Heat Shock Protein 72 Is Associated with the Hepatitis C Virus Replicase Complex and Enhances Viral RNA Replication

Chen, Yin‐Ju; Chen, Yu-Hsuan; Chow, Lu-Ping; Tsai, Yu-Chen; Chen, Pei Hong; Huang, Chi Ying F.; Chen, Wei Tzu; Hwang, Lih Hwa

doi:10.1074/jbc.m110.118323

Cited by 58 publications

(54 citation statements)

References 52 publications

(34 reference statements)

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“…Heat shock is a welldocumented experimental state that has been shown to cause viral reactivation (39,40). More importantly, heat shock at 43°C for 90 min causes viral reactivation of the replication incompetent CELO virus (lacking the Gam1 gene) (34).…”

Section: Socs Domain Containing An Intact Bc-box Motif Is Sufficient Tomentioning

confidence: 99%

BC-box protein domain-related mechanism for VHL protein degradation

Pozzebon

Varadaraj

Mattoscio

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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The tumor suppressor VHL (von Hippel-Lindau) protein is a substrate receptor for Ubiquitin Cullin Ring Ligase complexes (CRLs), containing a BC-box domain that associates to the adaptor Elongin B/C. VHL targets hypoxia-inducible factor 1α to proteasomedependent degradation. Gam1 is an adenoviral protein, which also possesses a BC-box domain that interacts with the host Elongin B/C, thereby acting as a viral substrate receptor. Gam1 associates with both Cullin2 and Cullin5 to form CRL complexes targeting the host protein SUMO enzyme SAE1 for proteasomal degradation. We show that Gam1 protein expression induces VHL protein degradation leading to hypoxia-inducible factor 1α stabilization and induction of its downstream targets. We also characterize the CRL-dependent mechanism that drives VHL protein degradation via proteasome. Interestingly, expression of Suppressor of Cytokine Signaling (SOCS) domain-containing viral proteins and cellular BC-box proteins leads to VHL protein degradation, in a SOCS domain-containing manner. Our work underscores the exquisite ability of viral domains to uncover new regulatory mechanisms by hijacking key cellular proteins.ubiquitylation | oncoviral proteins | hypoxia U biquitylation is a posttranslational modification that involves the tagging of protein substrates with ubiquitin moieties. Ubiquitin transfer requires the coordinated and subsequent activities of the ubiquitin-activating E1 enzyme, ubiquitin-conjugating E2 enzymes, and ubiquitin E3 ligases. E3 ligases show substrate specificity and can be grouped into three families based on their different E2-docking domains. CRLs (Cullin RING Ligases) are the largest subfamily of RING domain ligases (1). They display the same modular structure: a scaffold subunit (Cullin), a RING subunit, an adaptor, and a substrate-receptor subunit to recruit the specific protein target to be ubiquitylated (2). Each Cullin associates with a specific adaptor subunit, except for Cullin2 and Cullin5, which both interact with the Elongin B and Elongin C (hereafter EloB/C) heterodimer (3). The EloB/C heterodimer is specifically bound by substrate receptors that contain a minimal consensus sequence (called BC-box motif) (4-6) usually included in the SOCS (Suppressor of Cytokine Signaling) domain of several substrate-receptor subunits (7,8). The consensus sequence and the function of the BC-box motif were first described for SOCS proteins (5, 8, 9), a family of downstream effectors of cytokine signaling cascade, involved in the negative feedback regulation of this pathway.Several viruses encode for their own substrate receptors possessing the BC-box motif and are thus able to interact with EloB/ C cellular adaptor, thereby modifying the cellular ubiquitin E3 ligase complex and their target proteins selection (10-17). We have demonstrated that the CELO (Chicken Embryo Lethal Orphan) Adenovirus early protein Gam1 is one of such BC boxcontaining proteins that is able to associate with both Cullin2 and Cullin5 to reconstitute active E3 ligase complexes and tar...

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Section: Socs Domain Containing An Intact Bc-box Motif Is Sufficient Tomentioning

confidence: 99%

BC-box protein domain-related mechanism for VHL protein degradation

Pozzebon

Varadaraj

Mattoscio

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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“…Considering their pivotal roles in cells, it is not surprising that Hsp70 and Hsp90 are involved together with their cochaperones in virus infection (62). For instance, Hsp70 facilitates the assembly and disassembly of viral capsids (7,26,46), promotes the subcellular transport of tombusvirus replicase proteins and affects the activity or assembly of tombusvirus replicase complexes (71,90), controls potyvirus gene expression in cooperation with its cochaperone CPIP (17), and positively and negatively affects the genome replication of various viruses (5,45,91). Hsp90 affects the early stages of Bamboo mosaic virus (BaMV) infection by binding to the genomic RNA (20), increases the synthesis or stability of viral proteins (4,8), supports the assembly and nuclear import of influenza A virus RNA polymerase complex (59,63), and tightly regulates hepatitis C virus replication in cooperation with FKBP8 and hB-ind1 cochaperones (67,79,88).…”

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confidence: 99%

Differential Roles of Hsp70 and Hsp90 in the Assembly of the Replicase Complex of a Positive-Strand RNA Plant Virus

Mine

Hyodo

Tajima

et al. 2012

J Virol

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Japan bAssembly of viral replicase complexes of eukaryotic positive-strand RNA viruses is a regulated process: multiple viral and host components must be assembled on intracellular membranes and ordered into quaternary complexes capable of synthesizing viral RNAs. However, the molecular mechanisms underlying this process are poorly understood. In this study, we used a model virus, Red clover necrotic mosaic virus (RCNMV), whose replicase complex can be detected readily as the 480-kDa functional protein complex. We found that host heat shock proteins Hsp70 and Hsp90 are required for RCNMV RNA replication and that they interact with p27, a virus-encoded component of the 480-kDa replicase complex, on the endoplasmic reticulum membrane. Using a cell-free viral translation/replication system in combination with specific inhibitors of Hsp70 and Hsp90, we found that inhibition of p27-Hsp70 interaction inhibits the formation of the 480-kDa complex but instead induces the accumulation of large complexes that are nonfunctional in viral RNA synthesis. In contrast, inhibition of p27-Hsp90 interaction did not induce such large complexes but rendered p27 incapable of binding to a specific viral RNA element, which is a critical step for the assembly of the 480-kDa replicase complex and viral RNA replication. Together, our results suggest that Hsp70 and Hsp90 regulate different steps in the assembly of the RCNMV replicase complex. Most plant and animal viruses are positive-strand RNA viruses, which have single-stranded messenger-sense genomic RNAs. These viruses often induce host membrane rearrangements to form organelle-like compartments in which viral genomic RNAs are replicated via negative-strand RNA intermediates by the viral replicase complexes (10). Viral replicase complexes comprise multiple proteins, including viral auxiliary proteins, viral RNAdependent RNA polymerase (RdRP), and host proteins (61). Viral replicase complexes have been studied extensively by characterizing their RdRP activities and the functions of the viral and host components of the complexes. These studies have provided important information about the mechanisms regulating genome replication (15, 19, 47, 89), viral pathogenicity (68, 69), and virushost interactions (24,25,32,33). However, an important question remains: how do multiple viral and host components assemble properly into the replicase complex?Molecular chaperones are essential for cell viability by ensuring folding of newly synthesized proteins, refolding of misfolded or aggregated proteins, protein complex assembly and disassembly, membrane translocation of organellar and secretory proteins, protein degradation, and activities of regulatory proteins in signal transduction pathways (12,18,51). In eukaryotic cells, the abundant and highly conserved molecular chaperones heat shock proteins Hsp70 and Hsp90 play central roles in the biological processes mentioned above, and the activities of Hsp70 and Hsp90 are modulated by a variety of cochaperones (37,80). Considering their pivotal roles i...

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“…NS5A is an essential factor in the HCV life cycle which participates in both viral replication and assembly (3,4,45). It interacts with several viral proteins involved in both stages of the HCV life cycle, as well as with myriad host factors (1,7,11,13,53). The interaction between NS5A and CyPA depends on both the proline residues in NS5A domain II (21,22) and the peptidylprolyl isomerase (PPIase) active site of CyPA (51).…”

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confidence: 99%

Suppression of Viral RNA Binding and the Assembly of Infectious Hepatitis C Virus Particles In Vitro by Cyclophilin Inhibitors

Nag

Robotham

Tang

2012

J Virol

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Nonstructural protein 5A (NS5A) of hepatitis C virus (HCV) is an indispensable component of the HCV replication and assembly machineries. Although its precise mechanism of action is not yet clear, current evidence indicates that its structure and function are regulated by the cellular peptidylprolyl isomerase cyclophilin A (CyPA). CyPA binds to proline residues in the C-terminal half of NS5A, in a distributed fashion, and modulates the structure of the disordered domains II and III. Cyclophilin inhibitors (CPIs), including cyclosporine (CsA) and its nonimmunosuppressive derivatives, inhibit HCV infection of diverse genotypes, both in vitro and in vivo. Here we report a mechanism by which CPIs inhibit HCV infection and demonstrate that CPIs can suppress HCV assembly in addition to their well-documented inhibitory effect on RNA replication. Although the interaction between NS5A and other viral proteins is not affected by CPIs, RNA binding by NS5A in cell culture-based HCV (HCVcc)-infected cells is significantly inhibited by CPI treatment, and sensitivity of RNA binding is correlated with previously characterized CyPA dependence or CsA sensitivity of HCV mutants. Furthermore, the difference in CyPA dependence between a subgenomic and a full-length replicon of JFH-1 was due, at least in part, to an additional role that CyPA plays in HCV assembly, a conclusion that is supported by experiments with the clinical CPI alisporivir. The host-directed nature and the ability to interfere with more than one step in the HCV life cycle may result in a higher genetic barrier to resistance for this class of HCV inhibitors. Hepatitis C virus (HCV) is a positive-strand RNA virus encoding a polyprotein that is cleaved into 10 proteins, including three structural proteins (core, E1, and E2), a putative ion channel (p7), and six nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) (42). Propelled by the development of both subgenomic replicons (8, 31) and a cell culture-based infection system (HCVcc) (9,29,49,54), research has garnered a wealth of information on the role of each of these proteins in the life cycle of the virus. The structural proteins and p7 are needed for virion assembly but not RNA replication, whereas the nonstructural proteins are probably involved in both replication and infectious virus production. In addition to the requisite protease and polymerase activities, HCV also encodes proteins that perform specialized functions, such as deforming the membranes (to generate a microenvironment for RNA replication) or scaffolding (to bring the appropriate proteins together for virion assembly) (14,24,37,41).HCV infects ϳ3% of the world's population and is a major risk factor for liver cirrhosis and hepatocellular carcinoma (5, 47). Current treatment comprises pegylated interferon (IFN), the nucleoside analog ribavirin, and recently approved direct-acting antivirals (DAAs) that inhibit the HCV NS3 protease. Despite the success of this triple therapy, drug resistance against the DAAs develops quickly (38), presuma...

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Heat Shock Protein 72 Is Associated with the Hepatitis C Virus Replicase Complex and Enhances Viral RNA Replication

Cited by 58 publications

References 52 publications

BC-box protein domain-related mechanism for VHL protein degradation

BC-box protein domain-related mechanism for VHL protein degradation

Differential Roles of Hsp70 and Hsp90 in the Assembly of the Replicase Complex of a Positive-Strand RNA Plant Virus

Suppression of Viral RNA Binding and the Assembly of Infectious Hepatitis C Virus Particles In Vitro by Cyclophilin Inhibitors

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