Quantifying the Evolutionary Constraints and Potential of Hepatitis C Virus NS5A Protein

ABSTRACT RNA viruses, such as hepatitis C virus (HCV), influenza virus, and SARS-CoV-2, are notorious for their ability to evolve rapidly under selection in novel environments. It is known that the high mutation rate of RNA viruses can generate huge genetic diversity to facilitate viral adaptation. However, less attention has been paid to the underlying fitness landscape that represents the selection forces on viral genomes, especially under different selection conditions. Here, we systematically quantified the distribution of fitness effects of about 1,600 single amino acid substitutions in the drug-targeted region of NS5A protein of HCV. We found that the majority of nonsynonymous substitutions incur large fitness costs, suggesting that NS5A protein is highly optimized. The replication fitness of viruses is correlated with the pattern of sequence conservation in nature, and viral evolution is constrained by the need to maintain protein stability. We characterized the adaptive potential of HCV by subjecting the mutant viruses to selection by the antiviral drug daclatasvir at multiple concentrations. Both the relative fitness values and the number of beneficial mutations were found to increase with the increasing concentrations of daclatasvir. The changes in the spectrum of beneficial mutations in NS5A protein can be explained by a pharmacodynamics model describing viral fitness as a function of drug concentration. Overall, our results show that the distribution of fitness effects of mutations is modulated by both the constraints on the biophysical properties of proteins (i.e., selection pressure for protein stability) and the level of environmental stress (i.e., selection pressure for drug resistance). IMPORTANCE Many viruses adapt rapidly to novel selection pressures, such as antiviral drugs. Understanding how pathogens evolve under drug selection is critical for the success of antiviral therapy against human pathogens. By combining deep sequencing with selection experiments in cell culture, we have quantified the distribution of fitness effects of mutations in hepatitis C virus (HCV) NS5A protein. Our results indicate that the majority of single amino acid substitutions in NS5A protein incur large fitness costs. Simulation of protein stability suggests viral evolution is constrained by the need to maintain protein stability. By subjecting the mutant viruses to selection under an antiviral drug, we find that the adaptive potential of viral proteins in a novel environment is modulated by the level of environmental stress, which can be explained by a pharmacodynamics model. Our comprehensive characterization of the fitness landscapes of NS5A can potentially guide the design of effective strategies to limit viral evolution.

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Adaptive potential of a drug-targeted viral protein as a function of environmental stress

10.1101/078428 ◽

2016 ◽

Cited By ~ 1

Author(s):

Lei Dai ◽

Yushen Du ◽

Hangfei Qi ◽

Christian D. Huber ◽

Nicholas C. Wu ◽

...

Keyword(s):

Environmental Stress ◽

Rna Viruses ◽

Geometric Model ◽

Antiviral Drug ◽

Viral Fitness ◽

Single Amino Acid ◽

Adaptive Potential ◽

Beneficial Mutations ◽

Fitness Effects ◽

Ns5a Protein

AbstractRNA viruses are notorious for their ability to evolve rapidly under selection in novel environments. It is known that the high mutation rate of RNA viruses can generate huge genetic diversity to facilitate viral adaptation. However, less attention has been paid to the underlying fitness landscape that represents the selection forces on viral genomes. Here we systematically quantified the distribution of fitness effects (DFE) of about 1,600 single amino acid substitutions in the drug-targeted region of NS5A protein of Hepatitis C Virus (HCV). We found that the majority of non-synonymous substitutions incur large fitness costs, suggesting that NS5A protein is highly optimized in natural conditions. We characterized the adaptive potential of HCV by subjecting the mutant viruses to selection by the antiviral drug Daclatasvir. Both the selection coefficient and the number of beneficial mutations are found to increase with the level of environmental stress, which is modulated by the concentration of Daclatasvir. The changes in the spectrum of beneficial mutations in NS5A protein can be explained by a pharmacodynamics model describing viral fitness as a function of drug concentration. We test theoretical predictions regarding the distribution of beneficial fitness effects of mutations. We also interpret the data in the context of Fisher’s Geometric Model and find an increased distance to optimum as a function of environmental stress. Finally, we show that replication fitness of viruses is correlated with the pattern of sequence conservation in nature and viral evolution is constrained by the need to maintain protein stability.

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Pim Kinase Interacts with Nonstructural 5A Protein and Regulates Hepatitis C Virus Entry

Journal of Virology ◽

10.1128/jvi.01707-15 ◽

2015 ◽

Vol 89 (19) ◽

pp. 10073-10086 ◽

Cited By ~ 24

Author(s):

Chorong Park ◽

Saehong Min ◽

Eun-Mee Park ◽

Yun-Sook Lim ◽

Sangmin Kang ◽

...

Keyword(s):

Life Cycle ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Protein Stability ◽

Kinase Activity ◽

Cellular Proteins ◽

Physical Interaction ◽

Serine Threonine Kinase ◽

Threonine Kinase ◽

Ns5a Protein

ABSTRACTThe life cycle of hepatitis C virus (HCV) is highly dependent on host cellular proteins for virus propagation. In order to identify the cellular factors involved in HCV propagation, we performed protein microarray assay using the HCV nonstructural 5A (NS5A) protein as a probe. Of ∼9,000 human cellular proteins immobilized in a microarray, approximately 90 cellular proteins were identified as NS5A interactors. Of these candidates, Pim1, a member of serine/threonine kinase family composed of three different isoforms (Pim1, Pim2, and Pim3), was selected for further study. Pim kinases share a consensus sequence which overlaps with kinase activity. Pim kinase activity has been implicated in tumorigenesis. In the present study, we verified the physical interaction between NS5A and Pim1 by bothin vitropulldown and coimmunoprecipitation assays. Pim1 interacted with NS5A through amino acid residues 141 to 180 of Pim1. We demonstrated that protein stability of Pim1 was increased by NS5A protein and this increase was mediated by protein interplay. Small interfering RNA (siRNA)-mediated knockdown or pharmacological inhibition of Pim kinase abrogated HCV propagation. By employing HCV pseudoparticle entry and single-cycle HCV infection assays, we further demonstrated that Pim kinase was involved in HCV entry at a postbinding step. These data suggest that Pim kinase may represent a new host factor for HCV entry.IMPORTANCEPim1 is an oncogenic serine/threonine kinase. HCV NS5A protein physically interacts with Pim1 and contributes to Pim1 protein stability. Since Pim1 protein expression level is upregulated in many cancers, NS5A-mediated protein stability may be associated with HCV pathogenesis. Either gene silencing or chemical inhibition of Pim kinase abrogated HCV propagation in HCV-infected cells. We further showed that Pim kinase was specifically required at an early entry step of the HCV life cycle. Thus, we have identified Pim kinase not only as an HCV cell entry factor but also as a new anti-HCV therapeutic target.

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Nonstructural 5A Protein of Hepatitis C Virus Regulates Soluble Resistance-Related Calcium-Binding Protein Activity for Viral Propagation

Journal of Virology ◽

10.1128/jvi.02493-15 ◽

2015 ◽

Vol 90 (6) ◽

pp. 2794-2805 ◽

Cited By ~ 7

Author(s):

Giao V. Q. Tran ◽

Trang T. D. Luong ◽

Eun-Mee Park ◽

Jong-Wook Kim ◽

Jae-Woong Choi ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Protein Interaction ◽

Calcium Binding ◽

Crucial Role ◽

Binding Protein ◽

Binding Activity ◽

Calcium Binding Protein ◽

Virus Propagation ◽

Ns5a Protein

ABSTRACTHepatitis C virus (HCV) is a major cause of chronic liver disease and is highly dependent on cellular proteins for virus propagation. To identify the cellular factors involved in HCV propagation, we recently performed protein microarray assays using the HCV nonstructural 5A (NS5A) protein as a probe. Of 90 cellular protein candidates, we selected the soluble resistance-related calcium-binding protein (sorcin) for further characterization. Sorcin is a calcium-binding protein and is highly expressed in certain cancer cells. We verified that NS5A interacted with sorcin through domain I of NS5A, and phosphorylation of the threonine residue 155 of sorcin played a crucial role in protein interaction. Small interfering RNA (siRNA)-mediated knockdown of sorcin impaired HCV propagation. Silencing of sorcin expression resulted in a decrease of HCV assembly without affecting HCV RNA and protein levels. We further demonstrated that polo-like kinase 1 (PLK1)-mediated phosphorylation of sorcin was increased by NS5A. We showed that both phosphorylation and calcium-binding activity of sorcin were required for HCV propagation. These data indicate that HCV modulates sorcin activity via NS5A protein for its own propagation.IMPORTANCESorcin is a calcium-binding protein and regulates intracellular calcium homeostasis. HCV NS5A interacts with sorcin, and phosphorylation of sorcin is required for protein interaction. Gene silencing of sorcin impaired HCV propagation at the assembly step of the HCV life cycle. Sorcin is phosphorylated by PLK1 via protein interaction. We showed that sorcin interacted with both NS5A and PLK1, and PLK1-mediated phosphorylation of sorcin was increased by NS5A. Moreover, calcium-binding activity of sorcin played a crucial role in HCV propagation. These data provide evidence that HCV regulates host calcium metabolism for virus propagation, and thus manipulation of sorcin activity may represent a novel therapeutic target for HCV.

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The hepatitis C virus NS5A protein and response to interferon α: mutational analyses in patients with chronic HCV genotype 3a infection from India

Medical Microbiology and Immunology ◽

10.1007/s00430-006-0024-z ◽

2006 ◽

Vol 196 (1) ◽

pp. 11-21 ◽

Cited By ~ 7

Author(s):

Ankur Goyal ◽

Wolf P. Hofmann ◽

Eva Hermann ◽

Stella Traver ◽

Syed S. Hissar ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Interferon Α ◽

Hcv Genotype ◽

Ns5a Protein ◽

Chronic Hcv ◽

Genotype 3A

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TRIM14 inhibits hepatitis C virus infection by SPRY domain-dependent targeted degradation of the viral NS5A protein

Scientific Reports ◽

10.1038/srep32336 ◽

2016 ◽

Vol 6 (1) ◽

Cited By ~ 29

Author(s):

Shanshan Wang ◽

Yongzhi Chen ◽

Chunfeng Li ◽

Yaoxing Wu ◽

Lei Guo ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Virus Infection ◽

Hepatitis C Virus Infection ◽

Spry Domain ◽

Ns5a Protein

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Binding-Site Identification and Genotypic Profiling of Hepatitis C Virus Polymerase Inhibitors

Journal of Virology ◽

10.1128/jvi.01543-06 ◽

2007 ◽

Vol 81 (13) ◽

pp. 6909-6919 ◽

Cited By ~ 53

Author(s):

Frederik Pauwels ◽

Wendy Mostmans ◽

Ludo M. M. Quirynen ◽

Liesbet van der Helm ◽

Carlo W. Boutton ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Binding Site ◽

Antiviral Drug ◽

Variable Binding ◽

Polymerase Inhibitors ◽

Binding Site Identification ◽

Virus Genotypes ◽

Binding Pockets ◽

Virus Mutant

ABSTRACT The search for hepatitis C virus polymerase inhibitors has resulted in the identification of several nonnucleoside binding pockets. The shape and nature of these binding sites differ across and even within diverse hepatitis C virus genotypes. These differences confront antiviral drug discovery with the challenge of finding compounds that are capable of inhibition in variable binding pockets. To address this, we have established a hepatitis C virus mutant and genotypic recombinant polymerase panel as a means of guiding medicinal chemistry through the elucidation of the site of action of novel inhibitors and profiling against genotypes. Using a genotype 1b backbone, we demonstrate that the recombinant P495L, M423T, M414T, and S282T mutant enzymes can be used to identify the binding site of an acyl pyrrolidine analog. We assess the inhibitory activity of this analog and other nonnucleoside inhibitors with our panel of enzyme isolates generated from clinical sera representing genotypes 1a, 1b, 2a, 2b, 3a, 4a, 5a, and 6a.

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Escape from HLA-B*08-Restricted CD8 T Cells by Hepatitis C Virus Is Associated with Fitness Costs

Journal of Virology ◽

10.1128/jvi.00997-08 ◽

2008 ◽

Vol 82 (23) ◽

pp. 11803-11812 ◽

Cited By ~ 35

Author(s):

Shadi Salloum ◽

Cesar Oniangue-Ndza ◽

Christoph Neumann-Haefelin ◽

Laura Hudson ◽

Silvia Giugliano ◽

...

Keyword(s):

T Cells ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Viral Replication ◽

Consensus Sequence ◽

Acute Infection ◽

Spontaneous Clearance ◽

Fitness Costs ◽

Genotype 1B ◽

The Impact

ABSTRACT The inherent sequence diversity of the hepatitis C virus (HCV) represents a major hurdle for the adaptive immune system to control viral replication. Mutational escape within targeted CD8 epitopes during acute HCV infection has been well documented and is one possible mechanism for T-cell failure. HLA-B*08 was recently identified as one HLA class I allele associated with spontaneous clearance of HCV replication. Selection of escape mutations in the immunodominant HLA-B*08-restricted epitope HSKKKCDEL1395-1403 was observed during acute infection. However, little is known about the impact of escape mutations in this epitope on viral replication capacity. Their previously reported reversion back toward the consensus residue in patients who do not possess the B*08 allele suggests that the consensus sequence in this epitope is advantageous for viral replication in the absence of immune pressure. The aim of this study was to determine the impact of mutational escape from this immunodominant epitope on viral replication. We analyzed it with a patient cohort with chronic HCV genotype 1b infection and in a single-source outbreak (genotype 1b). Sequence changes in this highly conserved region are rare and selected almost exclusively in the presence of the HLA-B*08 allele. When tested in the subgenomic replicon (Con1), the observed mutations reduce viral replication compared with the prototype sequence. The results provide direct evidence that escape mutations in this epitope are associated with fitness costs and that the antiviral effect of HLA-B*08-restricted T cells is sufficiently strong to force the virus to adopt a relatively unfavorable sequence.

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Hepatitis C Virus NS5A Protein Promotes the Lysosomal Degradation of Hepatocyte Nuclear Factor 1α via Chaperone-Mediated Autophagy

Journal of Virology ◽

10.1128/jvi.00639-18 ◽

2018 ◽

Vol 92 (13) ◽

Cited By ~ 6

Author(s):

Chieko Matsui ◽

Lin Deng ◽

Nanae Minami ◽

Takayuki Abe ◽

Kazuhiko Koike ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Nuclear Factor ◽

Hepatocyte Nuclear Factor ◽

Lysosomal Degradation ◽

Selective Degradation ◽

Ns5a Protein ◽

Infected Cells ◽

Nuclear Factor 1 ◽

Chaperone Mediated Autophagy

ABSTRACT Hepatitis C virus (HCV) infection is closely associated with type 2 diabetes. We reported that HCV infection induces the lysosomal degradation of hepatocyte nuclear factor 1 alpha (HNF-1α) via interaction with HCV nonstructural protein 5A (NS5A) protein, thereby suppressing GLUT2 gene expression. The molecular mechanisms of selective degradation of HNF-1α caused by NS5A are largely unknown. Chaperone-mediated autophagy (CMA) is a selective lysosomal degradation pathway. Here, we investigated whether CMA is involved in the selective degradation of HNF-1α in HCV-infected cells and observed that the pentapeptide spanning from amino acid (aa) 130 to aa 134 of HNF-1α matches the rule for the CMA-targeting motif, also known as KFERQ motif. A cytosolic chaperone protein, heat shock cognate protein of 70 kDa (HSC70), and a lysosomal membrane protein, lysosome-associated membrane protein type 2A (LAMP-2A), are key components of CMA. Immunoprecipitation analysis revealed that HNF-1α was coimmunoprecipitated with HSC70, whereas the Q130A mutation (mutation of Q to A at position 130) of HNF-1α disrupted the interaction with HSC70, indicating that the CMA-targeting motif of HNF-1α is important for the association with HSC70. Immunoprecipitation analysis revealed that increasing amounts of NS5A enhanced the association of HNF-1α with HSC70. To determine whether LAMP-2A plays a role in the degradation of HNF-1α protein, we knocked down LAMP-2A mRNA by RNA interference; this knockdown by small interfering RNA (siRNA) recovered the level of HNF-1α protein in HCV J6/JFH1-infected cells. This result suggests that LAMP-2A is required for the degradation of HNF-1α. Immunofluorescence study revealed colocalization of NS5A and HNF-1α in the lysosome. Based on our findings, we propose that HCV NS5A interacts with HSC70 and recruits HSC70 to HNF-1α, thereby promoting the lysosomal degradation of HNF-1α via CMA. IMPORTANCE Many viruses use a protein degradation system, such as the ubiquitin-proteasome pathway or the autophagy pathway, for facilitating viral propagation and viral pathogenesis. We investigated the mechanistic details of the selective lysosomal degradation of hepatocyte nuclear factor 1 alpha (HNF-1α) induced by hepatitis C virus (HCV) NS5A protein. Using site-directed mutagenesis, we demonstrated that HNF-1α contains a pentapeptide chaperone-mediated autophagy (CMA)-targeting motif within the POU-specific domain of HNF-1α. The CMA-targeting motif is important for the association with HSC70. LAMP-2A is required for degradation of HNF-1α caused by NS5A. We propose that HCV NS5A interacts with HSC70, a key component of the CMA machinery, and recruits HSC70 to HNF-1α to target HNF-1α for CMA-mediated lysosomal degradation, thereby facilitating HCV pathogenesis. We discovered a role of HCV NS5A in CMA-dependent degradation of HNF-1α. Our results may lead to a better understanding of the role of CMA in the pathogenesis of HCV.

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