Domain III of NS5A contributes to both RNA replication and assembly of hepatitis C virus particles

Hughes, Mair; Griffin, Stephen; Harris, Mark

doi:10.1099/vir.0.009332-0

Cited by 94 publications

(104 citation statements)

References 25 publications

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“…These functions are associated with mutations in domains I and II which comprise complementation groups A, B, and C. NS5A also plays a critical role in virion assembly, and mutations affecting this process map to the C-terminal region of NS5A domain III (16,17,47,48). At least some of the NS5A mutations that affect virion assembly do not affect RNA replication, indicating that the assembly function can be genetically separated from RNA replication functions (16,17,47). These findings suggest the likelihood that the assembly mutations define a fourth NS5A complementation group.…”

Section: Discussionmentioning

confidence: 99%

Intragenic Complementation of Hepatitis C Virus NS5A RNA Replication-Defective Alleles

Fridell

Valera

Qiu

et al. 2013

J Virol

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Hepatitis C virus NS5A has three structural domains, is required for RNA replication and virion assembly, and exists in hypoand hyperphosphorylated forms. Accumulated data suggest that phosphorylation is involved in modulating NS5A functions. We performed a mutational analysis of highly conserved serine residues in the linker region between domains I and II of genotype 2a JFH1 NS5A. As with genotype 1b Con1 NS5A, we found that specific serine residues were important for efficient hyperphosphorylation of JFH1 NS5A. However, in contrast with Con1 replicons, we observed a positive correlation between hyperphosphorylation and JFH1 replicon replication. We previously demonstrated trans-complementation of a hyperphosphorylation-deficient, replication-defective JFH1 replicon. Our results suggested that the defective NS5A encoded by this replicon, while lacking one NS5A function, was capable of performing a separate replication function. In this report, we examined an additional set of replication-defective NS5A mutations in trans-complementation assays. While some behaved similarly to the S232I replicon, others displayed a unique trans-complementation phenotype, suggesting that NS5A trans-complementation can occur by two distinct modes. Moreover, we were able, for the first time, to demonstrate intragenic complementation of replication-defective NS5A alleles. Our results identified three complementation groups: group A, comprising mutations within NS5A domain I; group B, comprising mutations affecting serine residues important for hyperphosphorylation and a subset of the domain I mutations; and group C, comprising a single mutation within the C-terminal region of domain II. We postulate that these complementation groups define three distinct and genetically separable functions of NS5A in RNA replication. H epatitis C virus (HCV) is an enveloped positive-sense singlestranded RNA virus of the genus Hepacivirus in the familyFlaviviridae. The ϳ9.6-kb HCV genome encodes an ϳ3,000-amino-acid (aa) polyprotein that is cleaved by viral and cellular proteases into 10 mature proteins: core, E1, E2, P7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B (reviewed in references 1 and 2). Core is the viral capsid protein, while E1 and E2 are highly glycosylated envelope proteins important for viral entry. P7, a small transmembrane ion channel protein, and NS2, a membrane-associated protease, are required for assembly and release of infectious virions. The remaining nonstructural (NS) proteins are essential components of the HCV RNA replication complex. NS5B is an RNAdependent RNA polymerase that catalyzes synthesis of positiveand negative-strand RNA. NS3 and its cofactor NS4A function as a serine protease responsible for releasing mature NS proteins from the polyprotein precursor. NS3 also possesses RNA helicase and NTPase activities essential for RNA replication. NS4B is an integral membrane protein likely involved in formation of intracellular membranous compartments where viral replication occurs. The final viral component of the replication c...

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Section: Discussionmentioning

confidence: 99%

Intragenic Complementation of Hepatitis C Virus NS5A RNA Replication-Defective Alleles

Fridell

Valera

Qiu

et al. 2013

J Virol

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“…Recent publications describe how small compounds, which apparently target NS5A, inhibit replication with extreme potency, highlighting the validity of NS5A as a target for drug development (5)(6)(7). NS5A is a nonstructural protein that has no defined role in the virus life cycle but is absolutely required for RNA replication (8 -10) and particle assembly (11)(12)(13)(14). It has also been shown to interact with a large number of host proteins including CypA (cyclophilin A) (15)(16)(17)(18)(19)(20)(21)(22).…”

Section: Hepatitis C Virus (Hcv)mentioning

confidence: 99%

Correlation between NS5A Dimerization and Hepatitis C Virus Replication

Lim

Chatterji

Cordek

et al. 2012

Journal of Biological Chemistry

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Background: NS5A is critical for HCV replication, but its role is poorly understood. Results: Cysteines Cys-39, Cys-57, Cys-59, and Cys-80 are vital for NS5A dimerization, RNA binding, and viral replication. Conclusion: NS5A dimerization, RNA binding, and HCV replication are correlated. Significance: This study addresses an important issue in HCV research with NS5A being a major drug target with inhibitors in advanced stages of clinical development.

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“…To test this, intracellular infectious particles were quantified following lysis of transfected cells by repetitive freeze-thaw (Hughes et al, 2009). As shown in Fig.…”

Section: Inhibition Of Escrt Function Does Not Block the Accumulationmentioning

confidence: 99%

Vps4 and the ESCRT-III complex are required for the release of infectious hepatitis C virus particles

Corless

Crump

Griffin

et al. 2009

Journal of General Virology

102

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The mechanisms by which infectious hepatitis C virus (HCV) particles are assembled and released from infected cells remain poorly characterized. In this regard, many other enveloped viruses, notably human immunodeficiency virus type 1, have been shown to utilize the host vacuolar protein sorting machinery (also known as the endosomal sorting complex required for transport; ESCRT) to traffic through the cell and effect the membrane rearrangements required for the formation of enveloped particles. We postulated that this might also apply to HCV. To test this hypothesis, we established a method of conditional virus-like particle assembly involving transcomplementation of an envelope-deleted JFH-1 genome using plasmid transfection. This system reliably produced virus particles that were infectious and could be enumerated easily by focusforming assay in Huh7 cells. Following co-transfection with plasmids expressing various dominant-negative forms of either components of the ESCRT-III complex or Vps4 (the AAA ATPase that recycles the ESCRT complexes), a reduction in particle production was seen. No significant effect was observed after co-transfection of dominant-negative ESCRT-I or Alix, an ESCRT associated protein. Dominant-negative Vps4 or ESCRT-III components had no effect on either virus genome replication or the accumulation of intracellular infectious particles. These data were confirmed using cell culture infectious HCV and we conclude that HCV requires late components of the ESCRT pathway for release of infectious virus particles. INTRODUCTIONHepatitis C virus (HCV) represents a major global health burden. An estimated 170 million people are chronically infected worldwide and a significant proportion of these will go on to develop end stage liver disease. Current drug treatments are poorly tolerated and are only effective in approximately 50 % of individuals. There is, therefore, a pressing need for a greater understanding of the molecular mechanisms involved in HCV infection in order for novel drug targets to be identified.HCV is the prototype member of the genus Hepacivirus in the family Flaviviridae. It is a single-stranded, positive-sense RNA virus whose genome encodes a single ORF. This is translated in a cap-independent manner from an internal ribosome entry site (IRES) located in the 59 untranslated region, into a single polyprotein of approximately 3000 aa. The polyprotein is proteolytically cleaved by host and viral proteases to form 10 mature proteins; the structural proteins (core, E1 and E2), a viroporin p7 and the non-structural proteins (NS2, NS3, NS4A, NS4B, NS5A and NS5B).Although recent advances in the development of tissue culture systems for the study of the HCV life cycle (Lindenbach et al., 2005;Wakita et al., 2005;Zhong et al., 2005) have defined a critical role for lipid droplets in virus assembly (Miyanari et al., 2007), the precise mechanism by which infectious HCV particles are assembled and released remains poorly characterized. In particular, it has not been established how nascent...

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Domain III of NS5A contributes to both RNA replication and assembly of hepatitis C virus particles

Cited by 94 publications

References 25 publications

Intragenic Complementation of Hepatitis C Virus NS5A RNA Replication-Defective Alleles

Intragenic Complementation of Hepatitis C Virus NS5A RNA Replication-Defective Alleles

Correlation between NS5A Dimerization and Hepatitis C Virus Replication

Vps4 and the ESCRT-III complex are required for the release of infectious hepatitis C virus particles

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