Abstract
GB virus B (GBV-B) is closely related to hepatitis C virus (HCV), infects small non-human primates, and is thus a valuable surrogate for studying HCV. Despite significant differences, the 5′ nontranslated RNAs (NTRs) of these viruses fold into four similar structured domains (I-IV), with domains II-III-IV comprising the viral internal ribosomal entry site (IRES). We previously reported the in vivo rescue of a chimeric GBV-B (vGB/IIIHC) containing HCV sequence in domain III, an essential segment of the IRES. We show here that three mutations identified within the vGB/IIIHC genome (within the 3′NTR, upstream of the poly(U) tract, and NS5A coding sequence) are necessary and sufficient for production of this chimeric virus following intrahepatic inoculation of synthetic RNA in tamarins, and thus apparently compensate for the presence of HCV sequence in domain III. To assess the mechanism(s) underlying these compensatory mutations, and to determine whether 5′NTR subdomains participating in genome replication do so in a virus-specific fashion, we constructed and evaluated a series of chimeric subgenomic GBV-B replicons in which various 5′NTR subdomains were substituted with their HCV homologs. Domains I and II of the GBV-B 5′NTR could not be replaced with HCV sequence, indicating that they contain essential, virus-specific RNA replication elements. In contrast, domain III could be swapped with minimal loss of genome replication capacity in cell culture. The 3′NTR and NS5A mutations required for rescue of the related chimeric virus in vivo had no effect on replication of the subgenomic GBneoD/IIIHC RNA in vitro. The data suggest that in vivo fitness of the domain III chimeric virus is dependent on a cooperative interaction between the 5′NTR, 3′NTR and NS5A at a step in the viral life cycle subsequent to genome replication, most likely during particle assembly. Such a mechanism may be common to all hepaciviruses.
Highlights
Hepatitis C virus (HCV) infection is an important public health concern with about 170 million persons chronically infected worldwide who are at risk for developing liver cirrhosis and hepatocellular carcinoma
We previously reported that chimeric, genome-length GB virus B (GBV-B) RNAs in which domains I-II-III or II-III of the 59NTR were substituted by corresponding domains of HCV 59NTR were not infectious upon intrahepatic inoculation of synthetic RNAs in tamarins [3]
Analysis of second site mutations found in the genome of a domain III chimeric virus rescued in vivo: compensatory effect on chimeric genome infectivity in vivo In previous studies, we found that replacing domain III in an infectious molecular clone of GBV-B with the corresponding HCV domain (GB/IIIHC) resulted in an infectious genome with rescue of a chimeric virus following intrahepatic inoculation of RNAs into a susceptible tamarin, yet with an unusual delay in the onset of viremia
Summary
Hepatitis C virus (HCV) infection is an important public health concern with about 170 million persons chronically infected worldwide who are at risk for developing liver cirrhosis and hepatocellular carcinoma. The only available treatment is a combination therapy with pegylated interferon-a and ribavirin, which remains poorly effective for HCV genotype 1 infections, and is generally difficult to tolerate, leading to $50% therapeutic failures [reviewed in Ref. 1]. The development of specific, small molecule antiviral inhibitors that could be used to cure this infection is currently a high priority. Immunocompetent animal models susceptible to HCV infection would be useful to support these efforts and monitor potential antiviral resistance mechanisms. As a surrogate for chimpanzees, which represent the only immunocompetent animal model for human HCV infection but which are endangered and extraordinarily difficult to access, infection of small New World nonhuman primates from the Callithrichidae family (tamarins, marmosets) with a closely related, hepatotropic virus, GB virus B (GBV-B), may prove useful [2,3]. Experimental infection of tamarins or marmosets with GBV-B induces an acute hepatitis [2,4], but in limited cases described to date, experimental GBV-B infection has resulted in an extended viremia and chronic hepatitis in tamarins [5,6], mimicking closely chronic hepatitis C in humans
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