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Akt Phosphorylation of Hepatitis C Virus NS5B Regulates Polymerase Activity and Hepatitis C Virus Infection

ABSTRACT: Hepatitis C virus (HCV) is a single-stranded RNA virus of positive polarity [ssRNA(+)] that replicates its genome through the activity of one of its proteins, called NS5B. This viral protein is responsible for copying the positive-polarity RNA genome into a negative-polarity RNA strand, which will be the template for new positive-polarity RNA genomes. The NS5B protein is phosphorylated by cellular kinases, including Akt. In this work, we have identified several amino acids of NS5B that are phosphorylated by Akt, with positions S27, T53, T267, and S282 giving the most robust results. Site-directed mutagenesis of these residues to mimic (Glu mutants) or prevent (Ala mutants) their phosphorylation resulted in a reduced NS5B in vitro RNA polymerase activity, except for the T267E mutant, the only non-conserved position of all those that are phosphorylated. In addition, in vitro transcribed RNAs derived from HCV complete infectious clones carrying mutations T53E/A and S282E/A were transfected in Huh-7.5 permissive cells, and supernatant viral titers were measured at 6 and 15 days post-transfection. No virus was rescued from the mutants except for T53A at 15 days post-transfection whose viral titer was statistically lower as compared to the wild type. Therefore, phosphorylation of NS5B by cellular kinases is a mechanism of viral polymerase inactivation. Whether this inactivation is a consequence of interaction with cellular kinases or a way to generate inactive NS5B that may have other functions are questions that need further experimental work.

SUBMITTER: Sabariegos R

PROVIDER: S-EPMC8570118 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Viral diversity is an important feature of hepatitis C virus (HCV) infection and an important predictor of disease progression and treatment response. HIV/HCV co-infection is associated with enhanced HCV replication, increased fibrosis, and the development of liver disease. HIV also increases quasispecies diversity of HCV structural genes, although limited data are available regarding the impact of HIV on non-structural genes of HCV, particularly in the absence of direct-acting therapies. The genetic diversity and presence of drug resistance mutations within the RNA-dependent RNA polymerase (NS5B) gene were examined in 3 groups of women with HCV genotype 1a infection, including those with HCV mono-infection, antiretroviral (ART)-naïve women with HIV/HCV co-infection and CD4 cell count <350 cells/mm3, and ART-naïve women with HIV/HCV co-infection and CD4 cell count ≥350 cells/mm3. None had ever been treated for HCV infection. There was evidence of significant diversity across the entire NS5B gene in all women. There were several nucleotides and amino acids with distinct distributions across the three study groups, although no obvious clustering of NS5B sequences was observed based on HIV co-infection or CD4 cell count. Polymorphisms at amino acid positions associated with resistance to dasabuvir and sofosbuvir were limited, although the Q309R variant associated with ribavirin resistance was present in 12 individuals with HCV mono-infection, 8 HIV/HCV co-infected individuals with CD4 <350 cells/mm3, and 12 HIV/HCV co-infected individuals with CD4 ≥350 cells/mm3. Previously reported fitness altering mutations were rare. CD8+ T cell responses against the human leukocyte antigen (HLA) B57-restricted epitopes NS5B2629-2637 and NS5B2936-2944 are critical for HCV control and were completely conserved in 44 (51.8%) and 70 (82.4%) study participants. These data demonstrate extensive variation across the NS5B gene. Genotypic variation may have a profound impact on HCV replication and pathogenesis and deserves careful evaluation.

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Akt Phosphorylation of Hepatitis C Virus NS5B Regulates Polymerase Activity and Hepatitis C Virus Infection

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