Regulation of hepatitis C virus NS5A protein function by host-mediated phosphorylation

Open Access
Author:
Cordek, Daniel Girard
Graduate Program:
Biochemistry, Microbiology, and Molecular Biology
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
September 19, 2012
Committee Members:
  • Craig Eugene Cameron, Dissertation Advisor
  • Craig Eugene Cameron, Committee Chair
  • Paul Lee Babitzke, Committee Member
  • Philip C. Bevilacqua, Committee Member
  • Andrey S Krasilnikov, Committee Member
  • Lorraine C Santy, Committee Member
Keywords:
  • hepatitis C virus
  • HCV
  • NS5A
  • phosphorylation
  • intrinsic disorder
  • IDP
  • RNA-binding
Abstract:
We have established a paradigm for studying host-mediated phosphorylation of hepatitis C virus (HCV) nonstructural protein 5A (NS5A), from mapping phosphorylated residues on the viral protein, to biochemical analysis of these modifications in vitro, to verification of biological significance via cell culture- and human liver-based assays. Using this approach, we have determined protein kinase A (PKA)-mediated phosphorylation of NS5A impacts accumulation of HCV RNA and other viral proteins. Through the use of immunological reagents designed to specifically detect PKA-phosphorylated NS5A, we have shown this modified form of the protein is a subset of all NS5A present in cells stably replicating HCV RNA. Further, as the amount of PKA-phosphorylated NS5A correlated to the severity of liver fibrosis in biopsy samples, this may be a novel, noninvasive biomarker for the staging of HCV-induced liver disease. Our analysis of NS5A phosphorylation by casein kinase 1α (CK1α) has identified the modification of a previously unidentified serine residue as critical to the formation of the hyperphosphorylated form of NS5A in vitro, the first time this elusive species of the viral protein has ever been systematically and reproducibly generated solely with NS5A and CK1α. Loss of this phosphorylation via phosphoablative substitutions in the HCV subgenomic replicon permitted persistent replication in the absence of previously identified cell culture adaptive mutations. Finally, we provide biochemical analysis suggesting that domain I of the viral protein may serve as a kinase recruitment domain. Truncations of NS5A lacking domain I were phosphorylated less efficiently than full-length forms of NS5A, despite the absence of phospho-residues in this region of the viral protein. RNA-induced homodimerization of domain I further inhibited phosphorylation of full-length NS5A, perhaps by blocking residues involved in kinase recruitment.