6533b7d7fe1ef96bd1268453

RESEARCH PRODUCT

Modulation of Hepatitis C Virus NS5A Hyperphosphorylation by Nonstructural Proteins NS3, NS4A, and NS4B

subject

description

NS5A of the hepatitis C virus (HCV) is a highly phosphorylated protein involved in resistance against interferon and required most likely for replication of the viral genome. Phosphorylation of this protein is mediated by a cellular kinase(s) generating multiple proteins with different electrophoretic mobilities. In the case of the genotype 1b isolate HCV-J, in addition to the basal phosphorylated NS5A (designated pp56), a hyperphosphorylated form (pp58) was found on coexpression of NS4A (T. Kaneko, Y. Tanji, S. Satoh, M. Hijikata, S. Asabe, K. Kimura, and K. Shimotohno, Biochem. Biophys. Res. Commun. 205:320‐326, 1994). Using a comparative analysis of two full-length genomes of genotype 1b, competent or defective for NS5A hyperphosphorylation, we investigated the requirements for this NS5A modification. We found that hyperphosphorylation occurs when NS5A is expressed as part of a continuous NS3-5A polyprotein but not when it is expressed on its own or trans complemented with one or several other viral proteins. Results obtained with chimeras of both genomes show that single amino acid substitutions within NS3 that do not affect polyprotein cleavage can enhance or reduce NS5A hyperphosphorylation. Furthermore, mutations in the central or carboxy-terminal NS4A domain as well as small deletions in NS4B can also reduce or block hyperphosphorylation without affecting polyprotein processing. These requirements most likely reflect the formation of a highly ordered NS3-5A multisubunit complex responsible for the differential phosphorylation of NS5A and probably also for modulation of its biological activities. Hepatitis C virus (HCV) is the major causative agent of sporadic and transfusion-associated non-A, non-B hepatitis (10, 24). Although most infections are inapparent or initially associated with only mild symptoms, due to the high persistence the long-term effects are dramatic. About 50% of all infections lead to chronic liver disease, which can range from an apparently healthy carrier state to chronic active hepatitis, liver cirrhosis, or hepatocellular carcinoma. It is estimated that 100 million to 200 million people worldwide are infected with this insidious agent. HCV was classified as the distinct genus Hepacivirus together with the genera Flavivirus and Pestivirus in the family Flaviviridae (41). These viruses are characterized by an enveloped virion harboring a plus-strand RNA genome. In case of HCV, this genome has a length of ca. 9.6 kb and carries a single long open reading frame (ORF) flanked at the 59 and 39 ends by nontranslated regions required for RNA translation and replication (for reviews, see references 4, 9, and 45). The viral genes are expressed as a polyprotein, just 3,000 amino acids in length, which is cleaved co- and posttranslationally by host cell signal peptidases and two viral proteinases. At least 10 different cleavage products have been identified, which are ordered within the polyprotein (from the amino to the carboxy terminus): NH2-core-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5ANS5B-COOH. The structural proteins core, envelope protein 1 (E1), and E2 are the major constituents of the virion. The function of p7, a small highly hydrophobic peptide is not known. NS2 and the amino-terminal domain of NS3 constitute the NS2-3 proteinase responsible for cleavage at the NS2/3 junction (18, 20). NS3 is a bifunctional molecule. The aminoterminal domain carries a serine-type proteinase required for