The molecular virology of hepatitis C

Hepatitis C virus (HCV), first identified in 1989, is the structure. The proposed secondary structure is shown major causative agent of parenterally transmitted and comin Fig. 2A, the four stem-loops are indicated together with munity-acquired non-A, non-B hepatitis. Infection occurs the initiator AUG for translation of the polyprotein. The principally through blood or blood-derived products, but also structure was determined by first assigning conformational through inapparent parenteral exposure. It frequently leads contexts to regions of the HCV 5*UTR, which showed signifito chronic hepatitis and cirrhosis and is associated with the cant sequence homology to pestiviral nucleotide sequences. development of hepatocellular carcinoma. Over the past 5 This prediction was confirmed by analyzing the susceptibility years, considerable progress has been made in the molecular of synthetic RNA to cleavage with singleand double-strand characterization of this virus. specific RNases, and by the comparative analysis of other The viral particle consists of an envelope derived from host published HCV 5*UTR sequences. The 5*UTR is the most membranes, into which are inserted the virally encoded glyconserved portion of the HCV genome, although nucleotide coproteins (E1 and E2) surrounding a nucleocapsid and a variations characteristic of different HCV types exist that positive-sense, single-stranded RNA genome of approxihave been used in polymerase chain reaction (PCR)-based mately 9,500 nucleotides (nt). HCV has a similar genomic genotyping assays. However, these nucleotide variations organization to the pestiand flaviviruses (for review see ), are either within single-stranded regions or preserve the base and has now been classified, with these groups, as a separate pairing within the stem-loops, thereby maintaining the basic genus in the family Flaviviridae. A recent proposal for HCV secondary and tertiary structures. The extensive secnomenclature defined six major genotypes (1-6) based upon ondary structure and the presence of several AUG triplets phylogenetic analyses of the core, E1, and NS5 regions before the authentic initiator codon have led to comparisons with further divisions into subtypes (1a, 1b, 2c, etc.). This between the 5*UTR of HCV and that of picornaviruses, most nomenclature, which is the most widely accepted by researchimportantly the question of whether this region can act as ers in this field, will be employed in this review. an internal ribosome entry site (IRES). Such a feature, well The genome contains highly conserved untranslated recharacterized in picornaviruses, leads to the translation of gions (UTR) at both the 5* and 3* termini, which flank a the RNA genome through a mechanism of internal initiation large translational open reading frame encoding a polyproof an uncapped message, rather than ribosome scanning from tein of Ç3,000 amino acids (aa). This is processed by the 5* end of a capped molecule. Initially, there was conflictboth cellular and viral proteases to produce the specific viral ing data regarding the IRES function of the HCV 5*UTR, and gene products outlined in Fig. 1, the structural proteins, core, it is as yet unknown whether the HCV genome is capped. E1 and E2, are located in the N-terminal quarter with the Computer and enzyme analyses had shown that the 5*UTR nonstructural (NS) proteins (NS2, NS3, NS4A, NS4B, NS5A, contained secondary structures typical of IRES elements, and NS5B) in the remaining portion of the polyprotein. and Tsukiyama-Kohara et al. showed that this region could Despite the lack of an efficient tissue culture system that act as an IRES, although Yoo et al. found no evidence to allows the analysis of viral replication and gene function, a support the suggestion that internal initiation could take great deal of data have been accumulated through the use of place. More recently, several groups have confirmed a funcseveral established expression systems, leading to the identitional IRES occupying most of the HCV 5*UTR, as shown fication of functional regions and important interactions bein Fig. 2A. Additional evidence has suggested that there extween viral products. The purpose of this review is to summaists a unique requirement for HCV coding sequence from the rize the most recent information at the molecular level and core region, of Ç12 to 30 nt, for efficient IRES function, discuss its implications in viral replication and pathogenesis. although Tsukiyama-Kohara et al. and Wang et al. showed internal ribosome entry using constructs containing THE UTRs only reporter genes downstream of the HCV initiator AUG. The 5*UTR. The complete 5*UTR, consisting of 341 nt, To examine the discrepancies between these reports, Honda is longer than that of the flaviviruses, which have an average et al. altered the stability of stem-loop IV in the HCV length of 100 nt and more closely resembles the 5*UTR of 5*UTR, which contains the initiator AUG and several nucleopestiviruses with respect to length, sequence, and secondary tides from the core region (Fig. 2A). Translational activity was measured by HCV core production from almost genomelength transcripts. Mutations reducing the stability of stemloop IV did not reduce translation; however, mutations Abbreviations: HCV, hepatitis C virus; nt, nucleotide; UTR, untranslated region; aa, stabilizing the stem-loop led to significant reductions in IRES amino acid; NS, nonstructural; PCR, polymerase chain reaction; IRES, internal ribosome activity. entry site; p, protein; cDNA, complementary DNA; ER, endoplasmic reticulum; HVR, hyper-