The C terminus of NS5A domain II is a key determinant of hepatitis C virus genome replication, but is not required for virion assembly and release

The NS5A protein of hepatitis C virus (HCV) plays roles in both virus genome replication and the assembly of infectious virus particles. NS5A comprises three domains, separated by low-complexity sequences. Whilst the function of domain I appears to be predominantly involved with genome replication, the roles of domains II and III are less well defined. It has been reported previously that a deletion spanning the majority of domain II but retaining the C-terminal 35 residues had no effect on virus production; however, deletion of the entire domain II eliminated genome replication, pointing to a key role for the C terminus of this domain. Recent work has also highlighted this region as the potential binding site of the host factor cyclophilin A (CypA). To define this requirement for replication in more detail, and to investigate the involvement of CypA, we conducted a mutagenic study of the C-terminal 30 residues of domain II within the context of both the infectious JFH-1 virus and a JFH-1-derived subgenomic replicon. We showed that 12 of these residues were absolutely required for virus genome replication, whilst mutations of the remainder either had no phenotype or exhibited a partial reduction in genome replication. There was an absolute correlation between the datasets for virus and subgenomic replicon, indicating that this region is involved solely in the process of genome replication. Comparison of our data with a previously published analysis of the same region in genotype 1b revealed some important differences between the two genotypes of HCV.

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MicroRNA-135a Modulates Hepatitis C Virus Genome Replication through Downregulation of Host Antiviral Factors

Virologica Sinica ◽

10.1007/s12250-018-0055-9 ◽

2018 ◽

Vol 34 (2) ◽

pp. 197-210 ◽

Cited By ~ 6

Author(s):

Catherine Sodroski ◽

Brianna Lowey ◽

Laura Hertz ◽

T. Jake Liang ◽

Qisheng Li

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Virus Genome ◽

Genome Replication

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Involvement of Creatine Kinase B in Hepatitis C Virus Genome Replication through Interaction with the Viral NS4A Protein

Journal of Virology ◽

10.1128/jvi.02179-08 ◽

2009 ◽

Vol 83 (10) ◽

pp. 5137-5147 ◽

Cited By ~ 30

Author(s):

Hiromichi Hara ◽

Hideki Aizaki ◽

Mami Matsuda ◽

Fumiko Shinkai-Ouchi ◽

Yasushi Inoue ◽

...

Keyword(s):

Creatine Kinase ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Viral Genome ◽

Proteome Analysis ◽

Virus Genome ◽

Genome Replication ◽

Creatine Kinase B ◽

Efficient Replication

ABSTRACT Persistent infection with hepatitis C virus (HCV) is a major cause of chronic liver diseases. The aim of this study was to identify host cell factor(s) participating in the HCV replication complex (RC) and to clarify the regulatory mechanisms of viral genome replication dependent on the host-derived factor(s) identified. By comparative proteome analysis of RC-rich membrane fractions and subsequent gene silencing mediated by RNA interference, we identified several candidates for RC components involved in HCV replication. We found that one of these candidates, creatine kinase B (CKB), a key ATP-generating enzyme that regulates ATP in subcellular compartments of nonmuscle cells, is important for efficient replication of the HCV genome and propagation of infectious virus. CKB interacts with HCV NS4A protein and forms a complex with NS3-4A, which possesses multiple enzyme activities. CKB upregulates both NS3-4A-mediated unwinding of RNA and DNA in vitro and replicase activity in permeabilized HCV replicating cells. Our results support a model in which recruitment of CKB to the HCV RC compartment, which has high and fluctuating energy demands, through its interaction with NS4A is important for efficient replication of the viral genome. The CKB-NS4A association is a potential target for the development of a new type of antiviral therapeutic strategy.

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Evaluation of Canonical siRNA and Dicer Substrate RNA for Inhibition of Hepatitis C Virus Genome Replication – A Comparative Study

PLoS ONE ◽

10.1371/journal.pone.0117742 ◽

2015 ◽

Vol 10 (2) ◽

pp. e0117742 ◽

Cited By ~ 9

Author(s):

Bruno Carneiro ◽

Ana Cláudia Silva Braga ◽

Mariana Nogueira Batista ◽

Mark Harris ◽

Paula Rahal

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Comparative Study ◽

Virus Genome ◽

Genome Replication

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Mangosteen xanthones suppress hepatitis C virus genome replication

Virus Genes ◽

10.1007/s11262-014-1098-0 ◽

2014 ◽

Vol 49 (2) ◽

pp. 208-222 ◽

Cited By ~ 10

Author(s):

Moonju Choi ◽

Young-Mi Kim ◽

Sungjin Lee ◽

Young-Won Chin ◽

Choongho Lee

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Virus Genome ◽

Genome Replication

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Hydrophobic and Charged Residues in the C-Terminal Arm of Hepatitis C Virus RNA-Dependent RNA Polymerase Regulate Initiation and Elongation

Journal of Virology ◽

10.1128/jvi.01106-14 ◽

2014 ◽

Vol 89 (4) ◽

pp. 2052-2063 ◽

Cited By ~ 5

Author(s):

Amy L. Cherry ◽

Caitriona A. Dennis ◽

Andrew Baron ◽

Leslie E. Eisele ◽

Pia A. Thommes ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Rna Polymerase ◽

De Novo ◽

Structural Features ◽

Primer Extension ◽

Hcv Rna ◽

Genome Replication ◽

Rna Dependent Rna Polymerase ◽

Subgenomic Replicon

ABSTRACTThe RNA-dependent RNA polymerase (RdRp) of hepatitis C virus (HCV) is essential for viral genome replication. Crystal structures of the HCV RdRp reveal two C-terminal features, a β-loop and a C-terminal arm, suitably located for involvement in positioning components of the initiation complex. Here we show that these two elements intimately regulate template and nucleotide binding, initiation, and elongation. We constructed a series of β-loop and C-terminal arm mutants, which were used forin vitroanalysis of RdRpde novoinitiation and primer extension activities. All mutants showed a substantial decrease in initiation activities but a marked increase in primer extension activities, indicating an ability to form more stable elongation complexes with long primer-template RNAs. Structural studies of the mutants indicated that these enzyme properties might be attributed to an increased flexibility in the C-terminal features resulting in a more open polymerase cleft, which likely favors the elongation process but hampers the initiation steps. A UTP cocrystal structure of one mutant shows, in contrast to the wild-type protein, several alternate conformations of the substrate, confirming that even subtle changes in the C-terminal arm result in a more loosely organized active site and flexible binding modes of the nucleotide. We used a subgenomic replicon system to assess the effects of the same mutations on viral replication in cells. Even the subtlest mutations either severely impaired or completely abolished the ability of the replicon to replicate, further supporting the concept that the correct positioning of both the β-loop and C-terminal arm plays an essential role during initiation and in HCV replication in general.IMPORTANCEHCV RNA polymerase is a key target for the development of directly acting agents to cure HCV infections, which necessitates a thorough understanding of the functional roles of the various structural features of the RdRp. Here we show that even highly conservative changes, e.g., Tyr→Phe or Asp→Glu, in these seemingly peripheral structural features have profound effects on the initiation and elongation properties of the HCV polymerase.

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Regulation of hepatitis C virus genome replication by microRNA-122.

Uirusu ◽

10.2222/jsv.65.277 ◽

2015 ◽

Vol 65 (2) ◽

pp. 277-286

Author(s):

Takahiro MASAKI ◽

Stanley M. Lemon

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Virus Genome ◽

Genome Replication

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Development of full-length cell-culture infectious clone and subgenomic replicon for a genotype 3a isolate of hepatitis C virus

Journal of General Virology ◽

10.1099/jgv.0.001704 ◽

2021 ◽

Vol 102 (12) ◽

Author(s):

Mingxiao Chen ◽

Yi Xu ◽

Ni Li ◽

Ping Yin ◽

Qing Zhou ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Infectious Clone ◽

Virus Production ◽

Full Length ◽

Genotype 3 ◽

Subgenomic Replicon ◽

Hcv Genotype ◽

Hcv Genotype 3 ◽

Genotype 3A

Hepatitis C virus (HCV) genotype 3 is widely distributed, and genotype 3-infected patients achieve a lower cure rate in direct-acting antiviral (DAA) therapy and are associated with a higher risk of hepatic steatosis than patients with other genotypes. Thus, the study of the virology and pathogenesis of genotype 3 HCV is increasingly relevant. Here, we developed a full-length infectious clone and a subgenomic replicon for the genotype 3a isolate, CH3a. From an infected serum, we constructed a full-length CH3a clone, however, it was nonviable in Huh7.5.1 cells. Next, we systematically adapted several intergenotypic recombinants containing Core-NS2 and 5′UTR-NS5A from CH3a, and other sequences from a replication-competent genotype 2 a clone JFH1. Adaptive mutations were identified, of which several combinations facilitated the replication of CH3a-JFH1 recombinants; however, they failed to adapt to the full-length CH3a and the recombinants containing CH3a NS5B. Thus, we attempted to separately adapt CH3a NS5B-3′UTR by constructing an intragenotypic recombinant using 5′UTR-NS5A from an infectious genotype 3a clone, DBN3acc, from which L3004P/M in NS5B and a deletion of 11 nucleotides (Δ11nt) downstream of the polyU/UC tract of the 3′UTR were identified and demonstrated to efficiently improve virus production. Finally, we combined functional 5′UTR-NS5A and NS5B-3′UTR sequences that carried the selected mutations to generate full-length CH3a with 26 or 27 substitutions (CH3acc), and both revealed efficient replication and virus spread in transfected and infected cells, releasing HCV of 104.2 f.f.u. ml−1. CH3acc was inhibited by DAAs targeting NS3/4A, NS5A and NS5B in a dose-dependent manner. The selected mutations permitted the development of subgenomic replicon CH3a-SGRep, by which L3004P, L3004M and Δ11nt were proven, together with a single-cycle virus production assay, to facilitate virus assembly, release, and RNA replication. CH3acc clones and CH3a-SGRep replicon provide new tools for the study of HCV genotype 3.

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Nucleotide requirements at positions +1 to +4 for the initiation of hepatitis C virus positive-strand RNA synthesis

Journal of General Virology ◽

10.1099/vir.0.028423-0 ◽

2011 ◽

Vol 92 (5) ◽

pp. 1082-1086 ◽

Cited By ~ 3

Author(s):

Udvitha Nandasoma ◽

Christopher McCormick ◽

Stephen Griffin ◽

Mark Harris

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Rna Virus ◽

Hammerhead Ribozyme ◽

Rna Replication ◽

Genome Replication ◽

Wild Type ◽

Subgenomic Replicon ◽

Positive Strand ◽

Positive Strand Rna

RNA virus genome replication requires initiation at the precise terminus of the template RNA. To investigate the nucleotide requirements for initiation of hepatitis C virus (HCV) positive-strand RNA replication, a hammerhead ribozyme was inserted at the 5′ end of an HCV subgenomic replicon, allowing the generation of replicons with all four possible nucleotides at position 1. This analysis revealed a preference for a purine nucleotide at this position for initiation of RNA replication. The sequence requirements at positions 2–4 in the context of the J6/JFH-1 virus were also examined by selecting replication-competent virus from a pool containing randomized residues at these positions. There was strong selection for both the wild-type cytosine at position 2, and the wild-type sequence at positions 2–4 (CCU). An adenine residue was well tolerated at positions 3 and 4, which suggests that efficient RNA replication is less dependent on these residues.

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Regulation of Hepatitis C Virus Genome Replication by Xrn1 and MicroRNA-122 Binding to Individual Sites in the 5′ Untranslated Region

Journal of Virology ◽

10.1128/jvi.03631-14 ◽

2015 ◽

Vol 89 (12) ◽

pp. 6294-6311 ◽

Cited By ~ 49

Author(s):

Patricia A. Thibault ◽

Adam Huys ◽

Yalena Amador-Cañizares ◽

Julie E. Gailius ◽

Dayna E. Pinel ◽

...

Keyword(s):

Life Cycle ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Binding Site ◽

Binding Sites ◽

Untranslated Region ◽

Rna Stability ◽

Virus Genome ◽

Hcv Rna ◽

Genome Replication

ABSTRACTmiR-122 is a liver-specific microRNA (miRNA) that binds to two sites (S1 and S2) on the 5′ untranslated region (UTR) of the hepatitis C virus (HCV) genome and promotes the viral life cycle. It positively affects viral RNA stability, translation, and replication, but the mechanism is not well understood. To unravel the roles of miR-122 binding at each site alone or in combination, we employed miR-122 binding site mutant viral RNAs, Hep3B cells (which lack detectable miR-122), and complementation with wild-type miR-122, an miR-122 with the matching mutation, or both. We found that miR-122 binding at either site alone increased replication equally, while binding at both sites had a cooperative effect. Xrn1 depletion rescued miR-122-unbound full-length RNA replication to detectable levels but not to miR-122-bound levels, confirming that miR-122 protects HCV RNA from Xrn1, a cytoplasmic 5′-to-3′ exoribonuclease, but also has additional functions. In cells depleted of Xrn1, replication levels of S1-bound HCV RNA were slightly higher than S2-bound RNA levels, suggesting that both sites contribute, but their contributions may be unequal when the need for protection from Xrn1 is reduced. miR-122 binding at S1 or S2 also increased translation equally, but the effect was abolished by Xrn1 knockdown, suggesting that the influence of miR-122 on HCV translation reflects protection from Xrn1 degradation. Our results show that occupation of each miR-122 binding site contributes equally and cooperatively to HCV replication but suggest somewhat unequal contributions of each site to Xrn1 protection and additional functions of miR-122.IMPORTANCEThe functions of miR-122 in the promotion of the HCV life cycle are not fully understood. Here, we show that binding of miR-122 to each of the two binding sites in the HCV 5′ UTR contributes equally to HCV replication and that binding to both sites can function cooperatively. This suggests that active Ago2–miR-122 complexes assemble at each site and can cooperatively promote the association and/or function of adjacent complexes, similar to what has been proposed for translation suppression by adjacent miRNA binding sites. We also confirm a role for miR-122 in protection from Xrn1 and provide evidence that miR-122 has additional functions in the HCV life cycle unrelated to Xrn1. Finally, we show that each binding site may contribute unequally to Xrn1 protection and other miR-122 functions.

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A Conserved Proline between Domains II and III of Hepatitis C Virus NS5A Influences both RNA Replication and Virus Assembly

Journal of Virology ◽

10.1128/jvi.02406-08 ◽

2009 ◽

Vol 83 (20) ◽

pp. 10788-10796 ◽

Cited By ~ 33

Author(s):

Mair Hughes ◽

Sarah Gretton ◽

Holly Shelton ◽

David D. Brown ◽

Christopher J. McCormick ◽

...

Keyword(s):

Life Cycle ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Virus Assembly ◽

Infectious Clone ◽

Consensus Sequence ◽

Virus Production ◽

Rna Replication ◽

Subgenomic Replicon ◽

Genotype 1B

ABSTRACT We previously demonstrated that two closely spaced polyproline motifs, with the consensus sequence Pro-X-X-Pro-X-Lys/Arg, located between residues 343 to 356 of NS5A, mediated interactions with cellular SH3 domains. The N-terminal motif (termed PP2.1) is only conserved in genotype 1 isolates, whereas the C-terminal motif (PP2.2) is conserved throughout all hepatitis C virus (HCV) isolates, although this motif was shown to be dispensable for replication of the genotype 1b subgenomic replicon. In order to investigate the potential role of these motifs in the viral life cycle, we have undertaken a detailed mutagenic analysis of these proline residues in the context of both genotype 1b (FK5.1) or 2a subgenomic replicons and the genotype 2a infectious clone, JFH-1. We show that the PP2.2 motif is dispensable for RNA replication of all subgenomic replicons and, furthermore, is not required for virus production in JFH-1. In contrast, the PP2.1 motif is only required for genotype 1b RNA replication. Mutation of proline 346 within PP2.1 to alanine dramatically attenuated genotype 1b replicon replication in three distinct genetic backgrounds, but the corresponding proline 342 was not required for replication of the JFH-1 subgenomic replicon. However, the P342A mutation resulted in both a delay to virus release and a modest (up to 10-fold) reduction in virus production. These data point to critical roles for these proline residues at multiple stages in the HCV life cycle; however, they also caution against extrapolation of data from culture-adapted replicons to infectious virus.

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