Hepatitis C virus (HCV) is a major causative agent of posttransfusion non-A, non-B hepatitis, which often develops into malignant chronic diseases, including liver cirrhosis and hepatocellular carcinoma. We have cloned from human carriers overlapping cDNAs (9,416 bp) covering the entire coding region of the HCV genome. The latter encodes a 3,010-amino-acid polyprotein. In addition, there are 332 and 54 bases of 5' and 3' noncoding sequences, respectively. Our HCV strain has a 77% nucleic acid identity to the HCV strain cloned by workers at Chiron Corporation. The hydrophobicity profile of the putative polyprotein is similar to those of flaviviruses, but it has limited amino acid homology to polyproteins of flaviviruses and other viruses, indicating that HCV is at most distantly related to flaviviruses.
The NS3 protein of Japanese encephalitis virus (JEV) is a large multifunctional protein possessing protease, helicase, and nucleoside 5'-triphosphatase (NTPase) activities, and plays important roles in the processing of a viral polyprotein and replication. To clarify the enzymatic properties of NS3 protein from a structural point of view, an enzymatically active fragment of the JEV NTPase/helicase catalytic domain was expressed in bacteria and the crystal structure was determined at 1.8 A resolution. JEV helicase is composed of three domains, displays an asymmetric distribution of charges on its surface, and contains a tunnel large enough to accommodate single-stranded RNA. Each of the motifs I (Walker A motif), II (Walker B motif) and VI was composed of an NTP-binding pocket. Mutation analyses revealed that all of the residues in the Walker A motif (Gly(199), Lys(200) and Thr(201)), in addition to the polar residues within the NTP-binding pocket (Gln(457), Arg(461) and Arg(464)), and also Arg(458) in the outside of the pocket in the motif IV were crucial for ATPase and helicase activities and virus replication. Lys(200) was particularly indispensable, and could not be exchanged for other amino acid residues without sacrificing these activities. The structure of the NTP-binding pocket of JEV is well conserved in dengue virus and yellow fever virus, while different from that of hepatitis C virus. The detailed structural comparison among the viruses of the family Flaviviridae should help in clarifying the molecular mechanism of viral replication and in providing rationale for the development of appropriate therapeutics.
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