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The Virus–Chaperone Connection
... Hence, the identification of the cellular proteins and their role in replication and infection needs to be determined. It has been reported that viral infections induce cellular expression of stress response proteins like heat shock proteins (Hsps) [9]. Such induction of heat shock proteins have been reported for both the DNA and RNA viruses. ...
... However, the type of Hsp associated in a viral infection depends on the kind of pathogen and the nature of the host cells. [9,10].The Hsps are known as important molecular chaperones that modulate different cellular processes to maintain cellular homeostasis [11]. Chaperones bind to misfolded or unfolded polypeptides to assist in their correct folding and assembly, regulate protein transport and translocation and facilitate misfolded polypeptides for degradation by the ubiquitin-proteasome system to maintain cell viability [11][12][13][14]. ...
... Chaperones bind to misfolded or unfolded polypeptides to assist in their correct folding and assembly, regulate protein transport and translocation and facilitate misfolded polypeptides for degradation by the ubiquitin-proteasome system to maintain cell viability [11][12][13][14]. Although the assembly of cellular chaperones often increases with virus infection but it is still not clear whether this is a direct effect of infection or an indirect response to cellular stress induced by infection [9,15,16]. Moreover, any kind of stress or infection results in the induction of various Hsps like Hsp90, Hsp70, Hsp40 and several small Hsps [17,18]. ...
... Hence, the identification of the cellular proteins and their role in replication and infection needs to be determined. It has been reported that viral infections induce cellular expression of stress response proteins like heat shock proteins (Hsps) [9]. Such induction of heat shock proteins have been reported for both the DNA and RNA viruses. ...
... However, the type of Hsp associated in a viral infection depends on the kind of pathogen and the nature of the host cells. [9,10].The Hsps are known as important molecular chaperones that modulate different cellular processes to maintain cellular homeostasis [11]. Chaperones bind to misfolded or unfolded polypeptides to assist in their correct folding and assembly, regulate protein transport and translocation and facilitate misfolded polypeptides for degradation by the ubiquitin-proteasome system to maintain cell viability [11][12][13][14]. ...
... Chaperones bind to misfolded or unfolded polypeptides to assist in their correct folding and assembly, regulate protein transport and translocation and facilitate misfolded polypeptides for degradation by the ubiquitin-proteasome system to maintain cell viability [11][12][13][14]. Although the assembly of cellular chaperones often increases with virus infection but it is still not clear whether this is a direct effect of infection or an indirect response to cellular stress induced by infection [9,15,16]. Moreover, any kind of stress or infection results in the induction of various Hsps like Hsp90, Hsp70, Hsp40 and several small Hsps [17,18]. ...
Background
The high morbidity and socio-economic loss associated with the recent massive global outbreak of Chikungunya virus (CHIKV) emphasize the need to understand the biology of the virus for developing effective antiviral therapies.
Methods and Findings
In this study, an attempt was made to understand the molecular mechanism involved in Heat shock protein 90 (Hsp90) mediated regulation of CHIKV infection in mammalian cells using CHIKV prototype strain (S 27) and Indian outbreak strain of 2006 (DRDE-06). Our results showed that Hsp90 is required at a very early stage of viral replication and Hsp90 inhibitor Geldanamycin (GA) can abrogate new virus particle formation more effectively in the case of S 27 than that of DRDE-06. Further analysis revealed that CHIKV nsP2 protein level is specifically reduced by GA treatment as well as HSP90-siRNA transfection; however, viral RNA remains unaltered. Immunoprecipitation analysis showed that nsP2 interacts with Hsp90 during infection; however this interaction is reduced in the presence of GA. In addition, our analysis on Hsp90 associated PI3K/Akt/mTOR signaling pathway demonstrated that CHIKV infection stabilizes Raf1 and activates Hsp90 client protein Akt, which in turn phosphorylates mTOR. Subsequently, this phosphorylation leads to the activation of two important downstream effectors, S6K and 4EBP1, which may facilitate translation of viral as well as cellular mRNAs. Hence, the data suggests that CHIKV infection is regulated by Hsp90 associated Akt phosphorylation and DRDE-06 is more efficient than S 27 in enhancing the activation of host signaling molecules for its efficient replication and virus production.
Conclusion
Hsp90 positively regulates Chikungunya virus replication by stabilizing CHIKV-nsP2 through its interaction during infection. The study highlights the possible molecular mechanism of GA mediated inhibition of CHIKV replication and differential effect of this drug on S 27 and DRDE-06, which will be informative for developing effective anti-CHIKV therapies in future.
... Hence, the identification of the cellular proteins and their role in replication and infection needs to be determined. It has been reported that viral infections induce cellular expression of stress response proteins like heat shock proteins (Hsps) [9]. Such induction of heat shock proteins have been reported for both the DNA and RNA viruses. ...
... However, the type of Hsp associated in a viral infection depends on the kind of pathogen and the nature of the host cells. [9,10].The Hsps are known as important molecular chaperones that modulate different cellular processes to maintain cellular homeostasis [11]. Chaperones bind to misfolded or unfolded polypeptides to assist in their correct folding and assembly, regulate protein transport and translocation and facilitate misfolded polypeptides for degradation by the ubiquitin-proteasome system to maintain cell viability [11][12][13][14]. ...
... Chaperones bind to misfolded or unfolded polypeptides to assist in their correct folding and assembly, regulate protein transport and translocation and facilitate misfolded polypeptides for degradation by the ubiquitin-proteasome system to maintain cell viability [11][12][13][14]. Although the assembly of cellular chaperones often increases with virus infection but it is still not clear whether this is a direct effect of infection or an indirect response to cellular stress induced by infection [9,15,16]. Moreover, any kind of stress or infection results in the induction of various Hsps like Hsp90, Hsp70, Hsp40 and several small Hsps [17,18]. ...
Background: The high morbidity and socioeconomic loss associated with the recent massive global outbreak of Chikungunya virus (CHIKV) emphasize the need to understand the biology of the virus for developing effective antiviral therapies.
... Another susceptibility factor is heat shock protein 70 (Hsp70), a chaperone interacting with the CP of Potato virus A (PVA), necessary for viral gene expression and replication [35]. Two heat shock proteins, Hsp40 and Hsp90, are involved in the innate immunity of the plant [36][37][38][39]. Another susceptibility factor, plasma membrane-associated cation-binding protein 1 (PCap1), has been shown to be related to Potato virus Y (PVY) accumulation and cell-to-cell movement [40]. ...
... Potyviruses recruit the Hsp70 chaperone in the formation of replication complexes [35,75]. Another pair of Hsps-Hsp40 and Hsp90-appear to be involved in viral infections [36][37][38]. Hsp90 activates various cytosolic R proteins, such as the N tobacco protein, helping in the pathogen defense response [39]. The silencing of Hsp90 by means of a virus-induced gene silencing (VIGS) strategy in N. benthamiana caused the loss of resistance to P. syringae, the Tobacco mosaic virus (TMV), and the Potato X virus (PVX), confirming the key role of Hsp90 against pathogens [76]. ...
To uncover novel genes associated with the Sugarcane mosaic virus (SCMV) response, we used RNA-Seq data to analyze differentially expressed genes (DEGs) and transcript expression pattern clusters between a tolerant/resistant (CI-RL1) and a susceptible (B73) line, in addition to the F1 progeny (CI-RL1xB73). A Gene Ontology (GO) enrichment of DEGs led us to propose three genes possibly associated with the CI-RL1 response: a heat shock 90-2 protein and two ABC transporters. Through a clustering analysis of the transcript expression patterns (CTEPs), we identified two genes putatively involved in viral systemic spread: the maize homologs to the PIEZO channel (ZmPiezo) and to the Potyvirus VPg Interacting Protein 1 (ZmPVIP1). We also observed the complex behavior of the maize eukaryotic factors ZmeIF4E and Zm-elfa (involved in translation), homologs to eIF4E and eEF1α in A. thaliana. Together, the DEG and CTEPs results lead us to suggest that the tolerant/resistant CI-RL1 response to the SCMV encompasses the action of diverse genes and, for the first time, that maize translation factors are associated with viral interaction.
... Cells induce a stress response to virus infection, which results in the expression of stress response proteins like heat shock proteins (Hsps) (Sullivan and Pipas, 2001). It has been reported that both DNA and RNA viruses induce Hsps, independent of the cytoplasmic or nuclear location of their replication however the type of Hsp induced, depends on the virus or the cell type (Sedger and Ruby, 1994; Sullivan and Pipas, 2001). ...
... Cells induce a stress response to virus infection, which results in the expression of stress response proteins like heat shock proteins (Hsps) (Sullivan and Pipas, 2001). It has been reported that both DNA and RNA viruses induce Hsps, independent of the cytoplasmic or nuclear location of their replication however the type of Hsp induced, depends on the virus or the cell type (Sedger and Ruby, 1994; Sullivan and Pipas, 2001). Hsp family members including Hsp40, 70 and 90 (40-, 70-, 90-kDa heat shock proteins) are also important molecular chaperones which regulate various cellular processes such as protein folding, transport, cell viability etc (Welch, 1991). ...
Rotaviruses are the major cause of severe dehydrating gastroenteritis in children worldwide. In this study, we report a positive role of cellular chaperone Hsp90 during rotavirus infection. A highly specific Hsp90 inhibitor, 17-allylamono-demethoxygeldanamycin (17-AAG) was used to delineate the functional role of Hsp90. In MA104 cells treated with 17-AAG after viral adsorption, replication of simian (SA11) or human (KU) strains was attenuated as assessed by quantitating both plaque forming units and expression of viral genes. Phosphorylation of Akt and NFkappaB observed 2-4 hpi with SA11, was strongly inhibited in the presence of 17-AAG. Direct Hsp90-Akt interaction in virus infected cells was also reduced in the presence of 17-AAG. Anti-rotaviral effects of 17-AAG were due to inhibition of activation of Akt that was confirmed since, PI3K/Akt inhibitors attenuated rotavirus growth significantly. Thus, Hsp90 regulates rotavirus by modulating cellular signaling proteins. The results highlight the importance of cellular proteins during rotavirus infection and the possibility of targeting cellular chaperones for developing new anti-rotaviral strategies.
... However, it is uncertain if this directly results from the virus infection or indirect response to cellular stress generated by the virus. [64][65][66] The most diverse HSP40 family members are implicated in a wide array of additional functions, including modulation of polymerase activity, replication complex, and nuclear transportation. Earlier studies have reported that some HSP40 isoforms regulate the replication process of various viruses by interacting with cellular proteins or viral proteins. ...
HSP40/DNAJ family of proteins is the most diverse chaperone family, comprising about 49 isoforms in humans. Several reports have demonstrated the functional role of a few of these isoforms in the pathogenesis of various viruses, including HIV‐1. Our earlier study has shown that several isoforms of HSP40 get significantly modulated at the mRNA level during HIV‐1 infection in T cells. To explore the biological role of these significantly modulated isoforms, we analyzed their effect on HIV‐1 gene expression and virus production using knockdown and overexpression studies. Among these isoforms, DNAJA3, DNAJB1, DNAJB7, DNAJC4, DNAJC5B, DNAJC5G, DNAJC6, DNAJC22, and DNAJC30 seem to positively regulate virus replication, whereas DNAJB3, DNAJB6, DNAJB8, and DNAJC5 negatively regulate virus replication. Further investigation on the infectivity of the progeny virion demonstrated that only DNAJB8 negatively regulates the progeny virion infectivity. It was further identified that DNAJB8 protein is involved in the downregulation of Vif protein, required for the infectivity of HIV‐1 virions. DNAJB8 seems to direct Vif protein for autophagic‐lysosomal degradation, leading to rescue of the cellular restriction factor APOBEC3G from Vif‐mediated proteasomal degradation, resulting in enhanced packaging of APOBEC3G in budding virions and release of less infective progeny virion particles. Finally, our results also indicate that during the early stage of HIV‐1 infection, enhanced expression of DNAJB8 promotes the production of less infective progeny virions, but at the later stage or at the peak of infection, reduced expression of DNJAB8 protein allows the HIV‐1 to replicate and produce more infective progeny virion particles.
... Another role that has been attributed to ICP22 is involved in the host cell chaperone machinery by facilitating the formation of virus-induced chaperon-enriched (VICE) domains in the nucleus of some infected cells. Recent studies suggested that ICP22 mimics a cellular type II J protein, which is a co-chaperone in the nucleus [283][284][285]. The VICE domains are usually formed adjacent to the viral replication compartments, they contain several host chaperones (Hsp70, Hsp40, Hsp90), proteasomal components, ubiquitinated proteins, and at least one viral protein. ...
Viruses encode for structural proteins that participate in virion formation and include capsid and envelope proteins. In addition, viruses encode for an array of non-structural accessory proteins important for replication, spread, and immune evasion in the host and are often linked to virus pathogenesis. Most virus accessory proteins are non-essential for growth in cell culture because of the simplicity of the infection barriers or because they have roles only during a state of the infection that does not exist in cell cultures (i.e., tissue-specific functions), or finally because host factors in cell culture can complement their absence. For these reasons, the study of most nonessential viral factors is more complex and requires development of suitable cell culture systems and in vivo models. Approximately half of the proteins encoded by the herpes simplex virus 1 (HSV-1) genome have been classified as non-essential. These proteins have essential roles in vivo in counteracting antiviral responses, facilitating the spread of the virus from the sites of initial infection to the peripheral nervous system, where it establishes lifelong reservoirs, virus pathogenesis, and other regulatory roles during infection. Understanding the functions of the non-essential proteins of herpesviruses is important to understand mechanisms of viral pathogenesis but also to harness properties of these viruses for therapeutic purposes. Here, we have provided a comprehensive summary of the functions of HSV-1 non-essential proteins.
... *P < 0.05 was considered to represent a statistically significant difference. [32]. DNAJC5G significantly inhibits the replication efficiencies of adenovirus, vaccinia virus, and HIV-1 [33]. ...
Human papilloma virus (HPV)-associated oropharyngeal cancer (OPC) is an independent tumour type with regard to cellular, biological, and clinical features. The use of non-invasive biomarkers such as circulating tumour DNA (ctDNA) may be relevant in early diagnosis and eventually improve the outcomes of patients with head and neck squamous cell carcinoma (HNSCC). Genome-wide discovery using RNA sequencing and reduced representation bisulfite sequencing yielded 21 candidates for methylation-targeted genes. A verification study (252 HNSCC patients) using quantitative methylation-specific PCR (Q-MSP) identified 10 genes (ATP2A1, CALML5, DNAJC5G, GNMT, GPT, LY6D, LYNX1, MAL, MGC16275, and MRGPRF) that showed a significant increase recurrence in methylation groups with OPC. Further study on ctDNA using Q-MSP in HPV-associated OPC showed that three genes (CALML5, DNAJC5G, and LY6D) had a high predictive ability as emerging biomarkers for a validation set, each capable of discriminating between the plasma of the patients from healthy individuals. Among the the 42 ctDNA samples, methylated CALML5, DNAJC5G, and LY6D were observed in 31 (73.8%), 19 (45.2%), and 19 (45.2%) samples, respectively. Among pre-treatment ctDNA samples, methylated CALML5, DNAJC5G, and LY6D were observed in 8/8 (100%), 7/8 (87.5%), and 7/8 (87.5%) samples, respectively. Methylated CALML5, DNAJC5G, and LY6D were found in 2/8 (25.0%), 0/8 (0%), and 1/8 (12.5%) of the final samples in the series, respectively. Here, we present the relationship between the methylation status of three specific genes and cancer recurrence for risk classification of HPV-associated OPC cases. In conclusion, ctDNA analysis has the potential to aid in determining patient prognosis and real-time surveillance for disease recurrences and serves as an alternative method of screening for HPV-associated OPC.
... Other studies have stated that many HSP70 are induced during viral infection. Besides facilitating viral pathogenesis, increased expression of HSP70 induced by cellular stress also relieves stress caused by viral infection (Sullivan & Pipas, 2001;Neckers & Tatu, 2008;Roberts et al. 2010). The HSP70 causes KHV-infected tilapia to not experience mortality and be able to survive even though there is tissue damage within the gills. ...
Koi herpesvirus (KHV) not only can infect cyprinidae, but also infect other freshwater fishes. It causes KHV to spread widely throughout the world. Infection of KHV in fish has several symptoms and one of them is increased HSP70 expression. The purpose of this study is to detect HSP70 expression in KHV-infected tilapia. The study were carried out by qualitatively examining expression of HSP70 in the gill of tilapia, both normal tilapia and KHV-infected tilapia, and by detecting the tissue damage caused by KHV by means of histopathology. The results of this study show that HSP70 was detected in fish that were infected by KHV, while in control fish, HSP70 was not detected. This result indicates that tilapia infected by KHV experience an increase in HSP70 expression. Damage to the gill of tilapia is also one of the responses to KHV antigens in the host cell. Thus, this study presumed that KHV infection in tilapia is influenced by the expression of HSP70.
... The heat shock proteins (HSP) are molecular chaperones which bind and stabilize misfolded or unfolded polypeptides to ensure their proper folding and assembly with other polypeptides to decipher the normal protein function [40][41][42][43]. Induction of HSPs has been reported in both RNA and DNA viral infections, however, the type of HSP involved in a viral infection depends on the kind of virus and the type of host cells associated to the infection [44,45]. Recent studies have shown the functional requirement of HSP90 for Human cytomegalo virus (HCMV), Hepatitis C virus (HCV), Herpes Simplex virus-1 (HSV-1), Human Immunodeficiency virus-1 (HIV-1), Hepatitis E virus (HEV), Epstein Barr virus (EBV), Vaccinia virus and rotavirus infections [46][47][48][49][50][51][52][53]. ...
Chikungunya virus (CHIKV) infection has re-emerged as a major public health concern due to its recent worldwide epidemics and lack of control measures. Although CHIKV is known to infect macrophages, regulation of CHIKV replication, apoptosis and immune responses towards macrophages are not well understood. Accordingly, the Raw264.7 cells, a mouse macrophage cell line, were infected with CHIKV and viral replication as well as new viral progeny release was assessed by flow cytometry and plaque assay, respectively. Moreover, host immune modulation and apoptosis were studied through flow cytometry, Western blot and ELISA. Our current findings suggest that expression of CHIKV proteins were maximum at 8 hpi and the release of new viral progenies were remarkably increased around 12 hpi. The induction of Annexin V binding, cleaved caspase-3, cleaved caspase-9 and cleaved caspase-8 in CHIKV infected macrophages suggests activation of apoptosis through both intrinsic and extrinsic pathways. The pro-inflammatory mediators (TNF and IL-6) MHC-I/II and B7.2 (CD86) were also up-regulated during infection over time. Further, 17-AAG, a potential HSP90 inhibitor, was found to regulate CHIKV infection, apoptosis and pro-inflammatory cytokine/chemokine productions of host macrophages significantly. Hence, the present findings might bring new insight into the therapeutic implication in CHIKV disease biology.
... In the context of cellular chaperone activity, Hsc70 has been shown to play an important role in the virus life cycle by modulating infectivity [72,73] , serving as a receptor molecule [7,8] or participating in viral assembly and morphogenesis [74,75] . Hsc70 is a constitutively expressed molecular chaperone belonging to the Hsp70 family. ...
Rotaviruses are the single leading cause of life-threatening diarrhea affecting children under 5 years of age. Rotavirus entry into the host cell seems to occur by sequential interactions between virion proteins and various cell surface molecules. The entry mechanisms seem to involve the contribution of cellular molecules having binding, chaperoning and oxido-reducing activities. It appears to be that the receptor usage and tropism of rotaviruses is determined by the species, cell line and rotavirus strain. Rotaviruses have evolved functions which can antagonize the host innate immune response, whereas are able to induce endoplasmic reticulum (ER) stress, oxidative stress and inflammatory signaling. A networking between ER stress, inflammation and oxidative stress is suggested, in which release of calcium from the ER increases the generation of mitochondrial reactive oxygen species (ROS) leading to toxic accumulation of ROS within ER and mitochondria. Sustained ER stress potentially stimulates inflammatory response through unfolded protein response pathways. However, the detailed characterization of the molecular mechanisms underpinning these rotavirus-induced stressful conditions is still lacking. The signaling events triggered by host recognition of virus-associated molecular patterns offers an opportunity for the development of novel therapeutic strategies aimed at interfering with rotavirus infection. The use of N-acetylcysteine, non-steroidal anti-inflammatory drugs and PPARγ agonists to inhibit rotavirus infection opens a new way for treating the rotavirus-induced diarrhea and complementing vaccines.
... The proper folding of proteins within cells is mediated by the activities of molecular 5763)), in our work, we monitored polyprotein processing of YFV NS2A-5 587 polyprotein in DNAJC14-overexpressing cells. We found that NS3/4A/2K cleavage events were 588 particularly affected with an overall effect on uncleaved NS3-5 accumulation and reduced levels 589 of NS3-4A, NS3 and NS4B. ...
Importance:
Flaviviruses are single-stranded RNA viruses that cause a wide range of illnesses. Upon host cell entry, the viral genome is translated on ER membranes to produce a single polyprotein, which is cleaved by host and viral proteases to generate viral proteins required for genome replication and virion production. Several studies suggest a role for molecular chaperones during these processes. While the details of chaperone roles have been elusive, in this manuscript we show that overexpression of the ER-resident co-chaperone DNAJC14 affects YFV polyprotein processing at the NS3/4A site. This work reveals DNAJC14 modulation of NS3/4A site processing as an important mechanism to ensure virus replication. Our work highlights the importance of finely regulating flavivirus polyprotein processing. In addition, it suggests future studies addressing similarities and/or differences amongst flaviviruses and to interrogate precise mechanisms employed for polyprotein processing - a critical step that ultimately can be targeted for novel drug development.
... Conversely, the knockdown of HSP70 mediated by lentivirus or quercetin resulted in a significant decrease in virus production (Fig. 6), which was comparable to the reduction of virus observed by HSP70 knockdown in previous study (Gonzalez et al., 2009). HSP70 is involved in many viral life cycle activities, such as transcription, cellular transformation, viral genome replication, and increased virion assembly (Sullivan and Pipas, 2001). Hsp70 is a chaperone interacting with many proteins and might also interact with additional viral proteins. ...
The non-structural 5A (NS5A) protein of classical swine fever virus (CSFV) is proven to be involved in viral replication and can also modulate cellular signaling via to its ability to interact with various cellular proteins. Here, HSP70/NS5A complex formation is confirmed by coimmunoprecipitation and GST-pulldown studies. Additionally, the N-terminal amino acids (29-240) of NS5A were identified as the interaction region through in vivo deletion analyses, and confocal microscopy showed that NS5A and HSP70 colocalized in the cytoplasm. Overexpression of HSP70 via the eukaryotic expression plasmid pDsRED N1 or lentivirus significantly promoted viral RNA synthesis. Whereas the knockdown of HSP70 by lentivirus-mediated shRNA or inhibition by quercetin markedly decreased the viral load. These data suggest that HSP70 plays a critical role in the viral life cycle, particularly during the virus RNA replication period. The investigation of HSP70 protein functions may be beneficial for developing new strategies to treat CSFV infection.
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... The general function of the intracellular molecular chaperones has been suggested mainly to involve housekeeping and cytoprotection against various environmental stresses. The expression of many chaperones is induced during viral infection, either to facilitate viral pathogenesis or to participate in cellular response mechanisms to alleviate the stress caused by infection [48,49]. While increased expression of HSPs was suggested to be a biomarker for some viral infections [50], we have shown previously that TYLCV did not induce the expression of tomato HSP60, 70, 90, 100 and FtsH [8]. ...
In order to understand resistance to Tomato yellow leaf curl virus (TYLCV) we have performed a combined analysis of the metabolome and transcriptome of resistant (R) and susceptible (S) tomato plants both prior to and following TYLCV infection. Metabolites detected by gas chromatography–mass spectrometry (GC–MS) and liquid chromatography–mass spectrometry analysis, in leaves of R and S plants, at 1, 3, 7 and 14 days post infection and control plants, were used for the reconstruction of four independent metabolic networks. Measuring the network parameters revealed distinctive systemic metabolic responses to TYLCV infection between the R and S plants. Notably, the GC–MS metabolic network indicated that, following infection, the R plant exhibited tight coordination of the metabolome than the S plant. Clear differences in the level of specialized metabolites between the S and R plants were revealed; among them, substantial alteration in the abundance of amino acids and polyamines, phenolic and indolic metabolites, all leading to the synthesis of defense compounds. Integrating metabolome and transcriptome data highlighted differently regulated pathways in the R and S plants in response to TYLCV, including the phenylpropanoid, tryptophan/nicotinate and urea/polyamine pathways. Salicylic acid biosynthesis was additionally distinctively activated in R plants upon infection. Comparing the expression of genes of the urea and phenylpropanoid pathways in S, R and Solanum habrochaites, the resistance genitor wild species tomato, indicated a time-shift in the expression patterns, before and following infection, which on one hand reflected the genetic similarity between these plants, and on the other hand demonstrated that the resistant phenotype is intermediate between that of S and S. habrochaites.
... Host cell responses to metabolic or environmental stresses are generally mediated by inducing synthesis of a selected family of proteins, collectively known as stress proteins or heat-shock proteins (Hsps) (Morimoto, 1991). They are involved in many viral life cycle activities, such as transcription, cellular transformation, viral genome replication, and increased virion assembly (Sullivan and Pipas, 2001). Among them, the ubiquitous chaperone family of 70 kDa HSPs (Hsp70) plays a central role in protein homeostasis and protection against proteotoxic stresses. ...
The Hsp70 chaperone plays a central role in multiple processes within cells. Porcine circovirus type 2 (PCV2) is the essential causal agent of post-weaning multisystemic wasting syndrome (PMWS), which has spread worldwide. But the mechanism of PCV2 replication remains poorly understood. In this study, we firstly found the positive effect of heat stress on the replication of PCV2 in the continuous porcine monocytic cell line 3D4/31. Downregulation of Hsp70 by the specific chaperone inhibitor Quercetin or RNA interference and upregulation of Hsp70 by expression from a recombinant adenovirus showed that Hsp70 enhanced PCV2 genome replication and virion production. A specific interaction between Hsp70 and PCV2 Cap was confirmed by colocalization by confocal microscopy and co-immunoprecipitation. Furthermore, the NF-κB pathway was activated and caspase-3 activity was reduced when Hsp70 was overexpressed in PCV2-infected 3D4/31 cells. These data suggested that Hsp70 positively regulated PCV2 replication, which being helpful for understanding the molecular mechanism of PCV2 infection.
... The presence of CpHsp24 is required for appropriate adaptation of the fungus to both abiotic and biotic stresses. Although the Hsp has been implicated as a molecular chaperone for viral replication and assembly (Sullivan and Pipas 2001), persistent growth retardation of the virus-infected CpHsp24-null mutant without reverting phenotypes, such as rapidly growing sectors shown in the virusinfected pro1-null mutant (Sun et al. 2009), provided evidence of the stable maintenance of CHV1-EP713 in the CpHsp24- Fig. 7. Molecular changes in the accumulation of the lac3 gene transcript in response to tannic acid (TA) supplementation. Total RNA was extracted before and 24 h after TA induction. ...
A small heat shock protein gene CpHsp24 of Cryphonectria parasitica was selected based on its expression pattern, which showed that it was tannic acid-inducible and that its induction was severely hampered by a hypovirus. The predicted protein sequence of CpHsp24 consisted of a hallmark alpha-crystalline domain flanked by a variable N-terminal and a short C-terminal region. Disruption of CpHsp24 resulted in a slow growth rate under standard growth conditions. The CpHsp24-null mutant showed enhanced sensitivity to heat shock, which was consistent with Northern and Western analyses displaying the heat shock induction of the CpHsp24 gene and protein, respectively. Virulence tests on the excised bark revealed a severe decrease in the necrotic area of the CpHsp24-null mutant. When the hypovirus was transferred, virus-containing CpHsp24-null progeny displayed severely retarded growth patterns with hypovirulent characteristics of reduced pigmentation and sporulation. Since the tannic acid-inducible and hypoviral-suppressible expression and the severely impaired virulence are also characteristics of the laccase3 gene (lac3), lac3 expression in the CpHsp24-null mutant was also examined. The resulting lac3 induction was severely affected in the CpHsp24-null mutant suggesting that CpHsp24 is important for lac3 induction and that CpHsp24 may act as a molecular chaperone for the lac3 protein.
... In contrast, L1/L2 (17-36) predominantly formed protein aggregates, although the presence of larger amorphous VLP-like structures suggests there may be partial assembly of VLPs. L1/L2 (108-120), with the shortest L2 epitope, successfully assembled into regular chimaeric VLPs (cVLPs), in contrast to a similar chimaera expressed in insect cells (Varsani et al., 2003a)-possibly due to subtle differences in viral assembly chaperones such as heat shock protein 70 (Hsp70) and karyopherins (Sullivan and Pipas, 2001). ...
Cervical cancer is caused by infection with human papillomaviruses (HPV) and is a global concern, particularly in developing countries, which have ~80% of the burden. HPV L1 virus-like particle (VLP) type-restricted vaccines prevent new infections and associated disease. However, their high cost has limited their application, and cytological screening programmes are still required to detect malignant lesions associated with the nonvaccine types. Thus, there is an urgent need for cheap second-generation HPV vaccines that protect against multiple types. The objective of this study was to express novel HPV-16 L1-based chimaeras, containing cross-protective epitopes from the L2 minor capsid protein, in tobacco plants. These L1/L2 chimaeras contained epitope sequences derived from HPV-16 L2 amino acid 108-120, 56-81 or 17-36 substituted into the C-terminal helix 4 (h4) region of L1 from amino acid 414. All chimaeras were expressed in Nicotiana benthamiana via an Agrobacterium-mediated transient system and targeted to chloroplasts. The chimaeras were highly expressed with yields of ~1.2 g/kg plant tissue; however, they assembled differently, indicating that the length and nature of the L2 epitope affect VLP assembly. The chimaera containing L2 amino acids 108-120 was the most successful candidate vaccine. It assembled into small VLPs and elicited anti-L1 and anti-L2 responses in mice, and antisera neutralized homologous HPV-16 and heterologous HPV-52 pseudovirions. The other chimaeras predominantly assembled into capsomeres and other aggregates and elicited weaker humoral immune responses, demonstrating the importance of VLP assembly for the immunogenicity of candidate vaccines.
... Molecular chaperons were considered to be important for virus infection and involved in replication, transcription, translation and virion assembly (Sullivan and Pipas, 2001). Studies reported previously found that, when molecular chaperones of Hsp70 protein families were silenced by RNAi (Limjindaporn et al., 2009;Padwad et al., 2010) or destroyed by toxin (Wati et al., 2009), virus replication and proliferation in host cells will be significantly inhibited. ...
The interaction between dengue virus (DENV) and vector mosquitoes are still poorly understood at present. In this study, 2-D DIGE combined with MS was used to analyze the differential proteomes of Aedes albopictus salivary gland, midgut and C6/36 cells induced by DENV-2. Our results indicated that the virus infection regulated several functional classes of proteins. Among them, 26 were successfully analyzed by real-time RT-PCR. The mRNA levels of 15 were the highest in salivary gland, 2 in midgut and none in C6/36 cells, however, 18 were the least in fat body compared to other organs. Interestingly, the changes of differential proteins mRNA were the most obvious in fat body post-infection. Chaperone, cytoskeleton and energy metabolism enzyme were the most down- or up- regulated proteins after DENV-2 infection. The abundant expression of these proteins in salivary gland may relate to its high susceptibility.
... The host protein plays a critical role in various stages of the virus life cycle from entry, replication and assembly to egress of the virus particles. Hsc70 has been found to play a role in the life cycles of a variety of RNA and DNA viruses [68]. Inhibitor for Hsc70 mRNA/protein expression could inhibit HBV/HCV replication efficiently [69,70]. ...
Background
Hepatitis B virus (HBV) infection is a major health concern with more than two billion individuals currently infected worldwide. Because of the limited effectiveness of existing vaccines and drugs, development of novel antiviral strategies is urgently needed. Heat stress cognate 70 (Hsc70) is an ATP-binding protein of the heat stress protein 70 family. Hsc70 has been found to be required for HBV DNA replication. Here we report, for the first time, that combined siRNAs targeting viral gene and siHsc70 are highly effective in suppressing ongoing HBV expression and replication.
Methods
We constructed two plasmids (S1 and S2) expressing short hairpin RNAs (shRNAs) targeting surface open reading frame of HBV(HBVS) and one plasmid expressing shRNA targeting Hsc70 (siHsc70), and we used the EGFP-specific siRNA plasmid (siEGFP) as we had previously described. First, we evaluated the gene-silencing efficacy of both shRNAs using an enhanced green fluorescent protein (EGFP) reporter system and flow cytometry in HEK293 and T98G cells. Then, the antiviral potencies of HBV-specific siRNA (siHBV) in combination with siHsc70 in HepG2.2.15 cells were investigated. Moreover, type I IFN and TNF-α induction were measured by quantitative real-time PCR and ELISA.
Results
Cotransfection of either S1 or S2 with an EGFP plasmid produced an 80%–90% reduction in EGFP signal relative to the control. This combinational RNAi effectively and specifically inhibited HBV protein, mRNA and HBV DNA, resulting in up to a 3.36 log10 reduction in HBV load in the HepG2.2.15 cell culture supernatants. The combined siRNAs were more potent than siHBV or siHsc70 used separately, and this approach can enhance potency in suppressing ongoing viral gene expression and replication in HepG2.2.15 cells while forestalling escape by mutant HBV. The antiviral synergy of siHBV used in combination with siHsc70 produced no cytotoxicity and induced no production of IFN-α, IFN-β and TNF-α in transfected cells.
Conclusions
Our combinational RNAi was sequence-specific, effective against wild-type and mutant drug-resistant HBV strains, without triggering interferon response or producing any side effects. These findings indicate that combinational RNAi has tremendous promise for developing innovative therapy against viral infection.
... It is known that following infection with various eukaryotic viruses the synthesis of HSPs, including the chaperones Hsp27, Hsp70, Hsp40 and Hsp90 is induced (review in [20]). In some cases, the increased level of HSPs directly helps viral replication as it has been observed with the SV40, HIV-1 or CELO viruses [44][45][46]. Recently, Rawat and Mitra have shown that in human cell lines, the heat-shock factor 1 (HSF1), the major eukaryotic transcription factor that regulates transcription of the HSP genes in response to stress, is specifically induced during HIV-1 infection to directly drive viral gene expression and promote its own replication [44]. ...
The universally conserved J-domain proteins (JDPs) are obligate cochaperone partners of the Hsp70 (DnaK) chaperone. They stimulate Hsp70's ATPase activity, facilitate substrate delivery, and confer specific cellular localization to Hsp70. In this work, we have identified and characterized the first functional JDP protein encoded by a bacteriophage. Specifically, we show that the ORFan gene 057w of the T4-related enterobacteriophage RB43 encodes a bona fide JDP protein, named Rki, which specifically interacts with the Escherichia coli host multifunctional DnaK chaperone. However, in sharp contrast with the three known host JDP cochaperones of DnaK encoded by E. coli, Rki does not act as a generic cochaperone in vivo or in vitro. Expression of Rki alone is highly toxic for wild-type E. coli, but toxicity is abolished in the absence of endogenous DnaK or when the conserved J-domain of Rki is mutated. Further in vivo analyses revealed that Rki is expressed early after infection by RB43 and that deletion of the rki gene significantly impairs RB43 proliferation. Furthermore, we show that mutations in the host dnaK gene efficiently suppress the growth phenotype of the RB43 rki deletion mutant, thus indicating that Rki specifically interferes with DnaK cellular function. Finally, we show that the interaction of Rki with the host DnaK chaperone rapidly results in the stabilization of the heat-shock factor σ(32), which is normally targeted for degradation by DnaK. The mechanism by which the Rki-dependent stabilization of σ(32) facilitates RB43 bacteriophage proliferation is discussed.
... Previous findings have implicated members of the HSP70 family in the targeted transport of proteins to specific cellular domains (66,67,75). Furthermore, HSP70s were shown to play various roles in the life cycles of plant and animal viruses (33,74). Some plant viruses, such as closteroviruses, encode their own HSP70 homolog, which, in combination with the virus coat protein, facilitates transport through plasmodesmata and thus enables cell-to-cell movement (1,2,54). ...
The whitefly Bemisia tabaci (Gennadius) is a major cosmopolitan pest capable of feeding on hundreds of plant species and transmits several major plant
viruses. The most important and widespread viruses vectored by B. tabaci are in the genus Begomovirus, an unusual group of plant viruses owing to their small, single-stranded DNA genome and geminate particle morphology. B. tabaci transmits begomoviruses in a persistent circulative nonpropagative manner. Evidence suggests that the whitefly vector encounters
deleterious effects following Tomato yellow leaf curl virus (TYLCV) ingestion and retention. However, little is known about the molecular and cellular basis underlying these coevolved
begomovirus-whitefly interactions. To elucidate these interactions, we undertook a study using B. tabaci microarrays to specifically describe the responses of the transcriptomes of whole insects and dissected midguts following
TYLCV acquisition and retention. Microarray, real-time PCR, and Western blot analyses indicated that B. tabaci heat shock protein 70 (HSP70) specifically responded to the presence of the monopartite TYLCV and the bipartite Squash leaf curl virus. Immunocapture PCR, protein coimmunoprecipitation, and virus overlay protein binding assays showed in vitro interaction between TYLCV and HSP70. Fluorescence in situ hybridization and immunolocalization showed colocalization of TYLCV and the bipartite Watermelon chlorotic stunt virus virions and HSP70 within midgut epithelial cells. Finally, membrane feeding of whiteflies with anti-HSP70 antibodies and
TYLCV virions showed an increase in TYLCV transmission, suggesting an inhibitory role for HSP70 in virus transmission, a role
that might be related to protection against begomoviruses while translocating in the whitefly.
... Hsps are a diverse group of proteins classified into different families according to molecular size and play several vital roles in cells including folding of nascent proteins, protein translocation and degradation, prevention of aggregation and cell stress responses (for reviews see Gething and Sambrook 1992; Hartl 1996; Frydman et al. 1994; Frydman 2001; Young et al. 2004). There is substantial evidence to suggest that a wide variety of DNA and RNA viruses differing in host range and replication strategy require the assistance of one or more chaperone pathways to facilitate entry, genome replication and expression, and assembly (for reviews see Sullivan and Pipas 2001; Xiao et al. 2010). In the case of picornaviruses, Hsps 70 and 90 have been found to be essential for the folding and assembly of enteroviruses, as indicated by an association of these chaperones with the capsid P1 precursor (Macejak and Sarnow 1992; Geller et al. 2007). ...
Theiler's murine encephalomyelitis virus (TMEV) is a positive-sense RNA virus belonging to the Cardiovirus genus in the family Picornaviridae. In addition to other host cellular factors and pathways, picornaviruses utilise heat shock proteins (Hsps) to facilitate their propagation in cells. This study investigated the localisation of Hsps 70 and 90 in TMEV-infected BHK-21 cells by indirect immunofluorescence and confocal microscopy. The effect of Hsp90 inhibitors novobiocin (Nov) and geldanamycin (GA) on the development of cytopathic effect (CPE) induced by infection was also examined. Hsp90 staining was uniformly distributed in the cytoplasm of uninfected cells but was found concentrated in the perinuclear region during late infection where it overlapped with the signal for non-structural protein 2C within the viral replication complex. Hsp70 redistributed into the vicinity of the viral replication complex during late infection, but its distribution did not overlap with that of 2C. Inhibition of Hsp90 by GA and Nov had a negative effect on virus growth over a 48-h period as indicated by no observable CPE in treated compared to untreated cells. 2C was detected by Western analysis of GA-treated infected cell lysates at doses between 0.01 and 0.125 μM, suggesting that processing of viral precursors was not affected in the presence of this drug. In contrast, 2C was absent in cell lysates of Nov-treated cells at doses above 10 μM, although CPE was evident 48 hpi. This is the first study describing the dynamic behaviour of Hsps 70 and 90 in TMEV-infected cells and to identify Hsp90 as an important host factor in the life cycle of this virus.
... Viruses typically utilize host cell components to ensure efficient DNA replication, protein biosynthesis, and virion assembly, and it is generally considered that cellular chaperones facilitate the folding of virion capsid proteins and participate in virus budding (28). The host-independent tail development of ATV virions that occurs after their release inevitably requires a major rearrangement of the virion proteins, and we infer that chaperones could facilitate this reassembly process. ...
The crenarchaeal Acidianus two-tailed virus (ATV) undergoes a remarkable morphological development, extracellularly and independently of host cells,
by growing long tails at each end of a spindle-shaped virus particle. Initial work suggested that an intermediate filament-like
protein, p800, is involved in this process. We propose that an additional chaperone system is required, consisting of a MoxR-type
AAA ATPase (p618) and a von Willebrand domain A (VWA)-containing cochaperone, p892. Both proteins are absent from the other
known bicaudavirus, STSV1, which develops a single tail intracellularly. p618 exhibits ATPase activity and forms a hexameric
ring complex that closely resembles the oligomeric complex of the MoxR-like protein RavA (YieN). ATV proteins p387, p653,
p800, and p892 interact with p618, and with the exception of p800, all bind to DNA. A model is proposed to rationalize the
interactions observed between the different protein and DNA components and to explain their possible structural and functional
roles in extracellular tail development.
... Members of the HSP70 protein family have been identified as host factors involved in infection by numerous different viruses (Sullivan and Pipas, 2001; Mayer, 2005). HSP70 and a co-chaperone are central factors in this study and hence this subject is introduced more thoroughly. ...
Plus-stranded (plus) RNA viruses multiply within a cellular environment as tightly integrated units and rely on the genetic information carried within their genomes for multiplication and, hence, persistence. The minimal genomes of plus RNA viruses are unable to encode the molecular machineries that are required for virus multiplication. This sets requisites for the virus, which must form compatible interactions with host components during multiplication to successfully utilize primary metabolites as building blocks or metabolic energy, and to divert the protein synthesis machinery for production of viral proteins. In fact, the emerging picture of a virus-infected cell displays tight integration with the virus, from simple host and virus protein interactions through to major changes in the physiological state of the host cell. This study set out to develop a method for the identification of host components, mainly host proteins, that interact with proteins of Potato virus A (PVA; Potyvirus) during infection. This goal was approached by developing affinity-tag based methods for the purification of viral proteins complexed with associated host proteins from infected plants. Using this method, host membrane-associated viral ribonucleoprotein (RNP) complexes were obtained, and several host and viral proteins could be identified as components of these complexes. One of the host proteins identified using this strategy was a member of the heat shock protein 70 (HSP70) family, and this protein was chosen for further analysis. To enable the analysis of viral gene expression, a second method was developed based on Agrobacterium-mediated virus genome delivery into plant cells, and detection of virally expressed Renilla luciferase (RLUC) as a quantitative measure of viral gene expression. Using this method, it was observed that down-regulation of HSP70 caused a PVA coat protein (CP)-mediated defect associated with replication. Further experimentation suggested that CP can inhibit viral gene expression and that a distinct translational activity coupled to replication, referred to as replication-associated translation (RAT), exists. Unlike translation of replication-deficient viral RNA, RAT was dependent on HSP70 and its co-chaperone CPIP. HSP70 and CPIP together regulated CP turnover by promoting its modification by ubiquitin. Based on these results, an HSP70 and CPIP-driven mechanism that functions to regulate CP during viral RNA replication and/or translation is proposed, possibly to prevent premature particle assembly caused by CP association with viral RNA. Virus är obligata parasiter och kräver en cellulär miljö för att kopiera sig själv. Denna miljö kan erbjudas av bakterier eller högre organismer som växter och djur. Alla celler eller organismer fungerar inte som värd för alla virus, specifika virus klarar sig endast i specifika värdorganismer. Dessa kallas för kompatibla virus och värd kombinationer. Då en kompatibel samverkan mellan virus och värd existerar, uppstår en infektion, och under infektionens lopp samverkar virusets komponenter, dvs. virus genomet i sig och proteiner som kodas av det, med komponenter som erbjuds av värd cellen, inklusive proteiner och membran. Samtidigt utgör den cellulära miljön en resurs varifrån virus erhåller materia och energi för att kopiera sig själv och utvecklas. Denna studie har koncentrerat sig på att identifiera värd proteiner som deltar och reglerar en kompatibel infektion mellan modellväxten Nicotiana benthamiana och Potatis virus A. Ett av växtproteinerna som kunde identifieras, ser ut att delta i infektionen genom att destinera virusets höljes protein för nedbrytning, och denna process krävs för effektiv kopiering av viruset. Därmed föreslås att modellviruset i denna studie har inkorporerat växtkomponenter som fungerar i nedbrytning av växtens egna proteiner, för att degradera sitt höljes protein i ett tidigt skede av infektionen, och därigenom uppnå temporär kontroll över ackumulering av detta protein. Virusproteinet ifråga kan verka negativt på vissa kritiska processer i virusets infektionsförlopp, trots att det är absolut nödvändigt för viruset att spridas både i och emellan växter.
... 7 Infection by viruses, including human immunodeficiency virus 1, polyomaviridae, molluscum contagiosum, influenza, hepatitis B, human T-lymphotropic virus, papillomavirus, and adenovirus, has been shown to induce HSPs and facilitate viral production. 8 Two viruses actually encode their own HSP homologs. Closteroviridae encode an HSP70 homolog, and polyomaviridae encode a DNAJ homolog containing protein (T antigen) that activates HSP70 in a similar fashion to HSP40 (a DNAJ motif containing protein). ...
Unlabelled:
The hepatitis C viral (HCV) genome is translated through an internal ribosome entry site (IRES) as a single polyprotein precursor that is subsequently cleaved into individual mature viral proteins. Nonstructural protein 5A (NS5A) is one of these proteins that has been implicated in regulation of viral genome replication, translation from the viral IRES and viral packaging. We sought to identify cellular proteins that interact with NS5A and determine whether these interactions may play a role in viral production. Mass spectrometric analysis of coimmunoprecipitated NS5A complexes from cell extracts identified heat shock proteins (HSPs) 40 and 70. We confirmed an NS5A/HSP interaction by confocal microscopy demonstrating colocalization of NS5A with HSP40 and with HSP70. Western analysis of coimmunoprecipitated NS5A complexes further confirmed interaction of HSP40 and HSP70 with NS5A. A transient transfection, luciferase-based, tissue culture IRES assay demonstrated NS5A augmentation of HCV IRES-mediated translation, and small interfering RNA (siRNA)-mediated knockdown of HSP70 reduced this augmentation. Treatment with an inhibitor of HSP synthesis, Quercetin, markedly reduced baseline IRES activity and its augmentation by NS5A. HSP70 knockdown also modestly reduced viral protein accumulation, whereas HSP40 and HSP70 knockdown both reduced infectious viral particle production in an HCV cell culture system using the J6/JFH virus fused to the Renilla luciferase reporter. Treatment with Quercetin reduced infectious particle production at nontoxic concentrations. The marked inhibition of virus production by Quercetin may partially be related to reduction of HSP40 and HSP70 and their potential involvement in IRES translation, as well as viral morphogenesis or secretion.
Conclusion:
Quercetin may allow for dissection of the viral life cycle and has potential therapeutic use to reduce virus production with low associated toxicity.
... Hence, the Hsp70h gene was selected to be tested. Other molecular chaperones have also been shown to be involved in virus infections (reviewed by Sullivan & Pipas, 2001), e.g., Hsp90 is required for Hepatitis B virus replication (Hu & Seeger, 1996). The homologues of the putative SPCSV p60 protein (one of the closteroviral hallmark genes) in BYV and CTV were found to have some similarity with the Hsp90 family of chaperones (Agranovski, 1996), indicating that these proteins and their homologues in other clostero-and criniviruses may be a type of molecular chaperones. ...
Thesis (doctoral)-Swedish University of Agricultural Sciences, 2002. Includes bibliographical references.
... Similarly, the three Rb-proteins bind to an LXCXE motif located in the flexible linker between the J domain and the OBD. Finally, the J domain governs recruitment and activation of hsc70, a cellular chaperone [8][9][10]. T antigen has been shown to interact with another three targets, and these interactions could contribute to transformation as well, but they have been less studied. ...
The study of polyomavirus has benefited immensely from two scientific methodologies, cell culture and in vitro studies on one side and the use of transgenic mice as experimental models on the other. Both approaches allowed us to identify cellular products targeted by the viruses, the consequences of these interactions at the phenotypic and molecular level, and thus the potential roles of the targets within their normal cellular context. In particular, cell culture and in vitro reports suggest a model explaining partially how SV40 large T antigen contributes to oncogenic transformation. In most cases, T antigen induces cell cycle entry by inactivation of the Rb proteins (pRb, p130, and p107), thus activating E2F-dependent transcription and subsequent S-phase entry. Simultaneously, T antigen blocks p53 activity and therefore prevents the ensuing cell-cycle arrest and apoptosis. For the most part, studies of T antigen expression in transgenic mice support this model, but the use of T antigen mutants and their expression in different tissue and cell type settings have expanded our knowledge of the model system and raised important questions regarding tumorigenic mechanisms functioning in vivo.
Understanding the factors that shape viral evolution is critical for developing effective antiviral strategies, accurately predicting viral evolution, and preventing pandemics. One fundamental determinant of viral evolution is the interplay between viral protein biophysics and the host machineries that regulate protein folding and quality control. Most adaptive mutations in viruses are biophysically deleterious, resulting in a viral protein product with folding defects. In cells, protein folding is assisted by a dynamic system of chaperones and quality control processes known as the proteostasis network. Host proteostasis networks determine the fates of viral proteins with biophysical defects, either by assisting with folding or by targeting them for degradation. In this review, we discuss and analyze new discoveries revealing that host proteostasis factors can profoundly shape the sequence space accessible to evolving viral proteins. We also discuss the many opportunities for research progress proffered by the proteostasis perspective on viral evolution and adaptation.
Expected final online publication date for the Annual Review of Virology, Volume 10 is September 2023. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
Human papillomavirus (HPV) infection is a multi-step process that implies complex interactions of the viral particles with cellular proteins. The HPV capsid includes the two structural proteins L1 and L2, that play crucial roles on infectious viral entry. L2 is particularly relevant for the intracellular trafficking of the viral DNA towards the nucleus. Here, using proteomic studies we identified CCT proteins as novel interaction partners of HPV-16 L2. The CCT multimeric complex is an essential chaperonin which interacts with a large number of protein targets. We analysed the binding of different components of the CCT complex to L2. We confirmed the interaction of this structural viral protein with the CCT subunit 3 (CCT3) and we found that this interaction requires the N-terminal region of L2. Defects in HPV-16 pseudoviral particle (PsVs) infection were revealed by siRNA-mediated knockdown of some CCT subunits. While a substantial drop in the viral infection was associated with the ablation of CCT component 2, even more pronounced effects on infectivity were observed upon depletion of CCT component 3. Using confocal immunofluorescence assays, CCT3 co-localised with HPV PsVs at early times after infection, with L2 being required for this to occur. Further analysis showed the colocalization of several other subunits of CCT with the PsVs. Moreover, we observed a defect in capsid uncoating and a change in PsVs intracellular normal processing when ablating CCT3. Taken together, these studies demonstrate the importance of CCT chaperonin during HPV infectious entry.
Importance
Several of the mechanisms that function during the infection of target cells by HPV particles have been previously described. However, many aspects of this process remain unknown. In particular, the role of cellular proteins functioning as molecular chaperones during HPV infections has been only partially investigated. To the best of our knowledge, we describe here for the first time, a requirement of the CCT chaperonin for HPV infection. The role of this cellular complex seems to be determined by the binding of its component 3 to the viral structural protein L2. However, CCT’s effect on HPV infection most probably comprises the whole chaperonin complex. Altogether, these studies define an important role for the CCT chaperonin in the processing and intracellular trafficking of HPV particles and in subsequent viral infectious entry.
Drug reposition, or repurposing, has become a promising strategy in therapeutics due to its advantages in several aspects of drug therapy. General drug development is expensive and can take more than 10 years to go through the designing, development, and necessary approval steps. However, established drugs have already overcome these steps and thus a potential candidate may be already available decreasing the risks and costs involved. Viruses invade cells, usually provoking biochemical changes, leading to tissue damage, alteration of normal physiological condition in organisms and can even result in death. Inside the cell, the virus finds the machinery necessary for its multiplication, as for instance the protein quality control system, which involves chaperones and Hsps (heat shock proteins) that, in addition to physiological functions, help in the stabilization of viral proteins. Recently, many inhibitors of Hsp90 have been developed as therapeutic strategies against diseases such as the Hsp90 inhibitors used in anticancer therapy. Several shreds of evidence indicate that these inhibitors can also be used as therapeutic strategies against viruses. Therefore, since a drug treatment for COVID-19 is urgently needed, this review aims to discuss the potential use of Hsp90 inhibitors in the treatment of this globally threatening disease.
Murine norovirus (MNV), is a prevalent pathogen of laboratory mice closely related to human norovirus (HuNoV), a contagious pathogen known to cause gastroenteritis worldwide; however, the mechanism of norovirus replication remains poorly understood. Both heat shock protein 90 (Hsp90) and heat shock protein 70 (Hsp70) play an important role in viral genome replication and viral gene expression. In this study, we first found that heat stress exerted a positive effect on the replication of MNV in the murine macrophage RAW264.7 cell line. Inhibition of Hsp70 and Hsp90 by the specific inhibitors, KNK437 and 17-AGG, respectively showed that Hsp70 and Hsp90 enhanced MNV genome replication and virion production. In addition, we found that KNK437 and 17-AGG could decrease the level of IL-1β, IL-10, and TNF-α mRNA expression in MNV-infected cells. These data suggested that heat stress can positively regulate MNV replication, which advances our understanding of the molecular mechanism of MNV infection.
Viruses have evolved a variety of strategies to succeed in a hostile environment. The herpes simplex virus 1 (HSV-1) immediate early protein ICP22 plays several roles in the virus life cycle, including downregulation of cellular gene expression, upregulation of late viral gene expression, inhibition of apoptosis, prevention of aggregation of nonnative proteins, and the recruitment of a cellular heat shock protein, Hsc70, to nuclear domains. We present evidence that ICP22 functionally resembles a cellular J-protein/HSP40 family cochaperone, interacting specifically with Hsc70. We suggest that HSV has taken advantage of the adaptable nature of J proteins to evolve a multifunctional cochaperone that functions with Hsc70 to promote lytic infection.
Background/aims:
Bacteriophages (phages) are viruses of bacteria. Escherichia coli phage (T4) can potentially interfere with adsorption of HAdV-5 to cellular integrins by its KGD motif, while staphylococcal A5/80 phage does not possess this structure. The objective of this study was to investigate the effects of T4 and A5/80 phage preparations on type 5 human adenovirus (HAdV-5) DNA synthesis and the expression of HAdV-5 genes.
Methods:
Experiments were performed on the A549 cell line. HAdV-5 DNA synthesis was investigated with real-time PCR. Expression of HAdV-5 early (DBP) and late (hexon) genes was determined by quantitative real-time PCR in preincubation and coincubation experiments.
Results:
While both phage preparations significantly reduced the expression of HAdV-5 genes, synthesis of HAdV-5 DNA was inhibited only by T4.
Conclusion:
Phage preparations show promise as novel antiviral agents. However, further studies are required to investigate their antiviral effects.
Porcine circovirus type 2 (PCV2) is recognized as the causative agent of porcine circovirus-associated disease (PCVAD). However, the mechanism of PCV2 replication has not been understood completely. Heat shock protein 90 (Hsp90) plays an important role in viral genome replication, viral genes expression, and viral particle packaging. In this study, we firstly found that inhibition of Hsp90 by pretreatment of host cells with 17-AAG, a specific inhibitor of Hsp90, or blocking Hsp90α/Hsp90β with siRNA, resulted in significantly reduced viral replication in PK-15 cells. But inhibition of Hsp90 by 17-AAG did not affect PCV2 entry into the host cells. Meanwhile, over-expression of Hsp90α/Hsp90β enhanced PCV2 genome replication and virion production. In addition, Hsp90β was enriched in the nuclear zone in the cells infected with PCV2. But it did not interact with the viral Cap/Rep proteins. It suggested that Hsp90 is required for PCV2 production in PK-15 cells culture. It should be helpful for further evaluating the mechanism of replication and pathogenesis of PCV2 and developing novel antiviral therapies.
Importance:
Latency-associated nuclear antigen (LANA) is a conserved gamma-2-herpesvirus protein important for latency maintenance and pathogenesis. For MHV68 this includes regulating lytic replication and reactivation. While previous studies of KSHV LANA defined interactions with host cell proteins that impact latency, interactions that facilitate productive viral replication are not known. Thus, we performed a differential proteomics analysis to identify and prioritize cellular and viral proteins that interact with the MHV68 LANA homolog during lytic infection. Among the proteins identified was heat-shock cognate protein 70 (Hsc70), which we determined is recruited to host cell nuclei in an mLANA-dependent process. Moreover, Hsc70 facilitates MHV68 protein expression and DNA replication, thus contributing to efficient MHV68 lytic replication. These experiments expand the known LANA-binding proteins to include MHV68 lytic replication and demonstrate a previously unappreciated role for Hsc70 in regulating viral replication.
Porcine circovirus type 2 (PCV2) has been identified as the etiologic agent which causing postweaning multisystemic wasting syndrome in swine farms in the world. Some quantitative proteomic studies showed that many proteins significantly changed in PCV2-infected cells. To explore the role of cellular chaperones during PCV2 infection, cytoprotective chaperone Hsp27 was analyzed in PCV2-infected PK-15 cells in this study. The results showed that Hsp27 could up-regulate and accumulate in phosphorylated forms in the nuclear zone during PCV2 replication. Suppression of Hsp27 phosphorylation with specific chemical inhibitors or downregulation of all forms of Hsp27 via RNA interference significantly reduced the virus replication. Meanwhile, over-expression of Hsp27 enhanced PCV2 genome replication and virion production. It indicated that Hsp27 was required for PCV2 production in PK-15 cells culture. It should be helpful for understanding the mechanism of replication and pathogenesis of PCV2 and development of novel antiviral therapies in the future.
DNAJ/HSP40 is an evolutionarily conserved family of proteins bearing various functions. Historically, it has been emphasized that HSP40/DNAJ family proteins play a positive role in infection of various viruses. We identified DNAJ/HSP40B6 as a potential negative regulator of HIV-1 replication in our genetic screens. In this study, we investigated the functional interactions between HIV-1 and HSP40 family members.
We took genetic and comparative virology approaches to expand the primary observation.
Multiple HSP40/DNAJ proteins were tested for their ability to inhibit replication of adenovirus, herpes simplex virus type 1, HIV-1, and vaccinia virus. The mechanism of inhibition was investigated by using HSP40/DNAJ mutants and measuring the efficiencies of each viral replication steps.
HSP40A1, B1, B6, and C5, but not C3, were found to be able to limit HIV-1 production. This effect was specific to HIV-1 for such effects were not detected in adenovirus, herpes simplex virus type 1, and vaccinia virus. Genetic analyses suggested that the conserved DNAJ domain was responsible for the inhibition of HIV-1 production through which HSP40 regulates HSP70 ATPase activity. Interestingly, HSP40s lowered the levels of steady-state viral messenger RNA. This was not attributed to the inhibition of Tat/long terminal repeat-driven transcription but the downregulation of Rev expression.
This is the first report providing evidence that HSP70-HSP40 complex confers an innate resistance specific to HIV-1. For their interferon-inducible nature, HSP40 family members should account for the anti-HIV-1 function of interferon.
Introduction“Soluble” Hsp70s/J-proteins Function in General Protein Folding“Tethered” Hsp70s/J-proteins: Roles in Protein Folding on the Ribosome and in Protein TranslocationModulating of Protein Conformation by Hsp70s/J-proteinsCases of a Single Hsp70 Functioning With Multiple J-ProteinsHsp70s/J-proteins — When an Hsp70 Maybe Isn't Really a ChaperoneEmerging Concepts and Unanswered Questions
Via their interaction with client proteins, Hsp70 molecular chaperone machines function in a variety of cellular processes, including protein folding, translocation of proteins across membranes and assembly/disassembly of protein complexes. Such machines are composed of a core Hsp70, as well as a J‐protein and a nucleotide exchange factor as co‐chaperones. These co‐factors regulate the cycle of adenosine triphosphate (ATP) hydrolysis and nucleotide exchange, which is critical for Hsp70's interaction with client proteins. Cellular compartments often contain multiple Hsp70s, J‐proteins and nucleotide exchange factors. The capabilities of Hsp70s to carry out diverse cellular functions can result from either specialisation of an Hsp70 or by interaction of a multifunctional Hsp70 with a suite of J‐protein co‐chaperones. The well‐studied Hsp70 systems of mitochondria provide an example of such modes of diversification and specialisation of Hsp70 machinery, which are applicable to other cellular compartments.
Key Concepts
Fundamental biochemical properties of different Hsp70 systems are very similar, yet very adaptable.
Diversification of Hsp70 function is often due to multiple J‐protein partners.
Although most Hsp70s bind a broad array of peptide sequences, some have become specialised and have a very restricted binding specificity.
To survive in a hostile environment, viruses evolved proteinacious capsids and in many cases membranous envelopes that protect
their genome against a variety of impacts and retain infectivity. Since evolutionary survival requires efficient and correct
assembly of their capsids, many viruses use the cellular quality control system to ensure proper folding and assembly. This
chapter will review the interaction of the three major chaperone systems in the cytosol (Hsp60, Hsp70 and Hsp90) with viral
proteins that are involved in capsid assembly and morphogenesis. Most examples for the involvement of Hsp60 chaperones in
virus morphogenesis come from the investigation of bacteriophage propagation in Escherichia coli. The assistance of Hsp70 and Hsp90 chaperones in coat protein folding and capsid assembly was investigated in more detail
using eukaryotic viruses. Mechanistic aspects of the chaperone interactions with the viral proteins are emphasized whenever
sufficient evidence is available.
KeywordsCapsid assembly-Coat proteins-Molecular chaperones-Morphogenesis-Protein folding
Heat Shock Proteins (HSP) are important modulators of both anti-inflammatory and pro-inflammatory responses. In this chapter,
we address this apparent paradox by focusing on the effects of the highly heat inducible Hsp70 and its transcription machinery.
This transcription machinery may exert important effects on inflammation through pathways, which are independent of heat shock
proteins. We then discuss disease states where the balance between the anti-inflammatory and pro-inflammatory effectors is
critical to disease outcome
KeywordsCytokines-HSF-1-HSP-inflammatory response syndrome-heat stroke-inflammation-heat injury
Severe acute respiratory syndrome virus (SARS-CoV) that lacks the envelope (E) gene (rSARS-CoV-ΔE) is attenuated in vivo. To identify factors that contribute to rSARS-CoV-ΔE attenuation, gene expression in cells infected by SARS-CoV with or without E gene was compared. Twenty-five stress response genes were preferentially upregulated during infection in the absence of the E gene. In addition, genes involved in signal transduction, transcription, cell metabolism, immunoregulation, inflammation, apoptosis and cell cycle and differentiation were differentially regulated in cells infected with rSARS-CoV with or without the E gene. Administration of E protein in trans reduced the stress response in cells infected with rSARS-CoV-ΔE or with respiratory syncytial virus, or treated with drugs, such as tunicamycin and thapsigargin that elicit cell stress by different mechanisms. In addition, SARS-CoV E protein down-regulated the signaling pathway inositol-requiring enzyme 1 (IRE-1) of the unfolded protein response, but not the PKR-like ER kinase (PERK) or activating transcription factor 6 (ATF-6) pathways, and reduced cell apoptosis. Overall, the activation of the IRE-1 pathway was not able to restore cell homeostasis, and apoptosis was induced probably as a measure to protect the host by limiting virus production and dissemination. The expression of proinflammatory cytokines was reduced in rSARS-CoV-ΔE-infected cells compared to rSARS-CoV-infected cells, suggesting that the increase in stress responses and the reduction of inflammation in the absence of the E gene contributed to the attenuation of rSARS-CoV-ΔE.
Temperate bacteriophage lytic development is intrinsically related to the stress response in particular at the DNA replication
and virion maturation steps. Alternatively, temperate phages become lysogenic and integrate their genome into the host chromosome.
Under stressful conditions, the prophage resumes a lytic development program, and the phage DNA is excised before being replicated.
The KplE1 defective prophage of Escherichia coli K12 constitutes a model system because it is fully competent for integrative as well as excisive recombination and presents
an atypical recombination module, which is conserved in various phage genomes. In this work, we identified the host-encoded
stress-responsive molecular chaperone DnaJ (Hsp40) as an active participant in KplE1 prophage excision. We first show that
the recombination directionality factor TorI of KplE1 specifically interacts with DnaJ. In addition, we found that DnaJ dramatically
enhances both TorI binding to its DNA target and excisive recombination in vitro. Remarkably, such stimulatory effect by DnaJ was performed independently of its DnaK chaperone partner and did not require
a functional DnaJ J-domain. Taken together, our results underline a novel and unsuspected functional interaction between the
generic host stress-regulated chaperone and temperate bacteriophage lysogenic development.
A DnaJ-like gene, Cpdj1, a molecular chaperone and regulator of Hsp70 in Cryphonectria parasitica, was characterized. The protein product of Cpdj1 gene consists of 379 amino acids with a predicted molecular mass of 40.6 kDa and a pI of 7.79. The deduced protein sequence revealed preservation of the conserved hall-mark J-region and exhibited high homolo y to all known DnaJ-like proteins. Disruption of the Cpdj1 gene resulted in slow growth and produced colonies characterized by retarded growth and deep orange color. Accordingly, reduced virulence of the Cpdj1-null mutant was observed. This reduced growth rate was magnified when the Cpdj1-null mutant was cultured under heat-stress conditions. Reduced conidiation was also observed in the Cpdj1-null mutant, indicating that Cpdj1 gene, although not essential for cell viability, is required for appropriate cellular processes including growth and sporulation. Northern analysis showed that Cpdj1 was constitutively expressed, and when the culture was subject to high temperature, a strong induction of the transcript was observed. No significant difference in the expression and induction pattern of Cpdj1 was observed between virus-free EP155/2 and virus-infected hypovirulent UEP1 strains. However, further severe defects in mycelia growth and conidiation were observed in the hypovirus-infected Cpdj1-null mutant suggesting that the presence of Cpdj1 is required for mycelia growth and sporulation of the hypovirus-infected strain.
As essential effectors in protein quality control, molecular chaperones serve as the primary checkpoint to assist proper protein folding and prevent misfolded proteins from denaturation and aggregation. In addition, chaperones can function to direct terminally misfolded proteins to the proteolytic system for degradation. Viruses rely on host cell machineries for productive infection. Like for many other processes, various viruses have been shown to evolve mechanisms to utilize or subvert the host protein quality control machinery to support the completion of their life cycle. Furthermore, recent studies suggest that some viruses encode for their own chaperone-like proteins to enhance their infectivity. This review summarizes the current understanding of the interplay between molecular chaperones and viral proteins, highlights the chaperone activities of a number of viral proteins, and discusses potential antiviral therapeutic strategies targeting the virus-chaperone interactions.
Tobacco mosaic virus (TMV) derivatives are explored currently extensively with regard to nanotechnological applications. Since certain technically desired TMV mutants may not be accessible from plants, the utility of the fission yeast Schizosaccharomyces pombe for heterologous production of TMV coat protein (CP) variants was explored, including wild-type (wt) CP and two genetically engineered mutants: TMV-CP-His(6) containing a C-terminal hexahistidine (His(6)) tag, and TMV-CP-E50Q with enhanced lateral CP subunit interactions. After establishing expression clones and protocols for enrichment of the CP variants, their ability to reconstitute TMV-like nanostructures in the presence or absence of RNA was tested in comparison with the corresponding plant-derived CP variants, which were expressed from infectious TMV constructs. Both TMV-CP-E50Q and TMV-CP-wt yielded TMV-like rods, irrespective of the proteins' source. In contrast, His-tagged CP from plants produced only short rods in an inefficient manner, and no rods at all when expressed in yeast. This study introduces a novel approach to produce assembly competent TMV CP, but also demonstrates its limitations.
Hantavirus (HTV) infection is known to induce innate cellular response, a more specified cellular response in the host cells. However, whether it stimulates synthesis of stress proteins, particularly associations of viral proteins, is entirely unknown. The primary focus of this research is using Vero E6 cells infected with Hantaan 76-118 (HTNV) as an in vitro infection model to examine the individual contribution of HTV infection to heat shock response. This study shows that HTNV infection rapidly induced HSP70 expression in Vero E6 cells, which underwent a nucleo-cytoplasmic shuttle that lasted for more than 3 d. The increased HSP70 was preceded by induction of HSP70 mRNA. The physical association of HSP70 with viral nucleocapsid protein (NP) in infected cells was demonstrated by co-localization and immunoprecipation. Vero E6 cells that constitutively overexpress HSP70 after stable transfection with HSP70 gene, when infected with HTNV, showed selectively reduced NP synthesis. These findings suggest HSP70 is actively involved in the control of the expression level of viral structural proteins and possibly involved in virus assembly by binding of NP to HSP70. Overexpression of HSP70 does not favor viral propagation.
Increasing data indicate heat shock proteins (HSPs) including inducible HSP70 (HSP70i) are involved in the replicative cycles of various viruses including adenoviruses (Ads), polyomaviruses (PyVs), and some RNA viruses. Cell-free system studies implicate HSP70i in human papillomavirus type 11 (HPV11) genome replication with E1 and E2 proteins, and there is evidence that HSP70 is involved in capsid assembly and disassembly for PyVs and HPVs. HSP70 expression is increased in HPV16 E6/E7 gene transduced human primary keratinocytes, and frequently detected in early stage uterine cervical cancer at levels in conjunction with lesion severity. In this study we carry out analyses in the natural host epithelial tissues to assess the role of inducible HSP70 (HSP70i) in the HPV infectious life cycle. For these studies we used the organotypic (raft) culture system to recapitulate the full viral life cycle of the high-risk HPV31. Upon heat shock of HPV31-infected organotypic tissues, we find high and sustained expression of HSP70i coincident with enhanced HPV genome replication and virion production. Whereas there is no clear effect on L1 expression levels, we find HSP70i and L1 interact and HSP70i colocalizes with and enhances the nuclear localization of L1 in differentiated cells. Ad-mediated gene transfer was used to study the effects of HSP70i in naturally HPV-infected differentiating tissues and showed results similar to those in heat shocked rafts. These results indicate that increased HSP70i augments late activities in the viral life cycle. We conclude that HSP70i contributes directly to HPV replicative viral activities and the production of infectious virions.
The role of molecular chaperones in yeast cell wall integrity and identification of chaperone modulators that interfere with Simian virus 40 replication
p58(IPK), a member of the tetratricopeptide repeat and J-domain protein families, was first recognized for its ability to inhibit the double-stranded RNA-activated protein kinase, PKR. PKR is part of the interferon-induced host defense against viral infection, and down-regulates translation initiation via phosphorylation of eukaryotic initiation factor 2 on the alpha-subunit. P58(IPK) is activated in response to infection by influenza virus, and inhibits PKR through direct protein-protein interaction. Previously, we demonstrated that the molecular chaperone heat shock protein 40 (hsp40) was a negative regulator of p58(IPK). We could now report that influenza virus activates the p58(IPK) pathway by promoting the dissociation of hsp40 from p58(IPK) during infection. We also found that the P58(IPK)-hsp40 association was disrupted during recovery from heat shock, which suggested a regulatory role for P58(IPK) in the absence of virus infection. The PKR pathway is even more complex as we show in this report that the molecular chaperone, hsp/Hsc70, was a component of a trimeric complex with hsp40 and p58(IPK). Moreover, like other J-domain proteins, p58(IPK) stimulated the ATPase activity of Hsc70, Taken together, our data suggest that p58(IPK) is a co-chaperone, possibly directing hsp/Hsc70 to refold, and thus inhibit kinase function.
Heat shock cognate protein 70 (HSC70) has been shown to bind to the peptide corresponding to amino acids 197 to 216 of human T-cell lymphotropic virus type I (HTLV-I) envelope protein, gp46, and an anti-HSC70 monoclonal antibody (mAb) inhibits HTLV-I-induced syncytium formation. These findings suggest that HSC70 is necessary for the entry of HTLV-I into its target cells. Here we showed that HSC70 directly binds to gp46 by co-immunoprecipitation of HSC70 and gp46 from HTLV-I-producing human T-cell lysate. However, transduction of human HSC70 cDNA into BaF3 cells, which were found to be highly resistant to HTLV-I infection, did not support the HTLV-I entry, and HSC70 expressed in NIH3T3 cells, which were found to be almost resistant to syncytium formation upon cocultivation with HTLV-I-producing cells but sensitive to infection with cell-free HTLV-I, enhanced cell fusion induced by HTLV-I-producing cells, but did not enhance the entry of cell-free HTLV-I into these cells. The mAb against HSC70 inhibited syncytium formation in NIH3T3 cells expressing HSC70, but showed little effect on infection of these cells with cell-free HTLV-I. These findings indicate that HSC70 markedly enhances syncytium formation induced by HTLV-I but does not facilitate HTLV-I entry into target cells.
Tat activation of HIV-1 transcription is mediated by human transcription elongation factor P-TEFb, which interacts with Tat and phosphorylates the C-terminal domain of RNA polymerase II. The catalytic subunit of the P-TEFb complex, Cdk9, has been shown to interact with cyclin T and several other proteins of unknown identity. Consequently, the exact subunit composition of active P-TEFb has not been determined. Here we report the affinity purification and identification of the Cdk9-associated proteins. In addition to forming a heterodimer with cyclin T1, Cdk9 interacted with the molecular chaperone Hsp70 or a kinase-specific chaperone complex, Hsp90/Cdc37, to form two separate chaperone-Cdk9 complexes. Although the Cdk9/cyclin T1 dimer was exceptionally stable and produced slowly in the cell, free and unprotected Cdk9 appeared to be degraded rapidly. Several lines of evidence indicate the heterodimer of Cdk9/cyclin T1 to be the mature, active form of P-TEFb responsible for phosphorylation of the C-terminal domain of RNA polymerase II interaction with the Tat activation domain, and mediation of Tat activation of HIV-1 transcription. Pharmacological inactivation of Hsp90/Cdc37 function by geldanamycin revealed an essential role for the chaperone-Cdk9 complexes in generation of Cdk9/cyclin T1. Our data suggest a previously unrecognized chaperone-dependent pathway involving the sequential actions of Hsp70 and Hsp90/Cdc37 in the stabilization/folding of Cdk9 as well as the assembly of an active Cdk9/cyclin T1 complex responsible for P-TEFb-mediated Tat transactivation.
Expression of the human gene encoding the major heat shock protein, HSP70, was induced during cell growth by serum stimulation
and after infection with adenovirus 5. In this study we showed that HSP70 gene expression could be induced by adenovirus 5
infection, even in the absence of exogenous serum factors. Whereas serum stimulation induced the expression of the endogenous
HSP70 gene, it had no effect on early adenovirus promoters. However, expression of both the cellular HSP70 gene and the adenovirus
E3 promoter were activated during adenovirus infection. By using a collection of reconstructed mutant viruses, we identified
the 13S product of the E1A region as the specific transcriptional trans-activator of the HSP70 gene.
We have examined the transcriptional regulation of the 70-kDa (70K) heat shock gene family following infection of human and monkey cells with four different DNA viruses: adenovirus type 5 (Ad5), herpes simplex virus type 1 (HSV-1), simian virus 40, and vaccinia virus. Our results indicate that induction of these genes is not a general response to the stress of viral infection but is instead a highly specific response, both with regard to the inducing virus and with regard to the target gene. Of three 70K heat shock genes examined, only hsp70 was induced during viral infection, and induction occurred only after infection by Ad5 and HSV-1. As revealed by genomic footprinting analysis, the mechanism of transcriptional activation of hsp70 during Ad5 or HSV-1 infection does not involve changes in the avidity of binding of basal transcription factors to the hsp70 promoter. In HSV-1-infected HeLa cells, transcriptional activation of hsp70 was quite transient, following which transcription was rapidly repressed; this was accompanied by the release of bound factors from the hsp70 promoter. In addition to the selectivity which characterizes the viral activation of hsp70 transcription, our results indicate that the consequences of this activation, as measured by changes in hsp70 mRNA levels and protein synthesis, are also virus specific.
Simian virus 40 (SV40) mutant 5002 carries base pair substitutions of C-5109----T and C-5082----T. These mutations lie in a region of the genome that encodes amino acids common to the large and small viral tumor antigens (T and t antigens, respectively) and result in amino acid substitutions of Leu-19----Phe and Pro-28----Ser. In contrast to wild-type SV40, which produces large plaques that are clearly visible 8 days postinfection, mutant 5002 is defective for productive infection, producing tiny plaques that arise at around 21 days postinfection. However, 5002 is capable of replicating viral DNA and producing normal amounts of capsid proteins, indicating that the mutations alter an activity of T antigen that is required subsequent to DNA synthesis, such as maturation, viral assembly, or release of virions. The mutant T antigen has normal ATPase activity, is phosphorylated in a manner that is indistinguishable from that of the wild-type T antigen, and retains the ability to oligomerize. 5002 complements mutants defective in T antigen host range-adenovirus helper function for productive infection. Thus, T antigen encodes two activities that affect at least two different steps in viral infection other than DNA replication, one inactivated by mutations in the host range-adenovirus helper domain and one inactivated by the mutations present in 5002. The 5002-encoded T antigen is also defective for transformation of REF52 cells when expressed from the normal SV40 early promoter, although this defect can be partially overcome by expressing the protein from stronger promoters.
We have examined the promoter sequence requirements for E1a transactivation of the human HSP70 gene by using a transient cotransfection assay. A 5' deletion study has defined a basal transcription unit extending to -74 relative to the transcription initiation site which was fully E1a responsive. Further deletion, abolishing a CCAAT element at -67, drastically reduced basal and E1a-induced expression. A linker-scanner analysis has identified four functional elements within the basal transcription unit which may interact with CTF, SP1, TFIID, and an ATF/AP1-like factor. Sequences between -100 and -188 can partially compensate for mutations in these elements. No mutation specifically abolished E1a inducibility. Any reduction in absolute E1a-induced levels was accompanied by a corresponding reduction in absolute basal levels, thereby maintaining a constant relative fold induction. We conclude that E1a transactivation of the human HSP70 promoter does not require any single basal transcription element. We also examined an HSP70 promoter fragment, containing the CCAAT element at -67 and the purine-rich element at -54, out of its normal context by fusing it upstream of a transcriptionally inactive herpes simplex virus thymidine kinase deletion construct containing only the TATA box. The resulting chimeric promoter was fully E1a responsive. Mutagenesis of this promoter fusion demonstrated that the CCAAT element was essential for detectable basal and E1a-induced expression. Mutations in the purine-rich element resulted in an approximately 10-fold elevation in basal levels and rendered the promoter nonresponsive to E1a.
Based on previous in vivo genetic analysis of bacteriophage lambda growth, we have developed two in vitro lambda DNA replication systems composed entirely of purified proteins. One is termed 'grpE-independent' and consists of supercoiled lambda dv plasmid DNA, the lambda O and lambda P proteins, as well as the Escherichia coli dnaK, dnaJ, dnaB, dnaG, ssb, DNA gyrase and DNA polymerase III holoenzyme proteins. The second system includes the E.coli grpE protein and is termed 'grpE-dependent'. Both systems are specific for plasmid molecules carrying the ori lambda DNA initiation site. The major difference in the two systems is that the 'grpE-independent' system requires at least a 10-fold higher level of dnaK protein compared with the grpE-dependent one. The lambda DNA replication process may be divided into several discernible steps, some of which are defined by the isolation of stable intermediates. The first is the formation of a stable ori lambda-lambda O structure. The second is the assembly of a stable ori lambda-lambda O-lambda P-dnaB complex. The addition of dnaJ to this complex also results in an isolatable intermediate. The dnaK, dnaJ and grpE proteins destabilize the lambda P-dnaB interaction, thus liberating dnaB's helicase activity, resulting in unwinding of the DNA template. At this stage, a stable DNA replication intermediate can be isolated, provided that the grpE protein has acted and/or is present. Following this, the dnaG primase enzyme recognizes the single-stranded DNA-dnaB complex and synthesizes RNA primers. Subsequently, the RNA primers are extended into DNA by DNA polymerase III holoenzyme. The proposed model of the molecular series of events taking place at ori lambda is substantiated by the many demonstrable protein-protein interactions among the various participants.
Three Escherichia coli heat shock proteins, DnaJ, DnaK, and GrpE, are required for replication of the bacteriophage lambda chromosome in vivo. We show that the GrpE heat shock protein is not required for initiation of lambda DNA replication in vitro when the concentration of DnaK is sufficiently high. GrpE does, however, greatly potentiate the action of DnaK in the initiation process when the DnaK concentration is reduced to a subsaturating level. We demonstrate in the accompanying articles (Alfano, C. and McMacken, R. (1989) J. Biol. Chem. 264, 10699-10708; Dodson, M., McMacken, R., and Echols, H. (1989) J. Biol. Chem. 264, 10719-10725) that DnaJ and DnaK bind to prepriming nucleoprotein structures that are assembled at the lambda replication origin (ori lambda). Binding of DnaJ and DnaK completes the ordered assembly of an ori lambda initiation complex that also contains the lambda O and P initiators and the E. coli DnaB helicase. With the addition of ATP, the DnaJ and DnaK heat shock proteins mediate the partial disassembly of the initiation complex, and the P and DnaJ proteins are largely removed from the template. Concomitantly, on supercoiled ori lambda plasmid templates, the intrinsic helicase activity of DnaB is activated and DnaB initiates localized unwinding of the DNA duplex, thereby preparing the template for priming and DNA chain elongation. We infer from our results that DnaK and DnaJ function in normal E. coli metabolism to promote ATP-dependent protein unfolding and disassembly reactions. We also provide evidence that neither the lambda O and P initiators nor the E. coli DnaJ and DnaK heat shock proteins play a direct role in the propagation of lambda replication forks in vitro.
During the lytic infection of monkey and mouse cells with simian virus 40 and polyoma virus, respectively, the preferentially
increased synthesis of two host proteins of 92,000 and 72,000 Mr was observed by 15 to 20 h after infection besides the general
stimulation of most cellular proteins. The incubation of uninfected monkey and mouse cell cultures for 30 to 60 min at 43.5
degrees C induced the enhanced synthesis of at least three proteins of 92,000, 72,000 and 70,000 Mr, the last one being the
major heat shock protein of mammalian cells. Two-dimensional gel electrophoresis and partial proteolytic digestion confirmed
that the same 92,000- and 72,000-Mr proteins are stimulated by virus infection and thermal treatment. In simian virus 40-infected
CV-1 cells, we also observed the weak stimulation of a 70,000-Mr protein comigrating in gel electrophoresis with the major
heat shock protein. The 92,000-, 72,000- and 70,000-Mr proteins of monkey cells are structurally very similar to the corresponding
proteins of mouse cells. In immunoprecipitations, no specific association of these proteins to simian virus 40 T antigens
was noticed.
A monoclonal antibody, produced from mice immunized with a herpes simplex virus (HSV)-infected cell extract, reacts with a molecule which is present in uninfected cells and which accumulates in large amounts during HSV 2 infection. In uninfected cells this molecule is growth regulated, in that exponentially growing cells have intense nuclear immunofluorescence, whereas confluent quiescent cells have little. It has a mol. wt. of 57 000 (p57) in exponential cells, and one of 61 000 (p61) in quiescent cells. In HSV 2-infected cells, p57 accumulates and nuclear and cytoplasmic immunofluorescence increases. In uninfected cells, p57 also accumulates during heat-shock treatment, and this is associated with a new immunofluorescence throughout the cytoplasm. We suggest that HSV 2 infection induces a cellular stress response which is involved in the shut-off of host cell polypeptide synthesis.
A biological agent, Newcastle disease virus, stimulated the synthesis of stress proteins in cultured chicken embryo cells. Previously, only physical and chemical agents were known to induce these proteins. The levels of translatable stress mRNAs were elevated in cells infected with avirulent or virulent strains; however, stress protein synthesis was stimulated strongly only in cells infected by avirulent strains. As did several other paramyxoviruses, avirulent strains of Newcastle disease virus stimulated the synthesis of glucose-regulated proteins as well as stress proteins. Possible stimuli of the synthesis of these two sets of proteins in paramyxovirus-infected cells are considered.
Members of the 70-kDa family of cellular stress proteins assit in protein folding by preventing inappropriate intra- and intermolecular interactions during normal protein synthesis and transport and when cells are exposed to a variety of environmental stresses. During infection of A31 mouse fibroblasts with polyomavirus, the constitutive form of hsp70, hsc70, coimmunoprecipitated with all three viral capsid proteins (VP1, VP2, and VP3). In addition, the subcellular location of hsc70 changed from cytoplasmic to nuclear late in polyomavirus infection, coincident with the nuclear localization of the viral capsid proteins. VP1 and VP2 expressed in Sf9 insect cells with recombinant baculovirus vectors also coimmunoprecipitated with an hsp70-like protein, and VP1 expressed in Escherichia coli coimmunoprecipitated with the hsp70 homolog DnaK. Capsid proteins expressed by in vitro translation coimmunoprecipitated with the hsc70 protein present in the reticulocyte translation extract. Therefore, the polyomavirus capsid proteins associate with hsc70 during virus infection as well as in recombinant protein expression systems. This association may play a role in preventing the premature assembly of capsids in the cytosol and/or in facilitating the nuclear transport of capsid protein complexes.
Several bacteriophages use the Escherichia coli GroES and GroEL chaperonins for folding and assembly of their morphogenetic structures. Bacteriophage T4 is unusual in that it encodes a specialized protein (Gp31) that is thought to interact with the host GroEL and to be absolutely required for the correct assembly of the major capsid protein (Gp23) in vivo. Here we show that despite the absence of amino-acid sequence similarity between Gp31 and GroES, Gp31 can functionally substitute for the GroES co-chaperonin in the morphogenesis of bacteriophages lambda and T5, the in vivo and in vitro chaperonin-dependent assembly of ribulose bisphosphate carboxylase (Rubisco), as well as overall bacterial growth at the non-permissive temperature. Like GroES, the bacteriophage Gp31 protein forms a stable complex with the E. coli GroEL protein in the presence of Mg-ATP and inhibits the ATPase activity of GroEL in vitro.
We have investigated the induction of heat shock proteins (HSPs) in mice infected with vaccinia virus. Vaccinia virus replicates to high levels in the ovaries of infected mice and causes a significant inhibition of host cell DNA, RNA, and protein synthesis. Many HSPs are constitutively expressed in murine ovarian tissue at low levels, consistent with their obligatory role in normal physiological events. In contrast with these events, HSP expression was augmented in virus-infected mouse ovaries 6 days postinfection. In particular, there was a dramatic increase in the expression of a protein identified as the inducible 72-kDa HSP. Analysis of cellular mRNA confirmed this protein to be the major mouse inducible HSP70 and demonstrated its presence within virus-infected cells. Hence, we have demonstrated the expression of stress proteins during poxvirus infection in vivo.
The heat shock protein Hsp90 is known as an essential component of several signal transduction pathways and has now been identified as an essential host factor for hepatitis B virus replication. Hsp90 interacts with the viral reverse transcriptase to facilitate the formation of a ribonucleoprotein (RNP) complex between the polymerase and an RNA ligand. This RNP complex is required early in replication for viral assembly and initiation of DNA synthesis through a protein-priming mechanism. These results thus invoke a role for the Hsp90 pathway in the formation of an RNP.
A peptide encompassing the N-terminal 82 amino acids of simian virus 40 (SV40) large T antigen was previously shown to bind to the large subunit of DNA polymerase alpha-primase (I. Dornreiter, A. Höss, A. K. Arthur, and E. Fanning, EMBO J. 9:3329-3336, 1990). We report here that a mutant T antigen, T83-708, lacking residues 2 to 82 retained the ability to bind to DNA polymerase alpha-primase, implying that it carries a second binding site for DNA polymerase alpha-primase. The mutant protein also retained ATPase, helicase, and SV40 origin DNA-binding activity. However, its SV40 DNA replication activity in vitro was reduced compared with that of wild-type protein. The reduction in replication activity was accompanied by a lower DNA-binding affinity to SV40 origin sequences and aberrant oligomerization on viral origin DNA. Thus, the first 82 residues of SV40 T antigen are not strictly required for its interaction with DNA polymerase alpha-primase or for DNA replication function but may play a role in correct hexamer assembly and efficient DNA binding at the origin.
HSP72 is dramatically induced in the ovaries of vaccinia virus (VV)-infected mice and associates with VV proteins. In order to investigate the role of HSP72 during vaccinia virus replication, we have constructed a recombinant vaccinia virus encoding the major inducible cellular HSP72 (VV-HSP72+) and examined the replication characteristics of this virus. VV-HSP72+ exhibited growth kinetics identical to and peak titers very similar to those of control viruses, both in vitro and in vivo. In particular, replication of VV-HSP72+ was identical to that of control viruses in the HSP72-negative cell line Y3.Ag.1.2.3, and overexpression of HSP72 had no effect on the virulence of VV infection in normal or immunocompromised mice. We conclude that while VV infection results in the induction of the major inducible 72-kDa HSP, VV replication proceeds normally in the absence of this protein. It is unclear whether another celluar chaperone is required to facilitate virus replication in place of HSP72 in Y3.Ag.1.2.3 cells or whether HSP expression plays no role in virus replication, but is simply a component of the generalized stress response to virus infection.
Assembly of hepadnaviruses depends on the formation of a ribonucleoprotein (RNP) complex comprising the viral polymerase polypeptide and an RNA segment, epsilon, present on pregenomic RNA. This interaction, in turn, activates the reverse transcription reaction, which is primed by a tyrosine residue on the polymerase. We have shown recently that the formation of this RNP complex in an avian hepadnavirus, the duck hepatitis B virus, depends on cellular factors that include the heat shock protein 90 (Hsp90). We now report that RNP formation also requires ATP hydrolysis and the function of p23, a recently identified chaperone partner for Hsp90. Furthermore, we also provide evidence that the chaperone complex is incorporated into the viral nucleocapsids in a polymerase-dependent reaction. Based on these findings, we propose a model for hepadnavirus assembly and priming of viral DNA synthesis where a dynamic, energy-driven process, mediated by a multi-component chaperone complex consisting of Hsp90, p23 and, potentially, additional factors, maintains the reverse transcriptase in a specific conformation that is competent for RNA packaging and protein priming of viral DNA synthesis.
The amino-terminal domain of SV40 large tumor antigen (TAg) is required for efficient viral DNA replication. However, the biochemical activity associated with this domain has remained obscure. We show here that the amino-terminal domain of TAg shares functional homology with the J-domain of DnaJ/hsp40 molecular chaperones. DnaJ proteins function as cofactors by regulating the activity of a member of the 70-kD heat shock protein family. Genetic analyses demonstrated that amino-terminal sequences of TAg comprise a novel J-domain that mediates a specific interaction with the constitutively expressed hsc70 and show that the J-domain is also required for efficient viral DNA replication in vivo. Furthermore, we demonstrated that the J-domain of two human DnaJ homologs, HSJ1 or DNAJ2, could substitute functionally for the amino-terminus of TAg in promoting viral DNA replication. Together, our findings suggest that TAg uses its J-domain to support SV40 DNA replication in a manner that is strikingly similar to the use of Escherichia coli DnaJ by bacteriophage lambda in DNA replication. However, TAg has evolved a more efficient strategy of DNA replication through an intrinsic J-domain to associate directly with a partner chaperone protein. Our observations provide evidence of a role for chaperone proteins in the process of eukaryotic DNA replication.
The genome of Molluscum contagiosum virus (MCV) has a high G + C content, which largely differs from those of vaccinia virus (VAC) and other characterized poxviruses. This has precluded the use of DNA hybridization for the identification of MCV genes and the further establishment of the virus genetic map. To circumvent this problem, we have partially sequenced clones containing virus restriction endonuclease fragments, which were derived by either single or double-digestion of genomic DNA from the subtype I of MCV. The DNA sequences were translated and used to search protein data bases. This analysis resulted in the finding of high-scoring matches to data base entries, including forty-five VAC genes. In addition, MCV-specific sequences that encoded protein domains of known function (i.e. DNA J domain) were found. The locations of MCV clones were inferred from the presumed colinearity of both MCV and VAC genomes, and further confirmed by PCR technology. The data presented here led to the construction of a partial genetic map of MCVI, which revealed that the order and orientation of a large number of MCV genes were equivalent to those of their VAC homologues. The conserved gene arrangement was apparently disrupted in the terminal regions, where MCV sequences showing homologies with the VAC counterparts were not found.
Simian virus 40 (SV40) encodes two proteins, large T antigen and small t antigen that contribute to virus-induced tumorigenesis. Both proteins act by targeting key cellular regulatory proteins and altering their function. Known targets of the 708-amino-acid large T antigen include the three members of the retinoblastoma protein family (pRb, p107, and p130), members of the CBP family of transcriptional adapter proteins (cap-binding protein [CBP], p300, and p400), and the tumor suppressor p53. Small t antigen alters the activity of phosphatase pp2A and transactivates the cyclin A promoter. The first 82 amino acids of large T antigen and small t antigen are identical, and genetic experiments suggest that an additional target(s) important for transformation interacts with these sequences. This region contains a motif similar to the J domain, a conserved sequence found in the DnaJ family of molecular chaperones. We show here that mutations within the J domain abrogate the ability of large T antigen to transform mammalian cells. To examine whether a purified 136-amino-acid fragment from the T antigen amino terminus acts as a DnaJ-like chaperone, we investigated whether this fragment stimulates the ATPase activity of two hsc70s and discovered that ATP hydrolysis is stimulated four- to ninefold. In addition, ATPase-defective mutants of full-length T antigen, as well as wild-type small t antigen, stimulated the ATPase activity of hsc70. T antigen derivatives were also able to release an unfolded polypeptide substrate from an hsc70, an activity common to DnaJ chaperones. Because the J domain of T antigen plays essential roles in viral DNA replication, transcriptional control, virion assembly, and tumorigenesis, we conclude that this region may chaperone the rearrangement of multiprotein complexes.
Inactivation of the retinoblastoma tumor suppressor protein (pRB) contributes to tumorigenesis in a wide variety of cancers. In contrast, the role of the two pRB-related proteins, p130 and p107, in oncogenic transformation is unclear. The LXCXE domain of simian virus 40 large T antigen (TAg) specifically binds to pRB, p107, and p130. We have previously shown that the N terminus and the LXCXE domain of TAg cooperate to alter the phosphorylation state of p130 and p107. Here, we demonstrate that TAg promotes the degradation of p130 and that the N terminus of TAg is required for this activity. The N terminus of TAg has homology to the J domain of the DnaJ family of molecular chaperone proteins. Mutants with mutations in the J-domain homology region of TAg are defective for altering p130 and p107 phosphorylation and for p130 degradation. A heterologous J-domain from a human DnaJ protein can functionally substitute for the N terminus of TAg in the effect on p107 and p130 phosphorylation and p130 stability. We further demonstrate that the J-domain homology region of TAg confers a growth advantage to wild-type mouse embryo fibroblasts (MEFs) but is dispensable in the case of MEFs lacking both p130 and p107. This indicates that p107 and p130 have overlapping growth-suppressing activities whose inactivation is mediated by the J domain of TAg.
B. B. thanks members of his lab and J. Reinstein for critical reading of the manuscript and C. Gassler, T. Laufen, and S. Rudiger for figure preparation. A. H. thanks Wayne Fenton for critical reading and Zhaohui Xu for figure preparation. A. H. dedicates this work to Guenter Brueckner, always an inspiration.
The immunosuppressive and cytostatic agent 15-deoxyspergualin (DSG) binds to the Hsc70 class of molecular chaperones with a KD = 4 μM. Because Hsc70s represent a diverse group of cellular effectors and because Hsc70 function frequently requires a DnaJ molecular chaperone, the specificity of DSG for different Hsc70s and the ability of DSG to block the productive interaction between an Hsc70 and its DnaJ partner were examined. DSG stimulated the ATPase activity of a mammalian and yeast cytosolic Hsc70 from 20 to 40%, but was unable to elicit such a response in a homologous Hsc70, Binding Protein (BiP), that resides in the lumen of the endoplasmic reticulum. In addition, the DnaJ-stimulated Hsc70 ATPase activity and the DnaJ-mediated release of an unfolded polypeptide from an Hsc70 were unaffected by DSG. These results indicate that Hsc70s exhibit substrate selectivity for DSG and that DSG does not compromise Hsc70 functions that require DnaJs. Thus, the immunosuppressive and cytostatic effects of DSG may be specific for a subset of cellular Hsc70s and confined to DnaJ-independent Hsc70-mediated activities.
A monoclonal antibody produced from mice immunized with HSV-infected cell DNA binding proteins reacts with two cell-encoded polypeptides, p90 and p40, synthesized constitutively by many different cell types and expressed at high levels in transformed cells. In uninfected cells heat shock induces a cytoplasmic accumulation of p90 and in agreement with this we show that p90 is related to hsp90. During HSV-2 lytic productive infection, p90 accumulates to very high levels, such accumulation being detectable by 2 h p.i. In infected cells p90 is distributed throughout the cell, and in contrast to uninfected cells, is also located on the cell surface. Infection with HSV-1 strains causes an accumulation of p40, which has an intracellular location and is absent from the cell surface. These results show that HSV lytic infection results in an accumulation of at least two cell-encoded polypeptides, one of which is related to hsp90.
Inactivation of the retinoblastoma tumor suppressor protein (pRB) contributes to tumorigenesis in a wide variety of cancers. In contrast, the role of the two pRB-related proteins, p130 and p107, in oncogenic transformation is unclear. The LXCXE domain of simian virus 40 large T antigen (TAg) specifically binds to pRB, p107, and p130. We have previously shown that the N terminus and the LXCXE domain of TAg cooperate to alter the phosphorylation state of p130 and p107. Here, we demonstrate that TAg promotes the degradation of p130 and that the N terminus of TAg is required for this activity. The N terminus of TAg has homology to the J domain of the DnaJ family of molecular chaperone proteins. Mutants with mutations in the J-domain homology region of TAg are defective for altering p130 and p107 phosphorylation and for p130 degradation. A heterologous J-domain from a human DnaJ protein can functionally substitute for the N terminus of TAg in the effect on p107 and p130 phosphorylation and p130 stability. We further demonstrate that the J-domain homology region of TAg confers a growth advantage to wild-type mouse embryo fibroblasts (MEFs) but is dispensable in the case of MEFs lacking both p130 and p107. This indicates that p107 and p130 have overlapping growth-suppressing activities whose inactivation is mediated by the J domain of TAg.
A portion of the RNA genome of beet yellows closterovirus (BYV) has been sequenced encompassing a complete long open reading frame (ORF) potentially encoding a 65 kDa protein. The sequence of this putative protein was strikingly similar to those of HSP70-related heat shock proteins. The counterparts of all the eight segments strongly conserved in HSP70s could be confidently identified in the BYV 65 kDa protein. It is suggested that some of these segments might be the ATP-binding site(s) and that, similarly to the heat shock proteins, the 65 kDa is probably ATP-binding. Generally, however, the divergence between the 65 kDa sequence and the sequences of the HSP70s was much more pronounced than that between any two members of the latter family, allowing a clearer delineation of clusters of conserved residues that might be crucial for protein function. It is suggested that these observations will be helpful in functional dissection of the proteins of the HSP70 family. Analysis of the sequence of a portion of the ORF found upstream from the 65 kDa ORF showed that the C-terminal domain of the encoded protein could be an RNA-dependent RNA polymerase closely related to those of tricornaviruses, a family of RNA plant viruses with three component genomes.
Hsp90 is an abundant chaperone protein that assists the folding of specific proteins, such as steroid receptors, protein kinases, and so on, for their proper function. TP and RT domains of HBV polymerase have been also shown to be associated with Hsp90. Therefore, the identification of the binding sites within Hsp90, responsible for forming Hsp90/HBV Pol complex, is important for the understanding of HBV replication. In this study, cotransfection and immunoprecipitation experiments were performed to localize the binding sites of HBV pol to Hsp90. Our data show that HBV pol interact independently with both N-terminal and C-terminal fragments of Hsp90. Further analysis showed that N-terminal fragment (1–302) of Hsp90 interacts with both TP and RT domains of HBV pol, whereas C-terminal fragment (438–723) interacts with only RT domain. In conclusion, we showed that HBV pol independently interacts with N-terminal and C-terminal fragments, but not the middle fragment (327–438) of Hsp90.
Plant closteroviruses encode a homolog of the HSP70 (heat shock protein, 70 kDa) family of cellular proteins. To facilitate studies of the function of HSP70 homolog (HSP70h) in viral infection, the beet yellows closterovirus (BYV) was modified to express green fluorescent protein. This tagged virus was competent in cell-to-cell movement, producing multicellular infection foci similar to those formed by the wild-type BYV. Inactivation of the HSP70h gene by replacement of the start codon or by deletion of 493 codons resulted in complete arrest of BYV translocation from cell to cell. Identical movement-deficient phenotypes were observed in BYV variants possessing HSP70h that lacked the computer-predicted ATPase domain or the C-terminal domain, or that harbored point mutations in the putative catalytic site of the ATPase. These results demonstrate that the virus-specific member of the HSP70 family of molecular chaperones functions in intercellular translocation and represents an additional type of a plant viral-movement protein.
Initiation of DNA replication by phage lambda requires the ordered assembly and disassembly of a specialized nucleoprotein structure at the origin of replication. In the disassembly pathway, a set of Escherichia coli heat shock proteins termed the Hsp70 complex--DnaK, DnaJ, and GrpE--act with ATP to release lambda P protein from the nucleo-protein complex, freeing the DnaB helicase for its DNA-unwinding reaction. To investigate the mechanism of the release reaction, we have examined the interaction between P and the three heat shock proteins by glycerol gradient sedimentation and gel electrophoresis. We have discovered an ATP-dependent ternary interaction between P, DnaK, and DnaJ; this P.DnaK.DnaJ complex is dissociated by GrpE. We have concluded that the function of the Hsp70 complex in sequestering and releasing P protein provides for the critical step in the disassembly pathway. Based on our data and other work on protein folding, the formation of the P.DnaK.DnaJ complex might involve a conformational shift to a folding intermediate of P.
Beet yellows virus (BYV) genome encodes a 65 kDa protein homologous to the HSP70 family of cellular heat-shock proteins (Agranovsky, A.A., Boyko, V.P., Karasev, A.V., Koonin, E.V. and Dolja, V.V. (1991) J. Mol. Biol. 217, 603-610). The respective gene was cloned and expressed in vitro yielding a product of the expected size (p65). This product was found to bind to the purified microtubules with a binding constant of 4 x 10(-7) M. The binding of p65 was stimulated if ATP presented in the translation mixture was hydrolyzed by apyrase. Removal of the short C-terminal domains of alpha- and beta-tubulin by subtilisin digestion abolished the binding, demonstrating its specificity. The possible role of p65 association with microtubules in the movement of virus within and/or between plant cells is proposed.
Soon after penetration of adenovirus serotype 2 in BHK-21 and HeLa cells, HSP70 and HSC70 proteins become associated with the viral capsid. By analysis with a polyclonal antibody derived from a fusion protein containing the C-terminal domain, 290 amino acids of HSP70, and using both immunological methods and infected cells fractionation we observed that a significant amount of HSP70 proteins moved to the nucleus and colocalized with the adenovirus particles. HSP70 proteins of infected cells were isolated as a complex cross-linked with intracytoplasmic adenovirus type 2. By coprecipitation, using a polyclonal-specific antiserum derived from the fusion protein, or two different monoclonal-specific antisera, we showed that HSP70 and HSC70 proteins were associated with hexon, the major adenovirus capsid protein.
T antigen is able to transactivate gene expression from the simian virus 40 (SV40) late promoter and from several other viral and cellular promoters. Neither the mechanisms of transactivation by T antigen nor the regions of T antigen required for this activity have been determined. To address the latter point, we have measured the ability of a set of SV40 large T antigen mutants to stimulate gene expression in CV-1 monkey kidney cells from the SV40 late promoter and Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter. Transactivation, although reduced, was retained by an N-terminal 138-amino-acid fragment of T antigen. Mutants with alterations at various locations within the N-terminal 85 amino acids transactivated the RSV LTR promoter less well than did wild-type T antigen. Most of these were also partially defective in their ability to transactivate the SV40 late promoter. Two mutants with lesions in the DNA-binding domain that were unable to bind to SV40 DNA were completely defective for transactivation of both promoter, while a third mutant with a lesion in the DNA-binding domain which retained origin-binding activity transactivated both promoters as well as did wild-type T antigen. Only a low level of transactivation was seen with mutant T antigens which had lesions in or near the zinc finger region (amino acids 300 to 350). Mutations which caused defects in ATPase activity, host range/helper function, binding to p53, binding to the retinoblastoma susceptibility protein, or nuclear localization had little or no effect on transactivation. These results suggest that N-terminal portion of T antigen possesses an activation activity. The data are consistent with the idea that the overall conformation of T antigen is important for transactivation and that mutations in other regions that reduce or eliminate transactivation do so by altering the conformation or orientation of the N-terminal region so that its ability to interact with various targets is diminished or abolished.
A monoclonal antibody defines an antigen, p68, related to hsp70, which is located in nuclei of uninfected exponential cells. Nuclear p68 is released by DNase but not RNase treatment suggesting an association with DNA. Lytic productive infection of confluent quiescent BHK 21 cells with herpes simplex virus type-2 causes p68 to accumulate in nuclei. The effect is specific for HSV-2, and does not occur in HSV-1 infected cells. Maximum nuclear accumulation of p68 requires virus DNA synthesis although a significant accumulation occurs in the absence of such synthesis. It is suggested that the nuclear accumulation of p68 is an aspect of a cellular stress response to lytic infection with HSV-2.
We have dissected the human hsp70 promoter to define sequence elements allowing response to E1A. Alterations of sequence upstream of the TATA element, either with Bal 31 nuclease or by site-directed mutagenesis, had little or no effect on the response of the promoter to E1A. In general, the basal level was reduced, indicating that these sites interact with factors important for transcription, but regulation persisted. Although a CAT gene driven by just the hsp70 TATA (void of upstream sequences) could be stimulated by E1A, a similar construct containing the early SV40 TATA element was not. Analysis of several additional such constructions indicated that the specific sequence TATAA was crucial. Substitution of the TATAA sequence with the SV40 TATTTAT element in the context of the wild-type hsp70 promoter resulted in loss of E1A inducibility, but maintenance of heat inducibility. Replacement of this element with sequences not related to any TATA element resulted in loss of activity and inducibility. Thus, the SV40 TATA equivalent is functional in the context of the hsp70 promoter but cannot be induced by E1A. We conclude that the target for E1A induction of the hsp70 promoter is TATAA, and that multiple functionally distinct TATA elements, and presumably cognate transcription factors, can be distinguished in eukaryotic cells.
The late promoter of simian virus 40 (SV40) is activated in trans by the viral early gene product, T antigen. We inserted
the wild-type late-promoter region, and deletion mutants of it, into chloramphenicol acetyltransferase transient expression
vectors to identify promoter sequences which are active in the presence of T antigen. We defined two promoter activities.
One activity was mediated by a promoter element within simian virus 40 nucleotides 200 to 270. The activity of this element
was detectable only in the presence of an intact, functioning origin of replication and accounted for 25 to 35% of the wild-type
late-promoter activity in the presence of T antigen. The other activity was mediated by an element located within a 33-base-pair
sequence (simian virus nucleotides 168 to 200) which spans the junction of the 72-base-pair repeats. This element functioned
in the absence of both the origin of replication and the T-antigen-binding sites and appeared to be responsible for trans-activated
gene expression. When inserted into an essentially promoterless plasmid, the 33-base-pair element functioned in an orientation-dependent
manner. Under wild-type conditions in the presence of T antigen, the activity of this element accounted for 65 to 75% of the
late-promoter activity. The roles of the 33-base-pair element and T antigen in trans-activation are discussed.
A series of mutants of simian virus 40 has been constructed with deletions in the coding sequence for large T antigen. Nucleotide
sequence analysis indicates that 4 mutants have in-phase and 11 have out-of-phase deletions. Mutant DNAs were assayed for
the following activities: the ability to form plaques, the ability to produce T antigen as scored by indirect immunofluorescence,
viral DNA replication, and morphological transformation of rat cells. Two viable mutants were found, and these had deletions
confined to the carboxyl terminus of T antigen. Only those mutants coding for polypeptides greater than 40% of the length
of wildtype T antigen produced detectable nuclear fluorescence. The two viable mutants with deletions in the carboxyl terminus
of the protein retained the ability both to replicate their DNA, although at a reduced level, and to transform nonpermissive
cells. Mutants with sequence changes that result in the loss of more than 117 amino acids from the carboxyl terminus were
not viable and were also defective in the DNA replication and transformation functions of T antigen, although several produced
detectable nuclear fluorescence. These functions were also sensitive to the removal of amino acids near the amino terminus
and in the middle of the protein.
We have attempted to determine whether any cellular genes are activated as a result of the action of the adenoviral E1A gene. The proteins synthesized in uninfected HeLa cells have been compared to those produced in early adenovirus infected cells. At least one protein, absent from uninfected HeLa cells, was synthesized in large amounts following adenovirus infection. This 70 kd protein was not synthesized in cells infected with the E1A mutant dl312, even when the multiplicity of infection with the mutant was such that the only viral gene not expressed was the E1A gene. Thus the induction of the 70 kd protein requires the expression of the viral E1A gene. The 70 kd protein was also induced by heat shock in uninfected cells. The same 70 kd protein is synthesized in 293 cells, a line of human embryonic kidney cells transformed by a fragment of adenovirus DNA. These cells constitutively express the E1A and E1B genes.
Heat shock protein genes are induced by various kinds of stress. Besides stress, the heat shock family gene hsp70 has been shown to be induced by growth-stimulating agents such as the DNA virus oncoproteins and serum. Here, we report cloning of a novel cDNA that encodes a 100 kDa heat shock protein-related polypeptide as a human papillomavirus oncoprotein E7-inducible gene. E7 induces expression of this heat shock protein at the level of RNA synthesis. Moreover, the induction of this heat shock protein-mRNA was dependent on the conserved region 2 of the E7 protein, which is essential for binding to the proteins of the retinoblastoma family.
We have found that SV40 infection of CV1 cells induces the synthesis of a 72 kDa protein that upon molecular cloning was shown to be the product of the hsc70 gene. The above gene product was found to be mainly virus inducible, in contrast to the hsp70 gene product which was mainly heat inducible. The two genes were found to be cell cycle regulated in a distinctively different manner.
The SV40 large T antigen mutant 5002 has two amino acid substitutions (L19-F; P28-S) and is defective for productive viral infection as demonstrated by its small plaques that arise very late and by a 100-fold reduced yield of infectious progeny. 5002 replicates viral DNA at the same time postinfection as wild-type SV40, and the production of progeny DNA molecules is only marginally reduced. Furthermore, the viral capsid proteins accumulate to near normal levels following infection with 5002. In this manuscript we report evidence that 5002 infection is blocked at a specific stage of viral assembly. The SV40 viral assembly pathway involves conversion of 75S chromatin complexes to 240S virions. Unlike mutants within the T antigen host range (HR) domain, that are also defective for viral assembly and accumulate 75S particles (Spence and Pipas, 1994), 5002 particles are blocked as 150S previrions containing viral DNA and capsid proteins. We have previously shown that 5002 and HR mutants cooperate to produce viable progeny in trans complementation tests. Thus, by two criteria, SV40 large T antigen encodes two distinct activities that function at different steps in virion assembly.
The simian virus 40 (SV40) T antigen host range mutants dl1066 and dl1140 display a postreplicative block to plaque formation which suggests a novel role for T antigen late in the viral life cycle. The host range mutants dl1066 and dl1140 are able to grow in and plaque on BSC but not on CV1 monkey kidney cells, a normally permissive host. Previous work showed that in CV1 cells infected with dl1066 and dl1140, levels of viral DNA replication and of late capsid protein accumulation were only slightly reduced and the failure to accumulate agnoprotein was not likely to be the major factor responsible for the mutants' growth defect. Here we show that the host range mutants are defective in the assembly of viral particles. SV40 assembly proceeds as the progressive conversion of 75S viral chromatin complexes to 200S-240S assembled virions. When virus-infected cell extracts are separated on 5 to 40% sucrose gradients, wild-type extracts show the greatest accumulation of viral late protein in the 200S-240S fractions corresponding to the assembled virus peak and lesser amounts in the 75S-150S fractions corresponding to immature assembly intermediates. The host range mutants dl1066 and dl1140 grown in nonpermissive CV1 cells, however, failed to assemble any appreciable amounts of mature 200S-240S virions and accumulate 75S intermediates, whereas in permissive BSC cells, levels of assembly were more slightly reduced than those of the wild type. Analysis of the protein composition of gradient fractions suggests that SV40 assembly proceeds by a mechanism similar to that proposed for polyomavirus and suggests that the host range blockage may result from a failure of such mutants to add VP1 to 75S assembly intermediates.
Folding of newly synthesized polypeptides in the crowded cellular environment requires the assistance of so-called molecular chaperone proteins. Chaperones of the Hsp70 class and their partner proteins interact with nascent polypeptide chains on ribosomes and prevent their premature (mis)folding at least until a domain capable of forming a stable structure is synthesized. For many proteins, completion of folding requires the subsequent interaction with one of the large oligomeric ring-shaped proteins of the chaperonin family, which is composed of the GroEL-like proteins in eubacteria, mitochondria, and chloroplasts, and the TRiC family in eukaryotic cytosol and archaea. These proteins bind partially folded polypeptide in their central cavity and promote folding by ATP-dependent cycles of release and rebinding. In these reactions, molecular chaperones interact predominantly with the hydrophobic surfaces exposed by nonnative polypeptides, thereby preventing incorrect folding and aggregation.
The folding of many newly synthesized proteins in the cell depends on a set of conserved proteins known as molecular chaperones. These prevent the formation of misfolded protein structures, both under normal conditions and when cells are exposed to stresses such as high temperature. Significant progress has been made in the understanding of the ATP-dependent mechanisms used by the Hsp70 and chaperonin families of molecular chaperones, which can cooperate to assist in folding new polypeptide chains.
Hsc70s in the yeast endoplasmic reticulum (ER) and mitochondria interact with membrane-associated components of the translocation machinery and are required for post-translational protein import. Although it has been proposed that the mitochondrial and ER machines function similarly, a variety of experiments suggest that BiP, the ER hsc70, might play a more elaborate role.
The positive-strand RNA genome of beet yellows closterovirus (BYV) encodes a 65 kDa protein (p65) related to the HSP70 family of cell chaperones. The full-sized BYV p65, and N- and C-terminal fragments, with (His)6 tails, were overexpressed in bacteria and purified by metal-chelate chromatography. Using a polyclonal antiserum raised against the C-terminal fragment of p65, evidence was obtained for expression of the viral protein in planta. Purified recombinant p65 and its N-terminal 40 kDa fragment exhibited Mg2+-dependent ATPase activity in vitro. However, unlike its cellular HSP70 homologues, p65 was unable to bind to denatured protein and its ATPase activity was not stimulated by synthetic peptides which are known to stimulate HSP70 ATPases. Hence, the BYV p65, although being a chaperone-type ATPase, may have a distinct substrate specificity and function in BYV-infected cells.
Recent findings emphasize that different molecular chaperones cooperate during intracellular protein biogenesis. Mechanistic aspects of chaperone cooperation are now emerging from studies on the regulation of certain signal transduction pathways mediated by Hsc70 and Hsp90 in the eukaryotic cytosol. Efficient cooperation appears to be achieved through a defined regulation of Hsc70 activity by the chaperone cofactors Hip and Hop.