Hepatitis C Virus Co-Opts Ras-GTPase-Activating Protein-Binding Protein 1 for Its Genome Replication

Yi, Zhigang; Pan, Tingting; Wu, Xianfang; Song, Wuhui; Wang, Shanshan; Xu, Yan; Rice, Charles M.; MacDonald, Margaret R.; Yuan, Zhenghong

doi:10.1128/jvi.00013-11

Cited by 55 publications

(61 citation statements)

References 52 publications

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“…has also been identified in replication centers of rubella virus (32), again in the absence of TIA1 and PABP, and in those of HCV (56). G3BP1 is involved in recruiting proteins to SGs (52) and might play an analogous role in RTC formation.…”

Section: Figmentioning

confidence: 99%

Arenavirus Infection Induces Discrete Cytosolic Structures for RNA Replication

Baird

York

Nunberg

2012

J Virol

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Arenaviruses are responsible for acute hemorrhagic fevers with high mortality and pose significant threats to public health and biodefense. These enveloped negative-sense RNA viruses replicate in the cell cytoplasm and express four proteins. To better understand how these proteins insinuate themselves into cellular processes to orchestrate productive viral replication, we have identified and characterized novel cytosolic structures involved in arenavirus replication and transcription. In cells infected with the nonpathogenic Tacaribe virus or the attenuated Candid#1 strain of Junín virus, we find that newly synthesized viral RNAs localize to cytosolic puncta containing the nucleoprotein (N) of the virus. Density gradient centrifugation studies reveal that these replication-transcription complexes (RTCs) are associated with cellular membranes and contain full-length genomic-and antigenomic-sense RNAs. Viral mRNAs segregate at a higher buoyant density and are likewise scant in immunopurified RTCs, consistent with their translation on bulk cellular ribosomes. In addition, confocal microscopy analysis reveals that RTCs contain the lipid phosphatidylinositol-4-phosphate and proteins involved in cellular mRNA metabolism, including the large and small ribosomal subunit proteins L10a and S6, the stress granule protein G3BP1, and a subset of translation initiation factors. Elucidating the structure and function of RTCs will enhance our understanding of virus-cell interactions that promote arenavirus replication and mitigate against host cell immunity. This knowledge may lead to novel intervention strategies to limit viral virulence and pathogenesis.V iruses are wholly reliant on the host cell for replication. Viral proteins must access cellular systems to establish a productive environment for generating progeny virions while also evading the innate antiviral response. Viruses have therefore evolved diverse strategies and multifunctional proteins to coopt the host cell infrastructure. Elucidating these virus-cell interactions may suggest novel targets for antiviral intervention.Arenaviruses can cause acute hemorrhagic fevers with high mortality (34, 43). These viruses are endemic in rodent populations worldwide (47) and can be transmitted to humans by contact. Prototype arenavirus species include Lassa fever virus (LASV) in western Africa and Junín virus (JUNV) in the Pampas region of Argentina. These and other hemorrhagic fever arenaviruses pose ongoing threats to public health and raise concerns for biodefense planners. In the absence of licensed vaccines and specific antiviral therapies, these viruses are recognized as NIH category A priority pathogens (39). A critical understanding of the interactions of arenaviral proteins with the host cell will provide insight toward the development of effective therapies against arenavirus hemorrhagic fevers.The arenaviruses are a diverse family of enveloped negativesense RNA viruses that replicate entirely in the cell cytoplasm (9). The S and L RNA segments of the bipartite aren...

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Section: Figmentioning

confidence: 99%

Arenavirus Infection Induces Discrete Cytosolic Structures for RNA Replication

Baird

York

Nunberg

2012

J Virol

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“…It is overexpressed in human hepatocytes from patients with chronic hepatitis C (33) and in some colorectal cancers (46); also, its helicase activity is essential for efficient HCV replication (27). The RNA helicase DDX5 (growth-related nuclear 68 protein), which interacts with the C-terminal region of HCV NS5B, has been suggested to be part of the HCV replicase complex (47), whereas RasGTPase-activating protein-binding protein 1 (G3BP1) interacts with both HCV NS5B and the 5Ј end of the HCV minusstrand RNA, suggesting that it is part of HCV replication complex (48). Another RNA helicase DDX30 that we scored has been shown to have an inhibitory effect on HIV-1 packaging and to reduce viral infectivity (49).…”

Section: Discussionmentioning

confidence: 99%

Affinity Capture and Identification of Host Cell Factors Associated with Hepatitis C Virus (+) Strand Subgenomic RNA

Upadhyay

Dixit

Manvar

et al. 2013

Molecular & Cellular Proteomics

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Hepatitis C virus (HCV) infection leading to chronic hepatitis is a major factor in the causation of liver cirrhosis, hepatocellular carcinoma, and liver failure. This process may involve the interplay of various host cell factors, as well as the interaction of these factors with viral RNA and proteins. We report a novel strategy using a sequencespecific biotinylated peptide nucleic acid ( The hepatitis C virus, a blood-borne pathogen that causes chronic hepatitis, is the primary reason for liver transplantation in the United States. HCV 1 preferentially replicates in liver tissue without any direct cytopathic effect and thus is able to maintain long term, persistent infection. More than 50% of HCV-infected patients do not respond to treatment; instead, the majority of patients develop chronic hepatitis C, which leads to progressive liver fibrosis, cirrhosis, end-stage liver disease, and hepatocellular carcinoma. The hepatitis C virus is a positive single-stranded RNA virus of a 9.6-kb genome. After its entry into cells, the (ϩ) strand RNA first serves as a messenger RNA for the translation of viral proteins. Newly synthesized HCV replicase (NS5B) then copies the (ϩ) strand RNA genome into the (Ϫ) strand RNA, which serves as a template for the production of the viral genome. The conserved 5Ј-and 3Ј-nontranslated (5ЈNTR and 3ЈNTR) regions of the HCV genome have multiple regulatory elements that are essential for replication of HCV and translation of viral proteins. Although the 5ЈNTR of HCV contains the internal ribosomal entry site, which is required for capindependent translation of (ϩ) strand HCV RNA (1-4), it is also the 3Ј region of the (Ϫ) strand RNA, which functions as the initiation site for replication of the (ϩ) strand HCV RNA genome. The 3Ј regions of both (Ϫ) and (ϩ) strand HCV RNAs are highly structured and serve as the initiation sites for viral replication (5). Various cellular proteins have been shown to interact with 5ЈNTR of HCV RNA; these include La autoantigen (6) nuclear factors NF90, NF110, NF45, and RNA helicase A (7), as well as the polypyrimidine tract-binding protein (8 -10). Recently, we affinity-captured different cellular proteins interacting with HCV 3ЈNTR and identified them by LC/MS/ MS; some of these proteins were found to be essential for HCV replication as confirmed by siRNA (11).Another recent study using sequence-specific gene silencing of the RNAi screen has identified 26 human genes encoding proteins that physically interact with HCV RNA or protein and modulate HCV replication (12). A more direct approach would be to capture the replicating HCV RNA genome in situ under physiological conditions and then identify all the cellular From the §Department of Biochemistry and Molecular Biology and Centre for the Study of Emerging and Re-emerging Pathogens,

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“…To generate Jc1G-NS5B.HA, a nucleotide sequence encoding a hemagglutinin (HA) peptide (TAC CCA TAC GAT GTT CCA GAT TAC GCT) was inserted into the NS5B region by fusion PCR. HCV subgenomic replicon construct sgJFH1 was generated by replacing the fragment of Jc1G that encompasses the sequence extending from the 5= UTR to the structural protein region with a fragment from the BB7 subgenomic replicon encompassing the 5= UTR-BSD-EMCV IRES region (13). To generate sgJFH1-DU-NS5B.HA1, sgJFH1-DU-NS5B.HA2, and sgJFH1-DU-NS5B.His constructs, first an RsrII/XbaI fragment was transferred from Jc1G-DU into sgJFH1 to get the sgJFH1-DU construct and then the nucleotides encoding the HA peptide, the HA peptide with flanking amino acids (GAC GCT TAC CCA TAC GAT GTT CCA GAT TAC GCT AGC CTA), and the His 6 (CAT CAT CAT CAT CAT CAC) peptide were inserted into the NS5B region by fusion PCR to get sgJFH1-DU-NS5B.HA1, sgJFH1-DU-NS5B.HA2, and sgJFH1-DU-NS5B.His, respectively.…”

Section: Methodsmentioning

confidence: 99%

Affinity Purification of the Hepatitis C Virus Replicase Identifies Valosin-Containing Protein, a Member of the ATPases Associated with Diverse Cellular Activities Family, as an Active Virus Replication Modulator

Fang

Zou

et al. 2016

J Virol

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Like almost all of the positive-strand RNA viruses, hepatitis C virus (HCV) induces host intracellular membrane modification to form the membrane-bound viral replication complex (RC), within which viral replicases amplify the viral RNA genome. Despite accumulated information about how HCV co-opts host factors for viral replication, our knowledge of the molecular mechanisms by which viral proteins hijack host factors for replicase assembly has only begun to emerge. Purification of the viral replicase and identification of the replicase-associated host factors to dissect their roles in RC biogenesis will shed light on the molecular mechanisms of RC assembly. To purify the viral replicase in the context of genuine viral replication, we developed an HCV subgenomic replicon system in which two different affinity tags were simultaneously inserted in frame into HCV NS5A and NS5B. After solubilizing the replicon cells, we purified the viral replicase by two-step affinity purification and identified the associated host factors by mass spectrometry. We identified valosin-containing protein (VCP), a member of the ATPases associated with diverse cellular activities (AAA؉ATPase) family, as an active viral replication modulator whose ATPase activity is required for viral replication. A transient replication assay indicated that VCP is involved mainly in viral genome amplification. VCP associated with viral replicase and colocalized with a viral RC marker. Further, in an HCV replicase formation surrogate system, abolishing VCP function resulted in aberrant distribution of HCV NS5A. We propose that HCV may co-opt a host AAA؉ATPase for its replicase assembly. IMPORTANCEAlmost all of the positive-strand RNA viruses share a replication strategy in which viral proteins modify host membranes to form the membrane-associated viral replicase. Viruses hijack host factors to facilitate this energy-unfavorable process. Understanding of this fundamental process is hampered by the challenges of purifying the replicase because of the technical difficulties involved. In this study, we developed an HCV subgenomic replicon system in which two different affinity tags were simultaneously inserted in frame into two replicase components. Using this dual-affinity-tagged replicon system, we purified the viral replicase and identified valosin-containing protein (VCP) AAA؉ATPase as a pivotal viral replicase-associated host factor that is required for viral genome replication. Abolishing VCP function resulted in aberrant viral protein distribution. We propose that HCV hijacks a host AAA؉ATPase for its replicase assembly. Understanding the molecular mechanism of VCP regulates viral replicase assembly may lead to novel antiviral strategies targeting the most conserved viral replication step.T he positive-strand RNA viruses are the largest class of viruses and include many medically and economically important pathogens, including hepatitis C virus (HCV); picornaviruses, which cause hand, foot, and mouth disease; and flaviviruses, such as the West N...

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Hepatitis C Virus Co-Opts Ras-GTPase-Activating Protein-Binding Protein 1 for Its Genome Replication

Cited by 55 publications

References 52 publications

Arenavirus Infection Induces Discrete Cytosolic Structures for RNA Replication

Arenavirus Infection Induces Discrete Cytosolic Structures for RNA Replication

Affinity Capture and Identification of Host Cell Factors Associated with Hepatitis C Virus (+) Strand Subgenomic RNA

Affinity Purification of the Hepatitis C Virus Replicase Identifies Valosin-Containing Protein, a Member of the ATPases Associated with Diverse Cellular Activities Family, as an Active Virus Replication Modulator

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