Dataset Information

Dynamic imaging of the hepatitis C virus NS5A protein during a productive infection.

ABSTRACT:

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Hepatitis C virus (HCV) NS5A is essential for viral genome replication within cytoplasmic replication complexes and virus assembly at the lipid droplet (LD) surface, although its definitive functions are poorly understood. We developed approaches to investigate NS5A dynamics during a productive infection. We report here that NS5A motility and efficient HCV RNA replication require the microtubule network and the cytoplasmic motor dynein and demonstrate that both motile and relatively static NS5A-positive foci are enriched with host factors VAP-A and Rab5A. Pulse-chase imaging revealed that newly synthesized NS5A foci are small and distinct from aged foci, while further studies using a unique dual fluorescently tagged infectious HCV chimera showed a relatively stable association of NS5A foci with core-capped LDs. These results reveal new details about the dynamics and maturation of NS5A and the nature of potential sites of convergence of HCV replication and assembly pathways.

Importance

Hepatitis C virus (HCV) is a major cause of serious liver disease worldwide. An improved understanding of the HCV replication cycle will enable development of novel and improved antiviral strategies. Here we have developed complementary fluorescent labeling and imaging approaches to investigate the localization, traffic and interactions of the HCV NS5A protein in living, virus-producing cells. These studies reveal new details as to the traffic, composition and biogenesis of NS5A foci and the nature of their association with putative sites of virus assembly.

SUBMITTER: Eyre NS

PROVIDER: S-EPMC3993538 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature

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Dynamic imaging of the hepatitis C virus NS5A protein during a productive infection.

Eyre Nicholas S NS Fiches Guillaume N GN Aloia Amanda L AL Helbig Karla J KJ McCartney Erin M EM McErlean Christopher S P CS Li Kui K Aggarwal Anupriya A Turville Stuart G SG Beard Michael R MR

Journal of virology 20140115 7

<h4>Unlabelled</h4>Hepatitis C virus (HCV) NS5A is essential for viral genome replication within cytoplasmic replication complexes and virus assembly at the lipid droplet (LD) surface, although its definitive functions are poorly understood. We developed approaches to investigate NS5A dynamics during a productive infection. We report here that NS5A motility and efficient HCV RNA replication require the microtubule network and the cytoplasmic motor dynein and demonstrate that both motile and rela ...[more]

PMID: 24429364

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Project description: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.

Project description: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.

Dataset Information

Dynamic imaging of the hepatitis C virus NS5A protein during a productive infection.

Unlabelled

Importance

Publications

Dynamic imaging of the hepatitis C virus NS5A protein during a productive infection.

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