The existence and functional importance of RNA secondary structure in the replication of positive-stranded RNA viruses is increasingly recognized. We applied several computational methods to detect RNA secondary structure in the coding region of hepatitis C virus (HCV), including thermodynamic prediction, calculation of free energy on folding, and a newly developed method to scan sequences for covariant sites and associated secondary structures using a parsimony-based algorithm. Each of the prediction methods provided evidence for complex RNA folding in the core-and NS5B-encoding regions of the genome. The positioning of covariant sites and associated predicted stem-loop structures coincided with thermodynamic predictions of RNA base pairing, and localized precisely in parts of the genome with marked suppression of variability at synonymous sites. Combined, there was evidence for a total of six evolutionarily conserved stem-loop structures in the NS5B-encoding region and two in the core gene. The virus most closely related to HCV, GB virus-B (GBV-B) also showed evidence for similar internal base pairing in its coding region, although predictions of secondary structures were limited by the absence of comparative sequence data for this virus. While the role(s) of stem-loops in the coding region of HCV and GBV-B are currently unknown, the structure predictions in this study could provide the starting point for functional investigations using recently developed self-replicating clones of HCV.

We compared the ability of two closely related truncated E2 glycoproteins (E2 660 ) derived from hepatitis C virus (HCV) genotype 1a strains Glasgow (Gla) and H77c to bind a panel of conformationdependent monoclonal antibodies (MAbs) and CD81. In contrast to H77c, Gla E2 660 formed disulfide-linked high molecular mass aggregates and failed to react with conformation-dependent MAbs and CD81. To delineate amino acid (aa) regions associated with protein aggregation and CD81 binding, several Gla-H77c E2 660 chimeric glycoproteins were constructed. Chimeras C1, C2 and C6, carrying aa 525-660 of Gla E2 660 , produced disulfide-linked aggregates and failed to bind CD81 and conformation-dependent MAbs, suggesting that amino acids within this region are responsible for protein misfolding. The presence of Gla hypervariable region 1 (aa 384-406) on H77 E2 660 , chimera C4, had no effect on protein folding or CD81 binding. Chimeras C3 and C5, carrying aa 384-524 or 407-524 of Gla E2 660 , respectively, were recognized by conformationdependent MAbs and yet failed to bind CD81, suggesting that amino acids in region 407-524 are important in modulating CD81 interaction without affecting antigen folding. Comparison of Gla and H77c E2 660 aa sequences with those of genotype 1a and divergent genotypes identified a number of variant amino acids, including two putative N-linked glycosylation sites at positions 476 and 532. However, introduction of G476N-G478S and/or D532N in Gla E2 660 had no effect on antigenicity or aggregation.

To identify proteins that can bind the 3 untranslated region (UTR) of hepatitis C virus (HCV) we screened human cDNA libraries using the Saccharomyces cerevisiae three-hybrid system. Screening with an RNA sequence derived from the 3-terminal 98 nucleotides (3X region) of an infectious clone of HCV (H77c) yielded clones of human ribosomal proteins L22, L3, S3, and mL3, a mitochondrial homologue of L3. We performed preliminary characterization of the binding between the 3X region and these proteins by a three-hybrid mating assay using mutant 3X sequences. We have further characterized the interaction between 3X and L22, since this protein is known to be associated with two small Epstein-Barr virus (EBV)-encoded RNA species (EBERs) which are abundantly produced in cells latently infected with EBV. The EBERs, which have similar predicted secondary structure to the HCV 3X, assemble into ribonucleoprotein particles that include L22 and La protein. To confirm that L22 binds HCV 3X we performed in vitro binding assays using recombinant L22 (expressed as a glutathione S-transferase [GST] fusion protein) together with a 3X riboprobe. The 3X region binds to the GST-L22 fusion protein (but not to GST alone), and this interaction is subject to competition with unlabeled 3X RNA. To establish the functional role played by L22 in internal ribosome entry site (IRES)-mediated translation of HCV sequences we performed translational analysis in HuH-7 cells using monocistronic and bicistronic reporter constructs. The relative amount of core-chloramphenicol acetyltransferase reporter protein translated under the control of the HCV IRES was stimulated in the presence of L22 and La when these proteins were supplied in trans.It is estimated that 170 million people worldwide are chronically infected with hepatitis C virus (HCV) (36). HCV infection is a leading cause of liver cirrhosis and hepatocellular carcinoma. As there is no vaccine or effective treatment available, HCV poses a significant threat to public health and there is thus an urgent need to understand the virus better and to develop vaccines and therapeutic agents (50).HCV, a member of the Flaviviridae, is an enveloped virus containing a single stranded, approximately 9.6-kb genomic RNA molecule of positive polarity (10). The genome contains a single open reading frame flanked by 5Ј untranslated regions (5ЈUTRs) and 3ЈUTRs. There are at least six genotypes of HCV whose sequences differ from each other by up to 30% over the complete genome, and the genotypes are grouped into subtypes according to sequence similarities (57). Recently, several infectious cDNA clones of HCV genome have been isolated (31, 68, 70). The genome encodes a polypeptide of approximately 3,010 amino acids which is cotranslationally processed by the host-and virus-encoded proteases to produce at least 10 mature proteins (11,40,50). In contrast to the recent progress made in understanding the genome organization of HCV, the proteolytic processing of the polyprotein and the biochemical characterization of th...