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Translation of the open reading frames (ORF) of the hepatitis C virus (HCV) and closely related GB virus B (GBV-B) genomes is driven by internal ribosome entry site (IRES) elements located within the 5' non-translated RNA. The functioning of these IRES elements is highly dependent on primary and higher order RNA structures. We present here the solution structures of a common, critical domain within each of these IRESs, stem-loop IIIc. These ten-nucleotide hairpins have nearly identical sequences and similar overall tertiary folds. The final refined structure of each shows a stem with three G:C base-pairs and a novel tetraloop fold. Although the bases are buckled, the first and fourth nucleotides of both tetraloops form a Watson-Crick type base-pair, while the apical nucleotides are located in the major groove where they adopt C(2)-endo sugar puckering with B-form geometry. No hydrogen bonding interactions were observed involving the two apical residues of the tetraloop. Stability of the loops appears to be derived primarily from the stacking of bases, and the hydrogen bonding between the fourth and seventh residues. Mutational analysis shows that the primary sequence of stem-loop IIIc is important for IRES function and that the stem and first and fourth nucleotides of the tetraloop contribute to the efficiency of internal ribosome entry. Base-pair formation between these two positions is essential. In contrast, the apical loop nucleotides differ between HCV and GBV-B, and substitutions in this region of the hairpin are tolerated without major loss of function.

GB virus B (GBV-B), a flavivirus closely related to HCV, has previously been shown to infect and replicate to high titers in tamarins ( Saguinus sp.). This study describes the use of GBV-B infection and replication in the common marmoset ( Callithrix jacchus ) for the successful development and validation of a surrogate animal model for hepatitis C virus (HCV). Infection of marmosets with GBV-B produced a viremia that peaked at 10 8 to 10 9 genome copies/ml for a period of 40 to 60 days followed by viral clearance at 60 to 80 days postinfection. Passage of the initial tamarin-derived GBV-B in marmosets produced an infectious stock that gave a more reproducible and consistent infection in the marmoset. Titration of the virus stocks in vivo indicated that they contained 1 infectious unit for every 1,000 genome copies. Cultures of primary marmoset hepatocytes were also successfully infected with GBV-B, with high levels of virus detected in supernatants and cells for up to 14 days postinfection. Treatment of GBV-B-infected hepatocyte cultures with a novel class of HCV protease inhibitor (pyrrolidine 5,5 trans -lactams) reduced viral levels by more than 2 logs. Treatment of GBV-B-infected marmosets with one such inhibitor resulted in a 3-log drop in serum viral titer over 4 days of therapy. These studies provide the first demonstration of the in vivo efficacy of a small-molecule inhibitor for HCV in an animal model and illustrate the utility of GBV-B as a surrogate animal model system for HCV.

Although responsible for a major health problem worldwide, hepatitis C virus is difficult to study because of the absence of fully permissive cell cultures or experimental animal models other than the chimpanzee. GB virus B (GBV-B), a closely related hepatotropic virus that infects small New World primates and replicates efficiently in primary hepatocyte cultures, is an attractive surrogate model system. However, little is known about processing of the GBV-B polyprotein. Because an understanding of these events is critical to further development of model GBV-B systems, we characterized signal peptidase processing of the polyprotein segment containing the putative structural proteins. We identified the exact N termini of the mature GBV-B envelope proteins, E1 and E2, and the first nonstructural protein, NS2, by direct amino acid sequencing. Interestingly, these studies document the existence of a previously unrecognized 13-kDa protein (p13) located between E2 and NS2 within the polyprotein. We compared the sequence of the p13 protein to that of hepatitis C virus p7, a small membrane-spanning protein with a similar location in the polyprotein and recently identified ion channel activity. The C-terminal half of p13 shows clear homology with p7, suggesting a common function, but the substantially larger size of p13, with 4 rather than 2 predicted transmembrane segments, indicates a different structural organization and/or additional functions. The identification of p13 in the GBV-B polyprotein provides strong support for the hypothesis that ion channel-forming proteins are essential for the life cycle of flaviviruses, possibly playing a role in virion morphogenesis and/or virus entry into cells.Although responsible for a major health problem worldwide, hepatitis C virus is difficult to study because of the absence of fully permissive cell cultures or experimental animal models other than the chimpanzee. GB virus B (GBV-B), a closely related hepatotropic virus that infects small New World primates and replicates efficiently in primary hepatocyte cultures, is an attractive surrogate model system. However, little is known about processing of the GBV-B polyprotein. Because an understanding of these events is critical to further development of model GBV-B systems, we characterized signal peptidase processing of the polyprotein segment containing the putative structural proteins. We identified the exact N termini of the mature GBV-B envelope proteins, E1 and E2, and the first nonstructural protein, NS2, by direct amino acid sequencing. Interestingly, these studies document the existence of a previously unrecognized 13-kDa protein (p13) located between E2 and NS2 within the polyprotein. We compared the sequence of the p13 protein to that of hepatitis C virus p7, a small membrane-spanning protein with a similar location in the polyprotein and recently identified ion channel activity. The C-terminal half of p13 shows clear homology with p7, suggesting a common function, but the substantially larger size of p13, with 4 rather than 2 predicted transmembrane segments, indicates a different structural organization and/or additional functions. The identification of p13 in the GBV-B polyprotein provides strong support for the hypothesis that ion channel-forming proteins are essential for the life cycle of flaviviruses, possibly playing a role in virion morphogenesis and/or virus entry into cells.

The yield of G418-resistant Huh7 cell clones bearing subgenomic dicistronic GB virus B (GBV-B) is significantly affected by the insertion of a portion of the viral core gene between the GBV-B 5′ untranslated region and the exogenous neomycin phosphotransferase selector gene (A. De Tomassi, M. Pizzuti, R. Graziani, A. Sbardellati, S. Altamura, G. Paonessa, and C. Traboni, J. Virol. 76:7736-7746, 2002). In this report, we have dissected this phenomenon, examining the effects of the insertion of core sequences of different lengths on GBV-B IRES-dependent translation and RNA replication by using experimental approaches aimed at analyzing these two aspects independently. The results achieved indicate that an enhancement of translation efficiency does occur and that it correlates with the length of the inserted core sequences. Interestingly, the insertion of these sequences also has a direct similar effect on the efficiency of replication of the GBV-B replicon. These results suggest that in GBV-B replicon RNA and potentially in the complete viral genome, the core coding sequences not only are part of the IRES but also take part in the replication process, independently of the presence of the corresponding whole protein.

RNA-dependent RNA polymerases (RdRps) that initiate RNA synthesis by a de novo mechanism should specifically recognize the template initiation nucleotide, T1, and the substrate initiation nucleotide, the NTPi. The RdRps from hepatitis C virus (HCV), bovine viral diarrhea virus (BVDV), and GB virus-B all can initiate RNA synthesis by a de novo mechanism. We used RNAs and GTP analogs, respectively, to examine the use of the T1 nucleotide and the initiation nucleotide (NTPi) during de novo initiation of RNA synthesis. The effects of the metal ions Mg 2+ and Mn 2+ on initiation were also analyzed. All three viral RdRps require correct base pairing between the T1 and NTPi for efficient RNA synthesis. However, each RdRp had some distinct tolerances for modifications in the T1 and NTPi. For example, the HCV RdRp preferred an NTPi lacking one or more phosphates regardless of whether Mn 2+ was present or absent, while the BVDV RdRp efficiently used GDP and GMP for initiation of RNA synthesis only in the presence of Mn 2+ . These and other results indicate that although the three RdRps share a common mechanism of de novo initiation, each has distinct preferences.