scholarly journals Comparative Analysis of Hepatitis C Virus NS5A Dynamics and Localization in Assembly-Deficient Mutants

Pathogens ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 172
Author(s):  
Laura Riva ◽  
Corentin Spriet ◽  
Nicolas Barois ◽  
Costin-Ioan Popescu ◽  
Jean Dubuisson ◽  
...  
Keyword(s):  
Life Cycle ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Lipid Droplets ◽  
Fluorescent Protein ◽  
Intracellular Localization ◽  
Rna Replication ◽  
Genome Replication ◽  
Virion Assembly ◽  
Green Fluorescent

The hepatitis C virus (HCV) life cycle is a tightly regulated process, during which structural and non-structural proteins cooperate. However, the interplay between HCV proteins during genomic RNA replication and progeny virion assembly is not completely understood. Here, we studied the dynamics and intracellular localization of non-structural 5A protein (NS5A), which is a protein involved both in genome replication and encapsidation. An NS5A-eGFP (enhanced green fluorescent protein) tagged version of the strain JFH-1-derived wild-type HCV was compared to the corresponding assembly-deficient viruses Δcore, NS5A basic cluster 352–533 mutant (BCM), and serine cluster 451 + 454 + 457 mutant (SC). These analyses highlighted an increase of NS5A motility when the viral protein core was lacking. Although to a lesser extent, NS5A motility was also increased in the BCM virus, which is characterized by a lack of interaction of NS5A with the viral RNA, impairing HCV genome encapsidation. This observation suggests that the more static NS5A population is mainly involved in viral assembly rather than in RNA replication. Finally, NS4B exhibited a reduced co-localization with NS5A and lipid droplets for both Δcore and SC mutants, which is characterized by the absence of interaction of NS5A with core. This observation strongly suggests that NS5A is involved in targeting NS4B to lipid droplets (LDs). In summary, this work contributes to a better understanding of the interplay between HCV proteins during the viral life cycle.

scholarly journals Insertion of Green Fluorescent Protein into Nonstructural Protein 5A Allows Direct Visualization of Functional Hepatitis C Virus Replication Complexes

2004 ◽  
Vol 78 (14) ◽  
pp. 7400-7409 ◽  
Author(s):  
Darius Moradpour ◽  
Matthew J. Evans ◽  
Rainer Gosert ◽  
Zhenghong Yuan ◽  
Hubert E. Blum ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Green Fluorescent Protein ◽  
Hepatitis C ◽  
Fusion Protein ◽  
Fluorescent Protein ◽  
Nonstructural Protein ◽  
Rna Replication ◽  
Direct Visualization ◽  
Green Fluorescent ◽  
Nonstructural Protein 5A

ABSTRACT Hepatitis C virus (HCV) replicates its genome in a membrane-associated replication complex, composed of viral proteins, replicating RNA and altered cellular membranes. We describe here HCV replicons that allow the direct visualization of functional HCV replication complexes. Viable replicons selected from a library of Tn7-mediated random insertions in the coding sequence of nonstructural protein 5A (NS5A) allowed the identification of two sites near the NS5A C terminus that tolerated insertion of heterologous sequences. Replicons encoding green fluorescent protein (GFP) at these locations were only moderately impaired for HCV RNA replication. Expression of the NS5A-GFP fusion protein could be demonstrated by immunoblot, indicating that the GFP was retained during RNA replication and did not interfere with HCV polyprotein processing. More importantly, expression levels were robust enough to allow direct visualization of the fusion protein by fluorescence microscopy. NS5A-GFP appeared as brightly fluorescing dot-like structures in the cytoplasm. By confocal laser scanning microscopy, NS5A-GFP colocalized with other HCV nonstructural proteins and nascent viral RNA, indicating that the dot-like structures, identified as membranous webs by electron microscopy, represent functional HCV replication complexes. These findings reveal an unexpected flexibility of the C-terminal domain of NS5A and provide tools for studying the formation and turnover of HCV replication complexes in living cells.

scholarly journals Poly(rC)-binding protein 1 limits hepatitis C virus assembly and egress

2021 ◽  
Author(s):  
Sophie E. Cousineau ◽  
Selena M. Sagan
Keyword(s):  
Life Cycle ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Replication ◽  
Viral Life Cycle ◽  
Viral Rna ◽  
Virion Assembly ◽  
Rna Accumulation

ABSTRACTThe hepatitis C virus (HCV) co-opts a number of cellular elements – including proteins, lipids, and microRNAs – to complete its viral life cycle. The cellular RNA-binding protein poly(rC)-binding protein 1 (PCBP1) had previously been reported to bind the HCV genome 5’ untranslated region (UTR), but its importance in the viral life cycle has remained unclear. Herein, we aimed to clarify the role of PCBP1 in the HCV life cycle. Using the HCV cell culture (HCVcc) system, we found that endogenous PCBP1 knockdown decreased viral RNA accumulation yet increased extracellular virus titers. To dissect PCBP1’s specific role in the viral life cycle, we carried out assays for viral entry, translation, genome stability, RNA replication, virion assembly and egress. We found that PCBP1 did not affect viral entry, translation, RNA stability, or RNA replication in the absence of efficient virion assembly. To specifically examine virion assembly and egress, we inhibited viral RNA replication with an RNA-dependent RNA polymerase inhibitor and tracked both intracellular and extracellular viral titers over time. We found that when viral RNA accumulation was inhibited, knockdown of PCBP1 still resulted in an overall increase in HCV particle secretion. We therefore propose a model where endogenous PCBP1 limits virion assembly and egress, thereby indirectly enhancing viral RNA accumulation in infected cells. This model furthers our understanding of how cellular RNA-binding proteins modulate HCV genomic RNA utilization during the viral life cycle.IMPORTANCEHepatitis C virus (HCV) is a positive-sense RNA virus, and as such, its genome must be a template for multiple mutually exclusive steps of the viral life cycle, namely translation, RNA replication, and virion assembly. However, the mechanism(s) that regulate how the viral genome is used throughout the viral life cycle still remain unclear. A cellular RNA-binding protein – PCBP1 – had previously been reported to bind the HCV genome, but its precise role in the viral life cycle was not known. In this study, we found that depleting PCBP1 decreased viral RNA accumulation but increased virus secretion. We ruled out a role for PCBP1 in virus entry, translation, genome stability or RNA replication, and demonstrate that PCBP1 knockdown enhances virus secretion when RNA replication is inhibited. We conclude that PCBP1 normally prevents virus assembly and egress, which allows more of the viral genomic RNA to be available for translation and viral RNA replication.

scholarly journals Insertion and deletion analyses identify regions of non-structural protein 5A of Hepatitis C virus that are dispensable for viral genome replication

2006 ◽  
Vol 87 (2) ◽  
pp. 323-327 ◽  
Author(s):  
Shuanghu Liu ◽  
Israrul H. Ansari ◽  
Subash C. Das ◽  
Asit K. Pattnaik
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Viral Genome ◽  
Fluorescent Protein ◽  
Genome Replication ◽  
Structural Protein ◽  
Mutant Proteins ◽  
Viral Genome Replication ◽  
Green Fluorescent ◽  
Carboxy Terminal

Hepatitis C virus (HCV) non-structural protein 5A (NS5A) plays an essential role in viral genome replication. A series of transposon-mediated insertion mutants and deletion mutants of NS5A was used to examine the colony-forming ability of HCV subgenomic replicons encoding the mutant proteins. The results reveal that two regions of NS5A can tolerate insertions: one spanning residues 240–314, which contain the interferon sensitivity-determining region (ISDR), and the other spanning residues 349–417 at the carboxy terminus. The majority of these sites also tolerated insertion of enhanced green fluorescent protein. Furthermore, replicons encoding NS5A with deletions in ISDR or in the carboxy-terminal regions were replication-competent, indicating that these regions of NS5A are not necessary for replication. Taken together, the results suggest that the central region spanning the ISDR and the carboxy-terminal region of the molecule are dispensable for the functions of NS5A in viral genome replication.

773 HEPATITIS C VIRUS AND LIPID DROPLETS: ROLE OF SEIPIN IN LIPID DROPLET MATURATION AND VIRAL LIFE CYCLE

2011 ◽  
Vol 54 ◽  
pp. S311 ◽  
Author(s):  
S. Clement ◽  
C. Fauvelle ◽  
S. Pascarella ◽  
S. Conzelmann ◽  
V. Kaddai ◽  
...  
Keyword(s):  
Life Cycle ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Lipid Droplet ◽  
Lipid Droplets ◽  
Viral Life Cycle

scholarly journals Development of hepatitis C virus production reporter-assay systems using two different hepatoma cell lines

2012 ◽  
Vol 93 (7) ◽  
pp. 1422-1431 ◽  
Author(s):  
Midori Takeda ◽  
Masanori Ikeda ◽  
Yasuo Ariumi ◽  
Takaji Wakita ◽  
Nobuyuki Kato
Keyword(s):  
Cell Culture ◽  
Life Cycle ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Cell Lines ◽  
Hepatoma Cell ◽  
Rna Replication ◽  
Hcv Rna ◽  
Reporter Assay ◽  
Hepatoma Cell Lines

A hepatitis C virus (HCV) infection system was developed previously using the HCV JFH-1 strain (genotype 2a) and HuH-7 cells, and this cell culture is so far the only robust production system for HCV. In patients with chronic hepatitis C, the virological effects of pegylated interferon and ribavirin therapy differ depending on the HCV strain and the genetic background of the host. Recently, we reported the hepatoma-derived Li23 cell line, in which the JFH-1 life cycle is reproduced at a level almost equal to that in HuH-7-derived RSc cells. To monitor the HCV life cycle more easily, we here developed JFH-1 reporter-assay systems using both HuH-7- and Li23-derived cell lines. To identify any genetic mutations by long-term cell culture, HCV RNAs in HuH-7 cells were amplified 130 days after infection and subjected to sequence analysis to find adaptive mutation(s) for robust virus replication. We identified two mutations, H2505Q and V2995L, in the NS5B region. V2995L but not H2505Q enhanced JFH-1 RNA replication. However, we found that H2505Q but not V2995L enhanced HCV RNA replication of strain O (genotype 1b). We also selected highly permissive D7 cells by serial subcloning of Li23 cells. The expression levels of claudin-1 and Niemann–Pick C1-like 1 in D7 cells are higher than those in parental Li23 cells. In this study, we developed HCV JFH-1 reporter-assay systems using two distinct hepatoma cell lines, HuH-7 and Li23. The mutations in NS5B resulted in different effects on strains O and JFH-1 HCV RNA replication.

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

2015 ◽  
Vol 89 (12) ◽  
pp. 6294-6311 ◽  
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.

scholarly journals Tagging of NS5A expressed from a functional hepatitis C virus replicon

2006 ◽  
Vol 87 (3) ◽  
pp. 635-640 ◽  
Author(s):  
Christopher J. McCormick ◽  
Sophie Maucourant ◽  
Stephen Griffin ◽  
David J. Rowlands ◽  
Mark Harris
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Cell Lines ◽  
Magnetic Beads ◽  
Fluorescent Protein ◽  
Coding Region ◽  
Replicon Cell ◽  
Replication Complex ◽  
Propionibacterium Shermanii ◽  
Green Fluorescent

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.

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.

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