BIOLOGICAL AND BIOCHEMICAL STUDIES OF HCV NS5A PROTEIN: FUNCTIONS IN GENOME REPLICATION AND ANTAGONISM OF THE INNATE IMMUNE RESPONSE
Open Access
- Author:
- Hwang, Jungwook
- Graduate Program:
- Integrative Biosciences
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- November 13, 2007
- Committee Members:
- Craig Eugene Cameron, Committee Chair/Co-Chair
Philip C. Bevilacqua, Committee Member
Harriet C Isom, Committee Member
Kouacou Konan, Committee Member
Joseph C. Reese, Committee Member - Keywords:
- HCV
NS5A
PKA phosphorylation
RNA binding protein
PKR - Abstract:
- HCV is an enveloped positive-stranded RNA virus that infects 3% of the world population. It has four structural proteins and six non-structural proteins with untranslated regions at the 5’ and 3’ termini. Functions for all proteins in HCV have been reported except for p7 and non-structural protein (NS5A). NS5A is a phosphoprotein that has two phosphorylated forms (p56 and p58). Although the roles of NS5A are not completely understood, properties have been reported in numerous papers. NS5A has two roles; genomic replication and host cellular protein interactions. And these functions are switched by the level of phosphorylation on NS5A. The hyperphosphorylated form (p58) is suggested to interfere with cellular signaling and the un- or hypophosphorylated form (p56) is involved in genomic replication. In this study, we mapped the minimal domain of NS5A for RNA binding. The N-terminus of NS5A is required for RNA binding. The interface of the dimer has positive electrostatic potential that is sufficient to accommodate an RNA helix. NS5A-RNA binding is dependent on electrostatic interactions and ionic strength. NS5A binds RNA at neutral and low pH, but not at high pH or high salt concentration (300 mM). NS5A is able to form oligomer. Structural feature of dimerized NS5A has enough space for RNA binding and, interestingly, positive charged amino acids in the bottom and negative charged in the side of groove, suggesting that this space can be a putative groove for RNA binding. In addition, glutaraldehyde and UV crosslinking studies reveal that monomeric and dimeric NS5A bind RNA. NS5A is phosphorylated by cellular kinases including cAMP-dependent kinase A (PKA). We identified a PKA phosphorylation site on NS5A and examined the effects of PKA phosphorylation on HCV genomic replication. PKA phosphorylation enhances the accumulation of subgenomic replication during cell divisions and modulates cleaved NS5A subcellular localization. In order to prevent viral spread and expansion, an infected cell recognizes foreign agents through various mechanisms. One of the systems is the PKR pathway, which turns off cellular and viral translation by sensing and binding double-stranded RNA (dsRNA). Herein, we show that single-stranded RNA (ssRNA) is also able to activate PKR and this activation requires a 5’-triphosphate on the RNA. PKR cannot be activated by 7-methyl guanine capped or hydroxylated RNA, suggesting that PKR differentiates between cellular RNA and foreign RNA. HCV NS5A has the ability to prevent PKR autophosphorylation induced by dsRNA and ssRNA. Deciphering the NS5A functions in genomic replication and PKR responses to foreign RNA will provide significant practical applications as well as insight into replication and innate immune response mechanism. Furthermore, this study will contribute to developing strategies for antiviral drug targets.