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

2015 ◽  
Vol 89 (19) ◽  
pp. 10073-10086 ◽  
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.

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

mSystems ◽  
2021 ◽  
Vol 6 (2) ◽  
Author(s):  
Lei Dai ◽  
Yushen Du ◽  
Hangfei Qi ◽  
Christian D. Huber ◽  
Dongdong Chen ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Protein Stability ◽  
Viral Evolution ◽  
Antiviral Drug ◽  
Single Amino Acid ◽  
Fitness Costs ◽  
Adaptive Potential ◽  
Fitness Effects ◽  
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.

scholarly journals Phosphorylation of Serine 225 in Hepatitis C Virus NS5A Regulates Protein-Protein Interactions

2017 ◽  
Vol 91 (17) ◽  
Author(s):  
Niluka Goonawardane ◽  
Anna Gebhardt ◽  
Christopher Bartlett ◽  
Andreas Pichlmair ◽  
Mark Harris
Keyword(s):  
Life Cycle ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Molecular Mechanisms ◽  
Nonstructural Protein ◽  
Antiviral Drugs ◽  
Cellular Proteins ◽  
Proximity Ligation Assay ◽  
Genome Replication ◽  
Virus Life Cycle

ABSTRACT Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is a phosphoprotein that plays key, yet poorly defined, roles in both virus genome replication and virion assembly/release. It has been proposed that differential phosphorylation could act as a switch to regulate the various functions of NS5A; however, the mechanistic details of the role of this posttranslational modification in the virus life cycle remain obscure. We previously reported (D. Ross-Thriepland, J. Mankouri, and M. Harris, J Virol 89:3123–3135, 2015, doi:10.1128/JVI.02995-14) a role for phosphorylation at serine 225 (S225) of NS5A in the regulation of JFH-1 (genotype 2a) genome replication. A phosphoablatant (S225A) mutation resulted in a 10-fold reduction in replication and a perinuclear restricted distribution of NS5A, whereas the corresponding phosphomimetic mutation (S225D) had no phenotype. To determine the molecular mechanisms underpinning this phenotype we conducted a label-free proteomics approach to identify cellular NS5A interaction partners. This analysis revealed that the S225A mutation disrupted the interactions of NS5A with a number of cellular proteins, in particular the nucleosome assembly protein 1-like protein 1 (NAP1L1), bridging integrator 1 (Bin1, also known as amphiphysin II), and vesicle-associated membrane protein-associated protein A (VAP-A). These interactions were validated by immunoprecipitation/Western blotting, immunofluorescence, and proximity ligation assay. Importantly, small interfering RNA (siRNA)-mediated knockdown of NAP1L1, Bin1 or VAP-A impaired viral genome replication and recapitulated the perinuclear redistribution of NS5A seen in the S225A mutant. These results demonstrate that S225 phosphorylation regulates the interactions of NS5A with a defined subset of cellular proteins. Furthermore, these interactions regulate both HCV genome replication and the subcellular localization of replication complexes. IMPORTANCE Hepatitis C virus is an important human pathogen. The viral nonstructural 5A protein (NS5A) is the target for new antiviral drugs. NS5A has multiple functions during the virus life cycle, but the biochemical details of these roles remain obscure. NS5A is known to be phosphorylated by cellular protein kinases, and in this study, we set out to determine whether this modification is required for the binding of NS5A to other cellular proteins. We identified 3 such proteins and show that they interacted only with NS5A that was phosphorylated on a specific residue. Furthermore, these proteins were required for efficient virus replication and the ability of NS5A to spread throughout the cytoplasm of the cell. Our results help to define the function of NS5A and may contribute to an understanding of the mode of action of the highly potent antiviral drugs that are targeted to NS5A.

scholarly journals The hepatitis C virus NS5A protein binds to members of the Src family of tyrosine kinases and regulates kinase activity

2004 ◽  
Vol 85 (3) ◽  
pp. 721-729 ◽  
Author(s):  
Andrew Macdonald ◽  
Katherine Crowder ◽  
Andrew Street ◽  
Christopher McCormick ◽  
Mark Harris
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Tyrosine Kinases ◽  
Kinase Activity ◽  
Ns5a Protein

scholarly journals Multiple effects of toxins isolated from Crotalus durissus terrificus on the hepatitis C virus life cycle

PLoS ONE ◽  
2017 ◽  
Vol 12 (11) ◽  
pp. e0187857 ◽  
Author(s):  
Jacqueline Farinha Shimizu ◽  
Carina Machado Pereira ◽  
Cintia Bittar ◽  
Mariana Nogueira Batista ◽  
Guilherme Rodrigues Fernandes Campos ◽  
...  
Keyword(s):  
Life Cycle ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Crotalus Durissus Terrificus ◽  
Virus Life Cycle ◽  
Crotalus Durissus

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

2015 ◽  
Vol 90 (6) ◽  
pp. 2794-2805 ◽  
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.

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

2006 ◽  
Vol 196 (1) ◽  
pp. 11-21 ◽  
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

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

2016 ◽  
Vol 6 (1) ◽  
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

Increased amount of a 25-kilodalton phosphoprotein after v-mos transfection of CHO cells

1988 ◽  
Vol 8 (11) ◽  
pp. 4685-4691
Author(s):  
J K Mayo ◽  
K E Sampson ◽  
L D Adams ◽  
E R Crumm ◽  
S L Kelly ◽  
...  
Keyword(s):  
Growth Rate ◽  
Cho Cells ◽  
Kinase Activity ◽  
Chinese Hamster ◽  
Temperature Sensitive ◽  
Serine Threonine Kinase ◽  
Dependent Protein Kinase ◽  
Threonine Kinase ◽  
Dependent Protein ◽  
A Cell

We transfected Chinese hamster ovary (CHO) cells with a cloned v-mos gene (pHT25). The mos family of oncogenes has previously been shown to have serine-threonine kinase activity. This kinase activity may be required for oncogenic transformation, although its exact biological role is unknown. We found that the transfected cells had an altered morphology, a slower doubling time, and an apparent increase in the amount of a 25-kilodalton (kDa) phosphoprotein that appeared to be of low abundance. Transfection of CHO cells with a cloned temperature-sensitive mos gene (ts159) led to isolation of a cell line that showed the presence of the 25-kDa phosphoprotein at the permissive but not at the nonpermissive temperature, suggesting a direct relationship between mos activity and the presence of this phosphoprotein. The characteristics of altered morphology and depressed growth rate were reminiscent of changes seen after the activation of the cyclic AMP-dependent protein kinase (PKA) in CHO cells. However, PKA activation did not stimulate phosphorylation of this 25-kDa protein, nor was there a change in total PKA activity in these cells. We suggest that the increased presence of the 25-kDa phosphoprotein is a consequence of the v-mos transfection and that it may be involved in the change of morphology and growth rate seen in the CHO cells. Phosphorylation of this protein may be a useful marker of mos and have some functional importance in the transformation of cells by the v-mos oncogene.

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