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

2009 ◽  
Vol 90 (6) ◽  
pp. 1329-1334 ◽  
Author(s):  
Mair Hughes ◽  
Stephen Griffin ◽  
Mark Harris
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Particle Production ◽  
Critical Role ◽  
Rna Replication ◽  
Subgenomic Replicon ◽  
Virus Particles ◽  
C Terminus ◽  
Genotype 2 ◽  
Domain Iii

The hepatitis C virus (HCV) NS5A protein plays a critical role in viral RNA replication and has recently been shown to play a role in particle production in the infectious genotype 2a HCV clone (JFH-1). Here, we show that alanine substitutions of serines 2428/2430 within the C-terminal domain III of NS5A do not affect subgenomic replicon RNA replication but do reduce particle production. In contrast, substitution of serines 2390/2391 had no effect on either RNA replication or particle production. Relative to genotype 1, all genotype 2 HCV isolates contain a 19 residue insertion near the C terminus of domain III which, when deleted (▵2408–2426), resulted in a delay to both RNA replication and particle production. None of these mutations affected the ratio of basal to hyperphosphorylated NS5A, suggesting that serines between residues 2390 and 2430 are not phosphorylated. We propose that although domain III is dispensable for RNA replication, it nevertheless influences this process.

scholarly journals A Genetic Interaction between Hepatitis C Virus NS4B and NS3 Is Important for RNA Replication

2008 ◽  
Vol 82 (21) ◽  
pp. 10671-10683 ◽  
Author(s):  
Anne M. Paredes ◽  
Keril J. Blight
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Genetic Interaction ◽  
Nonstructural Protein ◽  
Rna Replication ◽  
Integral Membrane Protein ◽  
Single Amino Acid ◽  
Hcv Rna ◽  
Subgenomic Replicon ◽  
C Terminus

ABSTRACT Hepatitis C virus (HCV) nonstructural protein 4B (NS4B), a poorly characterized integral membrane protein, is thought to function as a scaffold for replication complex assembly; however, functional interactions with the other HCV nonstructural proteins within this complex have not been defined. We report that a Con1 chimeric subgenomic replicon containing the NS4B gene from the closely related H77 isolate is defective for RNA replication in a transient assay, suggesting that H77 NS4B is unable to productively interact with the Con1 replication machinery. The H77 NS4B sequences that proved detrimental for Con1 RNA replication resided in the predicted N- and C-terminal cytoplasmic domains as well as the central transmembrane region. Selection for Con1 derivatives that could utilize the entire H77 NS4B or hybrid Con1-H77 NS4B proteins yielded mutants containing single amino acid substitutions in NS3 and NS4A. The second-site mutations in NS3 partially restored the replication of Con1 chimeras containing the N-terminal or transmembrane domains of H77 NS4B. In contrast, the deleterious H77-specific sequences in the C terminus of NS4B, which mapped to a cluster of four amino acids, were completely suppressed by second-site substitutions in NS3. Collectively, these results provide the first evidence for a genetic interaction between NS4B and NS3 important for productive HCV RNA replication.

scholarly journals Palmitoylation and Polymerization of Hepatitis C Virus NS4B Protein

2006 ◽  
Vol 80 (12) ◽  
pp. 6013-6023 ◽  
Author(s):  
Guann-Yi Yu ◽  
Ki-Jeong Lee ◽  
Lu Gao ◽  
Michael M. C. Lai
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Membrane Structure ◽  
Rna Replication ◽  
Polymerization Process ◽  
Cysteine Residues ◽  
Hcv Rna ◽  
Subgenomic Replicon ◽  
Lipid Modifications ◽  
Ns4b Protein

ABSTRACT Hepatitis C Virus (HCV) NS4B protein induces a specialized membrane structure which may serve as the replication platform for HCV RNA replication. In the present study, we demonstrated that NS4B has lipid modifications (palmitoylation) on two cysteine residues (cysteines 257 and 261) at the C-terminal end. Site-specific mutagenesis of these cysteine residues on individual NS4B proteins and on an HCV subgenomic replicon showed that the lipid modifications, particularly of Cys261, are important for protein-protein interaction in the formation of the HCV RNA replication complex. We further demonstrated that NS4B can undergo polymerization. The main polymerization determinants were mapped in the N-terminal cytosolic domain of NS4B protein; however, the lipid modifications on the C terminus also facilitate the polymerization process. The lipid modification and the polymerization activity could be two properties of NS4B important for its induction of the specialized membrane structure involved in viral RNA replication.

scholarly journals Interactions between Viral Nonstructural Proteins and Host Protein hVAP-33 Mediate the Formation of Hepatitis C Virus RNA Replication Complex on Lipid Raft

2004 ◽  
Vol 78 (7) ◽  
pp. 3480-3488 ◽  
Author(s):  
Lu Gao ◽  
Hideki Aizaki ◽  
Jian-Wen He ◽  
Michael M. C. Lai
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Lipid Raft ◽  
Critical Role ◽  
Rna Replication ◽  
Dominant Negative ◽  
Host Protein ◽  
Hcv Rna ◽  
Replication Complex ◽  
Detergent Resistant Membranes

ABSTRACT The lipid raft membrane has been shown to be the site of hepatitis C virus (HCV) RNA replication. The mechanism of formation of the replication complex is not clear. We show here that the formation of the HCV RNA replication complex on lipid raft (detergent-resistant membranes) requires interactions among the HCV nonstructural (NS) proteins and may be initiated by the precursor of NS4B, which has the intrinsic property of anchoring to lipid raft membrane. In hepatocyte cell lines containing an HCV RNA replicon, most of the other NS proteins, including NS5A, NS5B, and NS3, were also localized to the detergent-resistant membranes. However, when individually expressed, only NS4B was associated exclusively with lipid raft. In contrast, NS5B and NS3 were localized to detergent-sensitive membrane and cytosolic fractions, respectively. NS5A was localized to both detergent-sensitive and -resistant membrane fractions. Furthermore, we show that a cellular vesicle membrane transport protein named hVAP-33 (the human homologue of the 33-kDa vesicle-associated membrane protein-associated protein), which binds to both NS5A and NS5B, plays a critical role in the formation of HCV replication complex. The hVAP-33 protein is partially associated with the detergent-resistant membrane fraction. The expression of dominant-negative mutants and small interfering RNA of hVAP-33 in HCV replicon cells resulted in the relocation of NS5B from detergent-resistant to detergent-sensitive membranes. Correspondingly, the amounts of both HCV RNA and proteins in the cells were reduced, indicating that hVAP-33 is critical for the formation of HCV replication complex and RNA replication. These results indicate that protein-protein interactions among the various HCV NS proteins and hVAP-33 are important for the formation of HCV replication complex.

scholarly journals Mutations in NS5B Polymerase of Hepatitis C Virus: Impacts on in Vitro Enzymatic Activity and Viral RNA Replication in the Subgenomic Replicon Cell Culture

Virology ◽  
2002 ◽  
Vol 297 (2) ◽  
pp. 298-306 ◽  
Author(s):  
I.Wayne Cheney ◽  
Suhaila Naim ◽  
Vicky C.H. Lai ◽  
Shannon Dempsey ◽  
Daniel Bellows ◽  
...  
Keyword(s):  
Cell Culture ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Enzymatic Activity ◽  
Rna Replication ◽  
Viral Rna ◽  
Subgenomic Replicon ◽  
Replicon Cell ◽  
Ns5b Polymerase

scholarly journals Hepatitis C virus NS5A: tales of a promiscuous protein

2004 ◽  
Vol 85 (9) ◽  
pp. 2485-2502 ◽  
Author(s):  
Andrew Macdonald ◽  
Mark Harris
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Virus Replication ◽  
Rna Replication ◽  
Viral Rna ◽  
Cell Physiology ◽  
Subgenomic Replicon ◽  
Cellular Environment ◽  
Cellular Context

The non-structural 5A (NS5A) protein of hepatitis C virus (HCV) has been the subject of intensive research over the last decade. It is generally accepted that NS5A is a pleiotropic protein with key roles in both viral RNA replication and modulation of the physiology of the host cell. Our understanding of the role of NS5A in the virus life cycle has been hampered by the lack of a robust in vitro system for the study of HCV replication, although the recent development of the subgenomic replicon has at least allowed us to begin to dissect the involvement of NS5A in the process of viral RNA replication. Early studies into the effects of NS5A on cell physiology relied on expression of NS5A either alone or in the context of other non-structural proteins; the advent of the replicon system has allowed the extrapolation of these studies to a more physiologically relevant cellular context. Despite recent progress, this field is controversial, and there is much work to be accomplished before we fully understand the many functions of this protein. In this article, the current state of our knowledge of NS5A, discussing in detail its direct involvement in virus replication, together with its role in modulating the cellular environment to favour virus replication and persistence, are reviewed. The effects of NS5A on interferon signalling, and the regulation of cell growth and apoptosis are highlighted, demonstrating that this protein is indeed of critical importance for HCV and is worthy of further investigation.

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

2011 ◽  
Vol 92 (5) ◽  
pp. 1082-1086 ◽  
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.

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

2009 ◽  
Vol 83 (20) ◽  
pp. 10788-10796 ◽  
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.

scholarly journals The NS3 Helicase and NS5B-to-3′X Regions Are Important for Efficient Hepatitis C Virus Strain JFH-1 Replication in Huh7 Cells

2007 ◽  
Vol 81 (15) ◽  
pp. 8030-8040 ◽  
Author(s):  
Asako Murayama ◽  
Tomoko Date ◽  
Kenichi Morikawa ◽  
Daisuke Akazawa ◽  
Michiko Miyamoto ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Virus Particle ◽  
Particle Production ◽  
Nonstructural Protein ◽  
Rna Replication ◽  
Genomic Rna ◽  
Protein Coding ◽  
Coding Regions ◽  
Huh7 Cells

ABSTRACT The JFH-1 strain of hepatitis C virus (HCV) is a genotype 2a strain that can replicate autonomously in Huh7 cells. The J6 strain is also a genotype 2a strain, but its full genomic RNA does not replicate in Huh7 cells. However, chimeric J6/JFH-1 RNA that has J6 structural-protein-coding regions and JFH-1 nonstructural-protein-coding regions can replicate autonomously and produce infectious HCV particles. In order to determine the mechanisms underlying JFH-1 RNA replication, we constructed various J6/JFH-1 chimeras and tested their RNA replication and virus particle production abilities in Huh7 cells. Via subgenomic-RNA-replication assays, we found that both the JFH-1 NS5B-to-3′X (N5BX) and the NS3 helicase (N3H) regions are important for the replication of the J6CF replicon. We applied these results to full-length genomic RNA replication and analyzed replication using Northern blotting. We found that a chimeric J6 clone with JFH-1 N3H and N5BX could replicate autonomously but that a chimeric J6 clone with only JFH-1 N5BX had no replication ability. Finally, we tested the virus production abilities of these clones and found that a chimeric J6 clone with JFH-1 N3H and N5BX could produce infectious HCV particles. In conclusion, the JFH-1 NS3 helicase and NS5B-to-3′X regions are important for efficient replication and virus particle formation of HCV genotype 2a strains.

scholarly journals Identification of Residues Required for RNA Replication in Domains II and III of the Hepatitis C Virus NS5A Protein

2007 ◽  
Vol 82 (3) ◽  
pp. 1073-1083 ◽  
Author(s):  
Timothy L. Tellinghuisen ◽  
Katie L. Foss ◽  
Jason C. Treadaway ◽  
Charles M. Rice
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Amino Acid ◽  
Rna Replication ◽  
Hcv Rna ◽  
Alanine Scanning Mutagenesis ◽  
Amino Terminal ◽  
Ns5a Protein ◽  
Amino Acid Region ◽  
Domain Iii

ABSTRACT The NS5A protein of hepatitis C virus (HCV) plays an important but undefined role in viral RNA replication. NS5A has been proposed to be a three-domain protein, and the crystal structure of the well-conserved amino-terminal domain I has been determined. The remaining two domains of NS5A, designated domains II and III, and their corresponding interdomain regions are poorly understood. We have conducted a detailed mutagenesis analysis of NS5A domains II and III using the genotype 1b HCV replicon system. The majority of the mutants containing 15 small (8- to 15-amino-acid) deletions analyzed were capable of efficient RNA replication. Only five deletion mutations yielded lethal phenotypes, and these were colinear, spanning a 56-amino-acid region within domain II. This region was further analyzed by combining triple and single alanine scanning mutagenesis to identify individual residues required for RNA replication. Based upon this analysis, 23 amino acids were identified that were found to be essential. In addition, two residues were identified that yielded a small colony phenotype while possessing only a moderate defect in RNA replication. These results indicate that the entire domain III region and large portions of domain II of the NS5A protein are not required for the function of NS5A in HCV RNA replication.

scholarly journals NS2 is dispensable for efficient assembly of hepatitis C virus-like particles in a bipartite trans-encapsidation system

2014 ◽  
Vol 95 (11) ◽  
pp. 2427-2441 ◽  
Author(s):  
Matthew J. Bentham ◽  
Najat Marraiki ◽  
Christopher J. McCormick ◽  
David J. Rowlands ◽  
Stephen Griffin
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Particle Production ◽  
Early Stage ◽  
Infectious Hepatitis ◽  
Structural Proteins ◽  
Structural Protein ◽  
Subgenomic Replicon ◽  
Particle Assembly ◽  
Baculovirus Expression

Infectious hepatitis C virus (HCV) particle production in the genotype 2a JFH-1-based cell culture system involves non-structural proteins in addition to canonical virion components. NS2 has been proposed to act as a protein adaptor, co-ordinating the early stages of virion assembly. However, other studies have identified late-acting roles for this protein, making its precise involvement in infectious particle production unclear. Using a robust, bipartite trans-encapsidation system based upon baculovirus expression of HCV structural proteins, we have generated HCV-like particles (HCV-LP) in the absence of NS2 with overt similarity to wild-type virions. HCV-LP could transduce naive cells with trans-encapsidated subgenomic replicon RNAs and shared similar biochemical and biophysical properties with JFH-1 HCV. Both genotype 1b and JFH-1 intracellular HCV-LP were produced in the absence of NS2, whereas restoring NS2 to the JFH-1 system dramatically enhanced secreted infectivity, consistent with a late-acting role. Our system recapitulated authentic HCV particle assembly via trans-complementation of bicistronic, NS2-deleted, chimeric HCV, which is otherwise deficient in particle production. This closely resembled replicon-mediated NS2 trans-complementation, confirming that baculovirus expression of HCV proteins did not unduly affect particle production. Furthermore, this suggests that separation of structural protein expression from replicating HCV RNAs that are destined to be packaged alleviates an early stage requirement for NS2 during particle formation. This highlights our current lack of understanding of how NS2 mediates assembly, yet comparison of full-length and bipartite systems may provide further insight into this process.

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