Identification of Residues in the Hepatitis C Virus Core Protein That Are Critical for Capsid Assembly in a Cell-Free System

Abstract

Significant advances have been made in understanding hepatitis C virus (HCV) replication through development of replicon systems. However, neither replicon systems nor standard cell culture systems support significant assembly of HCV capsids, leaving a large gap in our knowledge of HCV virion formation. Recently, we established a cell-free system in which over 60% of full-length HCV core protein synthesized de novo in cell extracts assembles into HCV capsids by biochemical and morphological criteria. Here we used mutational analysis to identify residues in HCV core that are important for capsid assembly in this highly reproducible cell-free system. We found that basic residues present in two clusters within the N-terminal 68 amino acids of HCV core played a critical role, while the uncharged linker domain between them was not. Furthermore, the aspartate at position 111, the region spanning amino acids 82 to 102, and three serines that are thought to be sites of phosphorylation do not appear to be critical for HCV capsid formation in this system. Mutation of prolines important for targeting of core to lipid droplets also failed to alter HCV capsid assembly in the cell-free system. In addition, wild-type HCV core did not rescue assembly-defective mutants. These data constitute the first systematic and quantitative analysis of the roles of specific residues and domains of HCV core in capsid formation.Hepatitis C virus (HCV) is a major public health concern worldwide. Two percent of the world's population is infected with the virus (54), which is the major cause of non-A, non-B hepatitis and which often leads to cirrhosis of the liver or hepatocellular carcinoma (42). While current treatments have improved, they are poorly tolerated, not completely efficacious, and not available in all settings (31). Development of better treatments and vaccines has been hampered by the lack of virus replication systems. Standard cell culture systems do not support HCV replication, and the chimpanzee is the only animal model capable of being infected and yielding high HCV titers (23). These limitations have been partly overcome by the development of HCV replicon systems, which support autonomous replication of HCV RNA (2,10,36,37). Replicon systems have allowed specific requirements of HCV RNA replication to be studied; however, HCV particles, or even nucleocapsids, are not produced in these systems (9,

A method for in vitro assembly of hepatitis C virus core protein and for screening of inhibitors

“…4C). This result is consistent with the previously reported observation that particle formation is decreased with a Δ39-64 mutant in a cell-free in vitro assembly system [24]. To investigate if specific regions in the 39-62 fragment are more important for assembly, we mutated several clusters of charged residues.…”

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Host–rabies virus protein–protein interactions as druggable antiviral targets

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