A cooperative interaction between nontranslated RNA sequences and NS5A protein promotes in vivo fitness of a chimeric hepatitis C/GB virus B.

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/III(HC)) containing HCV sequence in domain III, an essential segment of the IRES. We show here that three mutations identified within the vGB/III(HC) 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/III(HC) 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.

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

Only humans and chimpanzees are fully permissive for replication of hepatitis C virus (HCV), an important cause of liver cirrhosis and cancer worldwide. The absence of suitable animal models limits opportunities for in vivo evaluation of candidate hepatitis C therapeutics and slows progress in the field. Here, we describe a chimeric virus derived from GB virus B (GBV-B), an unclassified hepatotropic member of the family Flaviviridae that is closely related to HCV and infects tamarins (Saguinus sp.), in which a functionally important HCV regulatory sequence replaced an analogous sequence in the 5' nontranslated region (5'NTR) of the GBV-B genome. The transplanted sequence comprised domain III of the internal ribosome entry site (IRES), which directly binds the 40S ribosome subunit and is a target for candidate therapeutics. The chimeric 5'NTR retained ribosome binding activity and was competent in directing protein translation both in cell-free translation reactions and in transfected primary tamarin hepatocyte cultures. Virus rescued from the chimeric RNA replicated in the liver of tamarins, causing biochemical and histopathological changes typical of viral hepatitis. However, adaptive mutations were required elsewhere in the genome for efficient replication. Virus was not rescued from other, translationally competent, chimeric RNAs in which domain II of the IRES was exchanged. Thus, the 5'NTR appears to contain virus-specific replication signals that interact with other sites within the viral genome or with viral proteins. In conclusion, such novel chimeric flaviviruses offer opportunities for new insights into HCV replication mechanisms, while potentially facilitating the evaluation of candidate therapeutics in vivo.

Parenchymal expression of CD86/B7.2 contributes to hepatitis C virus-related liver injury.

Hepatitis C virus (HCV) infection is a major global health problem. Hepatic expression of immune costimulatory signaling molecules (e.g., B7) is known to be associated with ongoing liver injury in hepatitis C patients. However, due to the general lack of viral culture systems and adequate animal models, the function of these molecules in disease pathogenesis is poorly understood. To investigate the role of CD86 in HCV-related liver injury, we developed two transgenic mouse lineages with inducible expression of HCV structural proteins and constitutive expression of the costimulatory molecule CD86/B7.2 in the liver. Using a hydrodynamic-based, nonviral delivery protocol, we induced HCV transgene expression in the livers of HCV and CD86 single- and double-transgenic mice. We found that hepatic CD86 expression resulted in increased activation of and cytokine production (e.g., interleukin-2 and gamma interferon) by CD4+ T cells and that the retention of these cells was associated with more pronounced necroinflammatory lesions in the liver. Taken together, these data suggest that augmented, parenchymal antigen presentation conferred by hepatocyte CD86 expression alters homeostasis and effector functions of CD4+ T cells and contributes to liver injury. This study provides an additional rationale for exploring immunomodulation-based therapies that could reduce disease progression in individuals with chronic HCV infection.

Subgenomic hepatitis C virus replicons inducing expression of a secreted enzymatic reporter protein.

We constructed dicistronic, subgenomic hepatitis C virus (HCV) replicons in which the sequence encoding the human immunodeficiency virus (HIV) tat protein was placed in the upstream cistron, between the HCV 5'NTR and a picornaviral 2A proteinase sequence fused to the selectable marker Neo. Stably transformed Huh7 cells expressing secreted alkaline phosphatase (SEAP) under transcriptional control of the HIV LTR promoter actively secreted SEAP following transfection with these replicon RNAs. Extracellular SEAP activity correlated closely with intracellular HCV RNA levels, as determined by Northern blotting and real-time RT-PCR analysis. These RNAs replicated efficiently despite the absence of core-protein-coding sequence downstream of the HCV IRES. The replication efficiency of replicons derived from the HCV-N strain of HCV was significantly greater than those derived from Con1 in transiently transfected cells. Using this reporter system, we have demonstrated significant differences in the response to interferon alpha-2b in cell lines containing replicons derived from these two strains of HCV.

Virus-host cell interactions during hepatitis C virus RNA replication: impact of polyprotein expression on the cellular transcriptome and cell cycle association with viral RNA synthesis.

Considerable controversy surrounds the impact of hepatitis C virus (HCV) protein expression on viability of host cells and regulation of the cell cycle. Both promotion of cellular proliferation and apoptosis have been observed in different experimental systems. To determine whether expression of the entire complement of HCV proteins in the context of ongoing viral RNA replication significantly alters the host cell transcriptome and cell cycle regulatory processes, we carried out high-density oligonucleotide microarray studies and analyzed cell cycle distributions and S-phase entry in Huh7 cell clones harboring selectable, full-length, replicating HCV RNAs that express the entire genotype 1b, HCV-N polyprotein, and clonally related cells in which all viral RNA was eliminated by prior treatment with alpha interferon. Oligonucleotide microarray analyses revealed only subtle, coordinated differences in the mRNA profiles of cells containing replicating viral RNA and their interferon-cured progeny, with variation between different cell clones having a greater influence on the cellular transcriptome than the presence or absence of replicating HCV RNA. Flow cytometric analysis demonstrated no significant differences in cell cycle distribution among populations of asynchronously growing cells of both types. Cell lines containing replicating viral RNA and their interferon-cured progeny were able to reenter the cell cycle similarly after transient G(1) arrest. In contrast, although viral protein expression and genome replication did not alter cell cycle control in these cells, HCV genome replication was highly dependent on cellular proliferation, with viral RNA synthesis strongly decreased in poorly proliferating, confluent, or serum-starved cells and substantially enhanced in the S phase of the cell cycle.

Chronic hepatitis associated with GB virus B persistence in a tamarin after intrahepatic inoculation of synthetic viral RNA.

Progress in understanding the pathogenesis of hepatitis C virus (HCV) has been slowed by the absence of tractable small animal models. Whereas GB virus B (GBV-B, an unclassified flavivirus) shares a phylogenetic relationship and several biologic attributes with HCV, including hepatotropism, it is not known to cause persistent infection, a hallmark of HCV. Here, we document persistent GBV-B infection in one of two healthy tamarins (Saguinus oedipus) inoculated intrahepatically with infectious synthetic RNA. High-titer viremia (108 to 109 genome equivalents per ml) and transiently elevated serum alanine transaminase activities were present from weeks 4 to 12 postinoculation in both animals. However, whereas GBV-B was eliminated from one animal by 20 weeks, the second animal remained viremic (103 to 107 genome equivalents per ml) for >2 years, with alanine transaminase levels becoming elevated again before spontaneous resolution of the infection. A liver biopsy taken late in the course of infection demonstrated hepatitis with periportal mononuclear infiltrates, hepatocellular microvesicular changes, cytoplasmic lipid droplets, and disordered mitochondrial ultrastructure, findings remarkably similar to chronic hepatitis C. GBV-B-infected hepatocytes contained numerous small vesicular membranous structures resembling those associated with expression of HCV nonstructural proteins, and sequencing of GBV-B RNA demonstrated a rate of molecular evolution comparable to that of HCV. We conclude that GBV-B is capable of establishing persistent infections in healthy tamarins, a feature that substantially enhances its value as a model for HCV. Mitochondrial structural changes and altered lipid metabolism leading to steatosis are conserved features of the pathogenesis of chronic hepatitis caused by these genetically distinct flaviviruses.