scholarly journals Viral RNA Mutations Are Region Specific and Increased by Ribavirin in a Full-Length Hepatitis C Virus Replication System

2002 ◽  
Vol 76 (17) ◽  
pp. 8505-8517 ◽  
Author(s):  
Ana Maria Contreras ◽  
Yoichi Hiasa ◽  
Wenping He ◽  
Adam Terella ◽  
Emmett V. Schmidt ◽  
...  
Keyword(s):  
Genetic Variation ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Viruses ◽  
Full Length ◽  
Mutation Rates ◽  
Hcv Rna ◽  
Nucleotide Substitutions ◽  
Replication System ◽  
Error Catastrophe

ABSTRACT High rates of genetic variation ensure the survival of RNA viruses. Although this variation is thought to result from error-prone replication, RNA viruses must also maintain highly conserved genomic segments. A balance between conserved and variable viral elements is especially important in order for viruses to avoid “error catastrophe.” Ribavirin has been shown to induce error catastrophe in other RNA viruses. We therefore used a novel hepatitis C virus (HCV) replication system to determine relative mutation frequencies in variable and conserved regions of the HCV genome, and we further evaluated these frequencies in response to ribavirin. We sequenced the 5′ untranslated region (5′ UTR) and the core, E2 HVR-1, NS5A, and NS5B regions of replicating HCV RNA isolated from cells transfected with a T7 polymerase-driven full-length HCV cDNA plasmid containing a cis-acting hepatitis delta virus ribozyme to control 3′ cleavage. We found quasispecies in the E2 HVR-1 and NS5B regions of untreated replicating viral RNAs but not in conserved 5′ UTR, core, or NS5A regions, demonstrating that important cis elements regulate mutation rates within specific viral segments. Neither T7-driven replication nor sequencing artifacts produced these nucleotide substitutions in control experiments. Ribavirin broadly increased error generation, especially in otherwise invariant regions, indicating that it acts as an HCV RNA mutagen in vivo. Similar results were obtained in hepatocyte-derived cell lines. These results demonstrate the potential utility of our system for the study of intrinsic factors regulating genetic variation in HCV. Our results further suggest that ribavirin acts clinically by promoting nonviable HCV RNA mutation rates. Finally, the latter result suggests that our replication model may be useful for identifying agents capable of driving replicating virus into error catastrophe.

scholarly journals Role of the 5′-Proximal Stem-Loop Structure of the 5′ Untranslated Region in Replication and Translation of Hepatitis C Virus RNA

2003 ◽  
Vol 77 (5) ◽  
pp. 3312-3318 ◽  
Author(s):  
Guangxiang Luo ◽  
Shaojie Xin ◽  
Zhaohui Cai
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Replication ◽  
Hcv Rna ◽  
Loop Structure ◽  
Nucleotide Substitutions ◽  
Stem Loop ◽  
Stem Loop Structure ◽  
Compensatory Mutations ◽  
Cell Colony

ABSTRACT Sequences of the untranslated regions at the 5′ and 3′ ends (5′UTR and 3′UTR) of the hepatitis C virus (HCV) RNA genome are highly conserved and contain cis-acting RNA elements for HCV RNA replication. The HCV 5′UTR consists of two distinct RNA elements, a short 5′-proximal stem-loop RNA element (nucleotides 1 to 43) and a longer element of internal ribosome entry site. To determine the sequence and structural requirements of the 5′-proximal stem-loop RNA element in HCV RNA replication and translation, a mutagenesis analysis was preformed by nucleotide deletions and substitutions. Effects of mutations in the 5′-proximal stem-loop RNA element on HCV RNA replication were determined by using a cell-based HCV replicon replication system. Deletion of the first 20 nucleotides from the 5′ end resulted in elimination of cell colony formation. Likewise, disruption of the 5′-proximal stem-loop by nucleotide substitutions abolished the ability of HCV RNA to induce cell colony formation. However, restoration of the 5′-proximal stem-loop by compensatory mutations with different nucleotides rescued the ability of the subgenomic HCV RNA to replicate in Huh7 cells. In addition, deletion and nucleotide substitutions of the 5′-proximal stem-loop structure, including the restored stem-loop by compensatory mutations, all resulted in reduction of translation by two- to fivefold, suggesting that the 5′-proximal stem-loop RNA element also modulates HCV RNA translation. These findings demonstrate that the 5′-proximal stem-loop of the HCV RNA is a cis-acting RNA element that regulates HCV RNA replication and translation.

scholarly journals Nonrandom Distribution of Hepatitis C Virus Quasispecies in Plasma and Peripheral Blood Mononuclear Cell Subsets

1999 ◽  
Vol 73 (11) ◽  
pp. 9213-9221 ◽  
Author(s):  
Anne Marie Roque Afonso ◽  
Jiaji Jiang ◽  
François Penin ◽  
Claire Tareau ◽  
Didier Samuel ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Peripheral Blood Cell ◽  
Hcv Rna ◽  
Structural Constraints ◽  
Nucleotide Substitutions ◽  
Peripheral Blood Mononuclear ◽  
Cellular Compartments

ABSTRACT The existence of an extrahepatic reservoir of hepatitis C virus (HCV) is suggested by differences in quasispecies composition between the liver, peripheral blood mononuclear cells, and serum. We studied HCV RNA compartmentalization in the plasma of nine patients, in CD19+, CD8+, and CD4+ positively selected cells, and also in the negatively selected cell fraction (NF). HCV RNA was detected in all plasma samples, in seven of nine CD19+, three of eight CD8+, and one of nine CD4+ cell samples, and in seven of eight NF cells. Cloning and sequencing of HVR1 in two patients showed a sequence grouping: quasispecies from a given compartment (all studied compartments for one patient and CD8+ and NF for the other) were statistically more genetically like each other than like quasispecies from any other compartment. The characteristics of amino acid and nucleotide substitutions suggested the same structural constraints on HVR1, even in very divergent strains from the cellular compartments, and homogeneous selection pressure on the different compartments. These findings demonstrate the compartmental distribution of HCV quasispecies within peripheral blood cell subsets and have important implications for the study of extrahepatic HCV replication and interaction with the immune system.

scholarly journals A Recombinant Hepatitis C Virus RNA-Dependent RNA Polymerase Capable of Copying the Full-Length Viral RNA

1999 ◽  
Vol 73 (9) ◽  
pp. 7694-7702 ◽  
Author(s):  
Jong-Won Oh ◽  
Takayoshi Ito ◽  
Michael M. C. Lai
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Polymerase ◽  
Rna Synthesis ◽  
De Novo ◽  
Full Length ◽  
Viral Rna ◽  
Hcv Rna ◽  
Independent Manner ◽  
Virus Rna

ABSTRACT All of the previously reported recombinant RNA-dependent RNA polymerases (RdRp), the NS5B enzymes, of hepatitis C virus (HCV) could function only in a primer-dependent and template-nonspecific manner, which is different from the expected properties of the functional viral enzymes in the cells. We have now expressed a recombinant NS5B that is able to synthesize a full-length HCV genome in a template-dependent and primer-independent manner. The kinetics of RNA synthesis showed that this RdRp can initiate RNA synthesis de novo and yield a full-length RNA product of genomic size (9.5 kb), indicating that it did not use the copy-back RNA as a primer. This RdRp was also able to accept heterologous viral RNA templates, including poly(A)- and non-poly(A)-tailed RNA, in a primer-independent manner, but the products in these cases were heterogeneous. The RdRp used some homopolymeric RNA templates only in the presence of a primer. By using the 3′-end 98 nucleotides (nt) of HCV RNA, which is conserved in all genotypes of HCV, as a template, a distinct RNA product was generated. Truncation of 21 nt from the 5′ end or 45 nt from the 3′ end of the 98-nt RNA abolished almost completely its ability to serve as a template. Inclusion of the 3′-end variable sequence region and the U-rich tract upstream of the X region in the template significantly enhanced RNA synthesis. The 3′ end of minus-strand RNA of HCV genome also served as a template, and it required a minimum of 239 nt from the 3′ end. These data defined the cis-acting sequences for HCV RNA synthesis at the 3′ end of HCV RNA in both the plus and minus senses. This is the first recombinant HCV RdRp capable of copying the full-length HCV RNA in the primer-independent manner expected of the functional HCV RNA polymerase.

scholarly journals Sphingomyelin Is Essential for the Structure and Function of the Double-Membrane Vesicles in Hepatitis C Virus RNA Replication Factories

2020 ◽  
Vol 94 (23) ◽  
Author(s):  
Hossam Gewaid ◽  
Haruyo Aoyagi ◽  
Minetaro Arita ◽  
Koichi Watashi ◽  
Ryosuke Suzuki ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Protein Production ◽  
Rna Viruses ◽  
Ectopic Expression ◽  
Membrane Vesicles ◽  
Hcv Rna ◽  
Independent System ◽  
Double Membrane ◽  
Transfer Protein

ABSTRACT Some plus-stranded RNA viruses generate double-membrane vesicles (DMVs), one type of the membrane replication factories, as replication sites. Little is known about the lipid components involved in the biogenesis of these vesicles. Sphingomyelin (SM) is required for hepatitis C virus (HCV) replication, but the mechanism of SM involvement remains poorly understood. SM biosynthesis starts in the endoplasmic reticulum (ER) and gives rise to ceramide, which is transported from the ER to the Golgi by the action of ceramide transfer protein (CERT), where it can be converted to SM. In this study, inhibition of SM biosynthesis, either by using small-molecule inhibitors or by knockout (KO) of CERT, suppressed HCV replication in a genotype-independent manner. This reduction in HCV replication was rescued by exogenous SM or ectopic expression of the CERT protein, but not by ectopic expression of nonfunctional CERT mutants. Observing low numbers of DMVs in stable replicon cells treated with a SM biosynthesis inhibitor or in CERT-KO cells transfected with either HCV replicon or with constructs that drive HCV protein production in a replication-independent system indicated the significant importance of SM to DMVs. The degradation of SM of the in vitro-isolated DMVs affected their morphology and increased the vulnerability of HCV RNA and proteins to RNase and protease treatment, respectively. Poliovirus, known to induce DMVs, showed decreased replication in CERT-KO cells, while dengue virus, known to induce invaginated vesicles, did not. In conclusion, these findings indicated that SM is an essential constituent of DMVs generated by some plus-stranded RNA viruses. IMPORTANCE Previous reports assumed that sphingomyelin (SM) is essential for HCV replication, but the mechanism was unclear. In this study, we showed for the first time that SM and ceramide transfer protein (CERT), which is in the SM biosynthesis pathway, are essential for the biosynthesis of double-membrane vesicles (DMVs), the sites of viral replication. Low numbers of DMVs were observed in CERT-KO cells transfected with replicon RNA or with constructs that drive HCV protein production in a replication-independent system. HCV replication was rescued by ectopic expression of the CERT protein, but not by CERT mutants, that abolishes the binding of CERT to vesicle-associated membrane protein-associated protein (VAP) or phosphatidylinositol 4-phosphate (PI4P), indicating new roles for VAP and PI4P in HCV replication. The biosynthesis of DMVs has great importance to replication by a variety of plus-stranded RNA viruses. Understanding of this process is expected to facilitate the development of diagnosis and antivirus.

scholarly journals Cell-Free Replication of the Hepatitis C Virus Subgenomic Replicon

2002 ◽  
Vol 76 (23) ◽  
pp. 12001-12007 ◽  
Author(s):  
Naushad Ali ◽  
Keith D. Tardif ◽  
Aleem Siddiqui
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Cultured Cells ◽  
Rnase A ◽  
Hcv Rna ◽  
Entry Site ◽  
Subgenomic Replicon ◽  
Replication System ◽  
Ribonucleoprotein Complexes ◽  
A Cell

ABSTRACT The hepatitis C virus (HCV) contains a plus-strand RNA genome. The 5′ noncoding region (NCR) of the viral genome functions as an internal ribosome entry site, and its unique 3′ NCR is required for the assembly of the replication complex during initiation of HCV RNA replication. Lohmann et al. (V. Lohmann, F. Korner, J.-O. Koch, U. Herian, L. Theilman, and R. Batenschlager, Science 285:110-113, 1999) developed a subgenomic HCV replicon system, which represents an important tool in studying HCV replication in cultured cells. In this study, we describe a cell-free replication system that utilizes cytoplasmic lysates prepared from Huh-7 cells harboring the HCV subgenomic replicons. These lysates, which contain ribonucleoprotein complexes associated with cellular membranes, were capable of incorporating [α32P]CTP into newly synthesized RNA from subgenomic replicons in vitro. Replicative forms (RFs) and replicative intermediates (RIs) were synthesized from the endogenous HCV RNA templates. Consistent with previous observations, RFs were found to be resistant to RNase A digestion, whereas RIs were sensitive to RNase treatment. The radiolabeled HCV RF-RI complexes contained both minus and plus strands and were specific to the lysates derived from replicon-expressing cells. The availability of a cell-free replication system offers opportunities to probe the mechanism(s) of HCV replication. It also provides a novel assay for potential therapeutic agents.

scholarly journals Efficient Replication of Hepatitis C Virus Genotype 1a RNAs in Cell Culture

2003 ◽  
Vol 77 (5) ◽  
pp. 3181-3190 ◽  
Author(s):  
Keril J. Blight ◽  
Jane A. McKeating ◽  
Joseph Marcotrigiano ◽  
Charles M. Rice
Keyword(s):  
Cell Culture ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Replication ◽  
Full Length ◽  
Hcv Rna ◽  
Metabolic Labeling ◽  
Virus Genotype ◽  
Adaptive Mutations ◽  
Genotype 1A

ABSTRACT Hepatitis C virus (HCV) genotype 1 (subtypes 1a and 1b) is responsible for the majority of treatment-resistant liver disease worldwide. Thus far, efficient HCV RNA replication has been observed only for subgenomic and full-length RNAs derived from genotype 1b isolates. Here, we report the establishment of efficient RNA replication systems for genotype 1a strain H77. Replication of subgenomic and full-length H77 1a RNAs required the highly permissive Huh-7.5 hepatoma subline and adaptive amino acid substitutions in both NS3 and NS5A. Replication could be detected by RNA quantification, fluorescence-activated cell sorting, and metabolic labeling of HCV-specific proteins. Replication efficiencies were similar for subgenomic and full-length RNAs and were most efficient for HCV RNAs lacking heterologous RNA elements. Interestingly, both subtype 1a and 1b NS3 adaptive mutations are surface exposed and present on only one face of the NS3 structure. The cell culture-adapted subtype 1a replicons should be useful for basic replication studies and for antiviral development. These results are also encouraging for the development of adapted replicons for the remaining HCV genotypes.

scholarly journals Long-Term Follow-Up of Chimpanzees Inoculated with the First Infectious Clone for Hepatitis C Virus

1999 ◽  
Vol 73 (4) ◽  
pp. 3317-3325 ◽  
Author(s):  
Marian E. Major ◽  
Kathleen Mihalik ◽  
Javier Fernandez ◽  
Jessica Seidman ◽  
David Kleiner ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Amino Acid ◽  
Immune Escape ◽  
Inflammatory Responses ◽  
Infectious Clone ◽  
Full Length ◽  
Single Amino Acid ◽  
Clonal Variation ◽  
Hcv Rna

ABSTRACT Two chimpanzees (Ch1535 and Ch1536) became infected with hepatitis C virus (HCV) following intrahepatic inoculation with RNA transcribed from a full-length cDNA clone of the virus. Both animals were persistently infected and have been followed for 60 weeks. They showed similar responses to infection, with transient liver enzyme elevations and liver inflammatory responses, which peaked at weeks 17 (Ch1535) and 12 (Ch1536) postinoculation (p.i.). Antibody responses to structural and nonstructural proteins were first detected at weeks 13 (Ch1535) and 10 (Ch1536) p.i. Serum RNA titers increased steadily during the first 10 to 13 weeks but decreased sharply in both animals following antibody and inflammatory responses. Despite direct evidence of humoral immune responses to multiple viral antigens, including hypervariable region 1 (HVR1), both animals remained chronically infected. Detailed sequence analysis of serum HCV RNA revealed no change in the majority HVR1 sequence in Ch1535 and a single-amino-acid mutation in Ch1536, with very little clonal variation in either animal. Full-length genome analysis at week 60 revealed several amino acid substitutions localized to antigens E1, E2, p7, NS3, and NS5. Of these, 55.6 and 40% were present as the majority sequence in serum RNA isolated at week 26 p.i. (Ch1535) and week 22 p.i. (Ch1536), respectively, and could represent immune escape mutations. Mutations accumulated at a rate of 1.57 × 10−3 and 1.48 × 10−3nucleotide substitutions/site/year for Ch1535 and Ch1536, respectively. Taken together, these data indicate that establishment of a persistent HCV infection in these chimpanzees is not due to changes in HVR1; however, the possibility remains that mutations arising in other parts of the genome contributed to this persistence.

scholarly journals Visualization of Positive and Negative Sense Viral RNA for Probing the Mechanism of Direct-Acting Antivirals against Hepatitis C Virus

Viruses ◽  
2019 ◽  
Vol 11 (11) ◽  
pp. 1039
Author(s):  
Dandan Liu ◽  
Philip R. Tedbury ◽  
Shuiyun Lan ◽  
Andrew D. Huber ◽  
Maritza N. Puray-Chavez ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Viruses ◽  
Drug Efficacy ◽  
Confocal Imaging ◽  
Hcv Rna ◽  
Direct Acting Antivirals ◽  
Infected Cells ◽  
Positive Strand Rna ◽  
Direct Acting

RNA viruses are highly successful pathogens and are the causative agents for many important diseases. To fully understand the replication of these viruses it is necessary to address the roles of both positive-strand RNA ((+)RNA) and negative-strand RNA ((−)RNA), and their interplay with viral and host proteins. Here we used branched DNA (bDNA) fluorescence in situ hybridization (FISH) to stain both the abundant (+)RNA and the far less abundant (−)RNA in both hepatitis C virus (HCV)- and Zika virus-infected cells, and combined these analyses with visualization of viral proteins through confocal imaging. We were able to phenotypically examine HCV-infected cells in the presence of uninfected cells and revealed the effect of direct-acting antivirals on HCV (+)RNA, (−)RNA, and protein, within hours of commencing treatment. Herein, we demonstrate that bDNA FISH is a powerful tool for the study of RNA viruses that can provide insights into drug efficacy and mechanism of action.

scholarly journals Effects of Mutations of the Initiation Nucleotides on Hepatitis C Virus RNA Replication in the Cell

2004 ◽  
Vol 78 (7) ◽  
pp. 3633-3643 ◽  
Author(s):  
Zhaohui Cai ◽  
T. Jake Liang ◽  
Guangxiang Luo
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Colony Formation ◽  
Rna Viruses ◽  
Rna Replication ◽  
Hcv Rna ◽  
Positive Strand ◽  
Negative Strand ◽  
Huh7 Cells ◽  
Cell Colony

ABSTRACT Replication of nearly all RNA viruses depends on a virus-encoded RNA-dependent RNA polymerase (RdRp). Our earlier work found that purified recombinant hepatitis C virus (HCV) RdRp (NS5B) was able to initiate RNA synthesis de novo by using purine (A and G) but not pyrimidine (C and U) nucleotides (G. Luo et al., J. Virol. 74:851-863, 2000). For most human RNA viruses, the initiation nucleotides of both positive- and negative-strand RNAs were found to be either an adenylate (A) or guanylate (G). To determine the nucleotide used for initiation and control of HCV RNA replication, a genetic mutagenesis analysis of the nucleotides at the very 5′ and 3′ ends of HCV RNAs was performed by using a cell-based HCV replicon replication system. Either a G or an A at the 5′ end of HCV genomic RNA was able to efficiently induce cell colony formation, whereas a nucleotide C at the 5′ end dramatically reduced the efficiency of cell colony formation. Likewise, the 3′-end nucleotide U-to-C mutation did not significantly affect the efficiency of cell colony formation. In contrast, a U-to-G mutation at the 3′ end caused a remarkable decrease in cell colony formation, and a U-to-A mutation resulted in a complete abolition of cell colony formation. Sequence analysis of the HCV replicon RNAs recovered from G418-resistant Huh7 cells revealed several interesting findings. First, the 5′-end nucleotide G of the replicon RNA was changed to an A upon multiple rounds of replication. Second, the nucleotide A at the 5′ end was stably maintained among all replicon RNAs isolated from Huh7 cells transfected with an RNA with a 5′-end A. Third, initiation of HCV RNA replication with a CTP resulted in a >10-fold reduction in the levels of HCV RNAs, suggesting that initiation of RNA replication with CTP was very inefficient. Fourth, the 3′-end nucleotide U-to-C and -G mutations were all reverted back to a wild-type nucleotide U. In addition, extra U and UU residues were identified at the 3′ ends of revertants recovered from Huh7 cells transfected with an RNA with a nucleotide G at the 3′ end. We also determined the 5′-end nucleotide of positive-strand RNA of some clinical HCV isolates. Either G or A was identified at the 5′ end of HCV RNA genome depending on the specific HCV isolate. Collectively, these findings demonstrate that replication of positive-strand HCV RNA was preferentially initiated with purine nucleotides (ATP and GTP), whereas the negative-strand HCV RNA replication is invariably initiated with an ATP.

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