Mutational Analysis of the GB Virus B Internal Ribosome Entry Site

Abstract

GB virus B (GBV-B) is a recently discovered hepatotropic flavivirus that is distantly related to hepatitis C virus (HCV).We show here that translation of its polyprotein is initiated by internal entry of ribosomes on GBV-B RNA. We analyzed the translational activity of dicistronic RNA transcripts containing wild-type or mutated 5 nontranslated GBV-B RNA (5NTR) segments, placed between the coding sequences of two reporter proteins, in vitro in rabbit reticulocyte lysate and in vivo in transfected BT7-H cells. We related these results to a previously proposed model of the secondary structure of the GBV-B 5NTR (M. Honda, et al. RNA 2:955-968, 1996). We identified an internal ribosome entry site (IRES) bounded at its 5 end by structural domain II, a location analogous to the 5 limit of the IRES in both the HCV and pestivirus 5NTRs. Mutational analysis confirmed the structure proposed for domain II of GBV-B RNA, and demonstrated that optimal IRES-mediated translation is dependent on each of the putative RNA hairpins in this domain, including two stem-loops not present in the HCV or pestivirus structures. IRES activity was also absolutely dependent on (i) phylogenetically conserved, adenosine-containing bulge loops in domain III and (ii) the primary nucleotide sequence of stem-loop IIIe. IRES-directed translation was inhibited by a series of point mutations predicted to stabilize stem-loop IV, which contains the initiator AUG codon in its loop segment. A reporter gene was translated most efficiently when fused directly to the initiator AUG codon, with no intervening downstream GBV-B sequence. This finding indicates that the 3 limit of the GBV-B IRES is at the initiator AUG and that it does not require downstream polyprotein-coding sequence as suggested for the HCV IRES. These results show that the GBV-B IRES, while sharing a common general structure, differs both structurally and functionally from other flavivirus IRES elements.

A chimeric GB virus B with 5′ nontranslated RNA sequence from hepatitis C virus causes hepatitis in tamarins†

“…1A). [17][18][19] The 5ЈNTRs of these viruses each comprise 4 distinct structural domains (Fig. 1), of which domains II-IV (sequence extending to the initiator AUG) represent the IRES.…”

Characterization of a canine homolog of hepatitis C virus

“…Independent of phylogenetic information, the secondary structure of the CHCV 5′ UTR RNA was modeled with MFOLD. Labeling of the predicted structures in the 5′ UTR followed numbering used for reported homologous structures in HCV and GBV-B (10,34). The CHV genome sequence was analyzed for evidence of GORS by comparing folding energies of consecutive fragments of nucleotide sequence with random sequence-order controls (35).…”

“…Identification and characterization of animal virus homologs provide new insights into pathogenesis of human viruses, and, in some instances, models for investigating prevention and treatment of human disease must be pursued with related animal viruses (3)(4)(5). Comparative genetic analysis of closely related viruses can also identify genomic regions (RNA structures, amino acid motifs, and residues) important for virus receptor binding, entry, replication, immunity, and other biological functions (6)(7)(8)(9)(10).…”

Development of a Primary Tamarin Hepatocyte Culture System for GB Virus-B: a Surrogate Model for Hepatitis C Virus

“…GBV-B is the virus most closely related to HCV genetically (22). The polyproteins possess approximately 25 to 30% homology at the amino acid level (21), while the 5Ј and 3Ј untranslated regions are more distinct (6,21,24). The functional similarities between the viruses were demonstrated by the correct processing of the HCV polyprotein by the GBV-B NS3 protease and the creation of functional chimeric NS3 proteins between HCV and GBV-B (7, 25).…”