for ICP0-mediated degradation. By targeting these ligases, ICP0 expression results in loss of ubiquitinated forms of H2A, mobilization of DNA repair proteins and enhanced viral fitness. Our study raises the possibility that the ICP0-mediated control of histone ubiquitination may link DNA repair, relief of transcriptional repression, and activation of latent viral genomes.
The EMBO Journal 03/2010; 29(5):943-55. · 9.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The cellular surveillance network for sensing and repairing damaged DNA prevents an array of human diseases, and when compromised it can lead to genomic instability and cancer. The carefully maintained cellular response to DNA damage is challenged during viral infection, when foreign DNA is introduced into the cell. The battle between virus and host generates a genomic conflict. The host attempts to limit viral infection and protect its genome, while the virus deploys tactics to eliminate, evade, or exploit aspects of the cellular defense. Studying this conflict has revealed that the cellular DNA damage response machinery comprises part of the intrinsic cellular defense against viral infection. In this review we examine recent advances in this emerging field. We identify common themes used by viruses in their attempts to commandeer or circumvent the host cell's DNA repair machinery, and highlight potential outcomes of the conflict for both virus and host.
Annual review of microbiology 01/2010; 64:61-81. · 12.80 Impact Factor