Hepatitis C Virus (HCV) NS5A Binds RNA-dependent RNA Polymerase (RdRP) NS5B and Modulates RNA-dependent RNA Polymerase Activity

Shirota, Yukihiro; Luo, Hong; Qin, Wenxin; Kaneko, Shuichi; Yamashita, Tatsuya; Kobayashi, Kenichi; Murakami, Seishi

doi:10.1074/jbc.m111392200

Cited by 208 publications

(179 citation statements)

References 42 publications

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“…Previous reports have demonstrated that there is coassociation between NS5A and the other non-structural proteins (Dimitrova et al, 2003;Shirota et al, 2002). As the PSTCD system provided an ideal method to confirm this observation, lysates were prepared from naïve Huh-7 cells or lines harbouring the FK5.1, FK5.1-PSTCD or FK5.1-DPSTCD replicons and biotinylated proteins were isolated from these cell lysates by using streptavidin-coated magnetic (SM) beads.…”

mentioning

confidence: 95%

Tagging of NS5A expressed from a functional hepatitis C virus replicon

McCormick

Maucourant

Griffin

et al. 2006

Journal of General Virology

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Knowledge of how hepatitis C virus (HCV) proteins associate with components of the host cell to form a functional replication complex is still limited. To address this issue, HCV replicon constructs were generated where either green fluorescent protein (GFP) or the Propionibacterium shermanii transcarboxylase domain (PSTCD) was introduced into the NS5A coding region. Insertion of both GFP and PSTCD was tolerated well, allowing formation of stable replicon-containing cell lines that contained viral protein and transcript levels that were comparable to those of an unmodified parental replicon. Cell lines generated from the GFP-tagged NS5A replicon allowed live-cell visualization of the location of NS5A. Cell lines generated from the PSTCD-tagged replicons allowed rapid and efficient precipitation of the PSTCD-tagged NS5A, as well as other HCV non-structural proteins, using streptavidin-coated magnetic beads. Both replicons represent useful tools that offer different but complementary ways of examining replication-complex formation in cells.Hepatitis C virus (HCV) is an enveloped RNA virus responsible for significant global morbidity and mortality (Lavanchy et al., 1999). In common with other positivestrand RNA viruses, the six non-structural proteins (NS2-NS5B) are likely to form part of a macromolecular complex responsible for replication of the RNA genome. Whilst structural information pertaining to several of the NS proteins is now available (O'Farrell et al., 2003;Tellinghuisen et al., 2005;Yao et al., 1999), very little is known about the structure and composition of the RNA replication complex. Identifying both the viral and cellular components of this complex will prove important, not only in providing an insight into how replication of the viral genome is coordinated, but also in the rational design of therapeutics for treating HCV infection.Progress towards this goal will require the development of robust protocols for isolation of functional RNA replication complexes. This could potentially be achieved by affinity purification of either the viral RNA or the NS proteins. In this regard, RNA replication complexes were recently isolated from replicon-harbouring cells by a two-step purification process: following hybridization with biotinand digoxigenin-tagged oligonucleotides, complexes were purified by sequential avidin-agarose and anti-digoxigenin precipitation steps (Waris et al., 2004). This allowed the isolation of complexes that contained all of the NS proteins and were capable of in vitro RNA synthesis. In a separate study, it was shown that functional HCV replicons could be generated in which either a small epitope tag or the coding sequence for enhanced green fluorescent protein (EGFP) was inserted in frame close to the C terminus of NS5A. The NS5A-EGFP fusion protein could be directly visualized and was used to demonstrate that NS5A co-localized with both NS3 and the sites of viral RNA synthesis (Moradpour et al., 2004;Appel et al., 2005). Based on these studies, we speculated that it might be pos...

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confidence: 95%

Tagging of NS5A expressed from a functional hepatitis C virus replicon

McCormick

Maucourant

Griffin

et al. 2006

Journal of General Virology

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“…Even though recent evidence has indicated that NS5B associates with other HCV viral proteins, including NS3, NS4A, and NS5A (27,28), the direct influence of these proteins on the NS5B RdRp activity has been uncertain, with the exception that a recent report has indicated that NS5A modulates NS5B activity (28). NS3 possesses a serine protease domain in the NH 2 -terminal one-third of the protein and an NTPase/helicase domain that resides in the COOH-terminal 500 amino acid residues (5).…”

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confidence: 99%

Modulation of the Hepatitis C Virus RNA-dependent RNA Polymerase Activity by the Non-Structural (NS) 3 Helicase and the NS4B Membrane Protein

Piccininni¹,

Varaklioti²,

Nardelli³

et al. 2002

Journal of Biological Chemistry

135

124

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The hepatitis C virus (HCV) nonstructural protein 5B (NS5B) is believed to be the central catalytic enzyme responsible for HCV replication but there are many unanswered questions about how its activity is controlled. In this study we reveal that two other HCV proteins, NS3 (a protease/helicase) and NS4B (a hydrophobic protein of unknown function), physically and functionally interact with the NS5B polymerase. We describe a new procedure for generating highly pure NS4B, and use this protein in biochemical studies together with NS5B and NS3. To study the functional effects of the protein-protein interactions, we have developed an in vitro replication assay using the natural noncoding 3 regions of the respective positive ((؉)-3 -untranslated region) and negative ((؊)-3 -terminal region) RNA strands of the HCV genome. Our studies show that NS3 dramatically modulates template recognition by NS5B and changes the synthetic products generated by this enzyme. The use of an NTPase-deficient mutant form of NS3 demonstrates that the NTPase activity (and thus helicase activity) of this protein is specifically required for these effects. Moreover, NS4B is found to be a negative regulator of the NS3-NS5B replication complex. Overall, these results reveal that NS3, NS4B, and NS5B can interact to form a regulatory complex that could feature in the process of HCV replication.Hepatitis C virus (HCV) 1 is a major pathogen of parenterally transmitted non-A, non-B hepatitis (1) and often causes the development of malignant chronic disease, including liver cirrhosis and hepatocellular carcinoma (2). With nearly 3% of the population of the world infected with HCV and no protective vaccine available at present, this disease has emerged as a serious global health problem since the virus was first identified (3, 4). HCV is a positive-stranded RNA virus with a genome of ϳ9400 bp. This genomic RNA initially directs the synthesis of at least 10 structural and nonstructural viral proteins (5). Following that, it is utilized by the viral RNA-dependent RNA polymerase (the nonstructural protein 5B; NS5B) as template to generate a complementary negative-stranded RNA. Once synthesized, the negative strands are transcribed into new molecules of positive-stranded genomic RNA, which in turn provide additional templates for viral protein synthesis as well as genomic RNA for the production of progeny virus (5, 6). However, the molecular events that mediate this process remain largely unclear.Several attempts to dissect the mechanistic details of the viral replication cycle have been reported to date. The focal point of such investigations has been NS5B, which possesses an RNA-dependent RNA polymerase (RdRp) activity and is believed to be the key enzyme catalyzing HCV RNA synthesis (7-14). Its crystal structure reveals that it contains the classical finger, palm, and thumb subdomains of the polymerases with the unique feature of a more fully enclosed active site tunnel (15)(16)(17), and a recent report by Bressanelli and colleagues (18) has provided add...

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“…It is still remained to be answered whether the NS3 helicase can unwind the heavily structured X-RNA at the 3'-end of the HCV genome and the double-stranded replication intermediate generated during HCV RNA replication to allow the NS5B to efficiently copy the RNA template in a cyclic manner. HCV NS5A, which can also form a complex with NS5B, was also tested in RdRp reaction, but it only slightly enhanced RNA synthesis efficiency (Shirota et al, 2002). These results all together suggest that individual HCV non-structural protein might not be sufficient for enabling NS5B to support cyclic replication.…”

Section: Discussionmentioning

confidence: 99%

Biochemical properties of full-length hepatitis C virus RNA-dependent RNA polymerase expressed in insect cells

Choi

Kim

Oh³

2003

Exp Mol Med

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A bstractThe hepatitis C virus (HCV) RNA-dependent RNA polym erase, NS5B protein, is the key viral enzym e responsible for replication of the HCV viral RNA genom e. Although several full-length and truncated form s of the HCV NS5B proteins have been expressed previously in insect cells, contam ination of host term inal transferase (TNTase) has ham pered analysis of the RNA synthesis initiation m echanism using natural HCV RNA tem plates. W e have expressed the HCV NS5B protein in insect cells using a recom binant baculovirus and purified it to near hom ogeneity w ithout contam inated TNTase. The highly purified recom binant HCV NS5B w as capable of copying 9.6-kb full-length HCV RNA tem plate, and m ini-HCV RNA carrying both 5'-and 3'-untranslated regions (UTRs) of the HCV genom e. In the absence of a prim er, and other cellular and viral factors, the NS5B could elongate over HCV RNA tem plates, but the synthesized products were prim arily in the double stranded form , indicating that no cyclic replication occurred with NS5B alone. RNA synthesis using RNA tem plates representing the 3'-end region of HCV m inus-strand RNA and the X-RNA at the 3'-end of HCV RNA genom e w as also initiated de novo. N o form ation of dim er-size self-prim ed RNA products resulting from extension of the 3'-end hydroxyl group w as observed. Despite the internal de novo initiation from the X-RNA, the NS5B could not initiate RNA synthesis from the internal region of oligouridylic acid (U)20, suggesting that HCV RNA polym erase initiates RNA synthesis from the selected region in the 3'-UTR of HCV genom e.

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Hepatitis C Virus (HCV) NS5A Binds RNA-dependent RNA Polymerase (RdRP) NS5B and Modulates RNA-dependent RNA Polymerase Activity

Cited by 208 publications

References 42 publications

Tagging of NS5A expressed from a functional hepatitis C virus replicon

Tagging of NS5A expressed from a functional hepatitis C virus replicon

Modulation of the Hepatitis C Virus RNA-dependent RNA Polymerase Activity by the Non-Structural (NS) 3 Helicase and the NS4B Membrane Protein

Biochemical properties of full-length hepatitis C virus RNA-dependent RNA polymerase expressed in insect cells

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