The question of whether hepatitis C virus (HCV) RNA is translated by a mechanism of internal ribosome entry has been examined by testing whether insertion of HCV sequences between the two cistrons of a dicistronic mRNA promotes translation of the downstream cistron in rabbit reticulocyte lysates. Deletion analysis showed that efficient internal initiation required a segment of the HCV genome extending from about nucleotides 40–370 and that deletions from the 3′‐end of this element were highly deleterious. As the authentic initiation codon for HCV polyprotein synthesis is at nucleotide 342, this demonstrates that, besides 5′‐UTR sequences, a short length of HCV coding sequences is required for internal initiation. This finding was confirmed in transfection assays of BT7‐H cells and was shown to be independent of the nature of the downstream reporter cistron. The strong requirement for coding sequences is in sharp contrast to internal initiation of picornavirus RNA translation. As a probable correlate with this, it was also found that the efficiency of internal initiation was only marginally compromised when the authentic initiation codon was mutated to a non‐AUG codon, again in sharp contrast with the picornaviruses. The finding that coding sequences are required for internal initiation has important implications for the design of experiments to test for internal initiation of translation of cellular mRNAs.

The helicase domain of hepatitis C virus NS3 (genotype 1b) was expressed in Escherichia coli and purified to homogeneity. The purified protein catalyzed the hydrolysis of nucleoside triphosphates (NTP) and the unwinding of duplex RNA in the presence of divalent metal ion. The enzyme was not selective for the NTP substrate. For example, UTP and acyclovir triphosphate were hydrolyzed efficiently by the enzyme. The rate of NTP hydrolysis was stimulated up to 27-fold by oligomeric nucleic acids (NA). Furthermore, NA bound to the enzyme with concomitant quenching of the intrinsic protein fluorescence. The dissociation constants of the enzyme for selected NA in the absence of NTP were between 10 and 35 M at pH 7.0 and 25°C. ؊1 ), respectively. These data were consistent with a random kinetic mechanism. Hepatitis C virus (HCV)1 is the principle agent responsible for non-A, non-B hepatitis (1, 2). Approximately 1% of the human population is infected with HCV. Standard interferon therapies are effective for some patients, but the majority do not clear the virus, resulting in relapse (3). Consequently, an urgent medical need for an effective antiviral agent exists.Development of an effective therapeutic agent has been hindered by the lack of reliable cell culture systems for propagating HCV. Consequently, efforts to characterize potential therapeutic agents have relied on surrogate expression systems and the techniques of molecular biology. For example, the NS3 protein of hepatitis C virus has several enzymatic activities necessary for viral replication that make it an attractive antiviral target. The N-terminal 20 kDa of NS3 is a serine proteinase that cleaves the HCV-encoded polyprotein at a minimum of four specific sites (4). The C-terminal 50 kDa of NS3 has NTPase (5) and RNA helicase activity (6). A detailed understanding of either of these enzymatic activities could facilitate identification of potent antiviral agents. Consequently, we have initiated a program to characterize the interaction of the HCV helicase domain with nucleoside triphosphates (NTP) and polymeric nucleic acids (NA). HCV helicase catalyzes the three reactions shown in Scheme I.dsNA ϩ NTP 3 ssNA ϩ NDP ϩ P i (c) SCHEME I. Reactions catalyzed by HCV helicase where NA is single stranded (ss) or double stranded (ds) polynucleic acid and NTP is a nucleoside triphosphate.The physiologically relevant reaction for viral replication is probably unwinding of double-stranded NA (reaction c). The kinetic studies presented herein, which represent the first comprehensive kinetic analysis of a RNA helicase, have focused on the NTPase activities of the HCV helicase in the presence or absence of NA (reactions a and b in Scheme I). We have chosen to work with HCV genotype 1b, which is a major subtype found in both the Japanese and American populations (7). In summary, we found that 1) the NTPase activity was nonselective for the nucleobase and sugar of the NTP substrate, 2) the NTPase activity was stimulated up to 25-fold by selected NA in a reaction that was rela...

Cap-independent translation of hepatitis C virus (HCV) RNA is mediated by an internal ribosomal entry segment (IRES) located within the 5' nontranslated RNA (5'NTR), but previous studies provide conflicting views of the viral sequences which are required for translation initiation. These discrepancies could have resulted from the inclusion of less than full-length 5'NTR in constructs studied for translation or destabilization of RNA secondary structure due to fusion of the 5'NTR to heterologous reporter sequences. In an effort to resolve this confusion, we constructed a series of mutations within the 5'NTR of a nearly full-length 9.5-kb HCV cDNA clone and examined the impact of these mutations on HCV translation in vitro in rabbit reticulocyte lysates and in transfected Huh-T7 cells. The inclusion of the entire open reading frame in HCV transcripts did not lead to an increase in IRES-directed translation of the capsid and E1 proteins, suggesting that the nonstructural proteins of HCV do not include a translational transactivator. However, in reticulocyte lysates programmed with full-length transcripts, there were multiple aberrent translation initiation sites resembling those identified in some picornaviruses. The deletion of nucleotides (nt) 28-69 of the 5'NTR (stem-loop IIa) sharply reduced capsid translation both in vitro and in vivo. A small deletion mutation involving nt 328-334, immediately upstream of the initiator AUG at nt 342, also resulted in a nearly complete inhibition of translation, as did the deletion of multiple intervening structural elements. An in-frame 12-nt insertion placed within the capsid-coding region 9 nt downstream of the initiator AUG strongly inhibited translation both in vitro and in vivo, while multiple silent mutations within the first 42 nt of the open reading frame also reduced translation in reticulocyte lysates. Thus, domains II and III of the 5'NTR are both essential to activity of the IRES, while conservation of sequence downstream of the initiator AUG is required for optimal IRES-directed translation.

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Synthetic vaccines for viral diseases can use defined regions of viral proteins as immunogens: the peptide sequence of amino acids 141-160 of the VP1 protein of foot and mouth disease virus (FMDV) elicits virus-neutralizing antibodies to protect guinea pigs, cattle and pigs either when coupled to a carrier protein or when administered in liposomes or in incomplete Freund's adjuvant. The immune response to these peptides is much lower than that to complete virus particles and the same sequence fused to the N terminus of beta-galactosidase did not produce a more potent immunogen than synthetic peptide alone. We report here an expression system for immunogenic epitopes linked to a carrier protein, hepatitis B core antigen, to form part of a virus-like complex which can present these epitopes to the immune system at high density. The immunogenicity of these structures approaches that of FMDV particles.