A direct comparison of the inhibitory effects of alpha, beta, and gamma interferons (IFNs) on replication of a hepatitis C virus subgenomic replicon in a hepatoma cell line revealed similarities in antiviral potency. However, alternate IFN-induced antiviral mechanisms were suggested following observations of striking differences between IFN-␥ and IFN-␣/␤ with respect to strength and durability of the antiviral response and the magnitude and pattern of IFN-mediated gene expression.Interferons (IFNs) are a family of cytokines that share the property of inducing proteins that inhibit virus replication (23). IFNs are not constitutively synthesized by cells but respond to external stimuli, such as virus infections, that trigger their transient synthesis and secretion. Binding of IFN to its receptor elicits signals that induce the transcription of a specific set of genes. These genes encode proteins that carry out the diverse functions of the IFN system including mediating antiviral activity. IFNs are classified as either alpha/beta IFN (IFN-␣/␤) or IFN-␥. IFN-␣ and -␤ are readily induced by double-stranded viral RNA, primarily in leukocytes and fibroblasts. These IFNs then induce some of the known IFN-responsive antiviral effector mechanisms, including (i) 2Ј,5Јoligoadenylate synthetase (2Ј,5Ј-OAS) (which then activates the constitutively expressed RNase L to degrade viral RNAs), (ii) double-stranded RNAactivated protein kinase (which inhibits viral protein synthesis), and (iii) MxA (a GTPase that blocks transport of viral ribonucleoproteins to the nucleus) (23). The IFN-␣/␤-regulated genes induce direct antiviral activity as part of the early or innate host immune response to limit viral infection. The host adaptive antiviral response mediated by CD8 ϩ cytotoxic T lymphocytes and natural killer cells and by virus-specific neutralizing antibodies then eradicates virus and virus-infected cells. IFN-␥ is induced as part of the adaptive immune response by a wide variety of stimuli and is restricted to T cells and natural killer cells (4). Little is known about the direct antiviral activity of IFN-␥, particularly with respect to the mechanism, magnitude, and kinetics of its antiviral activity in comparison with IFN-␣ and -␤.Hepatitis C virus (HCV), a member of the Flaviviridae family, has a single-stranded positive-sense RNA genome encoding a polyprotein of at least 10 distinct gene products (5). Infections caused by HCV are a major medical concern since as much as 3% of the worldwide population is estimated to be HCV seropositive (1). At present, the most widely prescribed HCV antiviral therapy is the combination of polyethylene glycol-modified IFN-␣2b (PEG-Intron) and ribavirin (18), a treatment capable of inducing sustained virologic responses in 54 to 56% of chronically infected patients (11a, 18). Although this is a significant improvement from the sustained response rate of IFN-␣ monotherapy (6 to 16%) (7, 21, 24) and IFN-␣ plus ribavirin (40%) (8, 19), improvement in sustained response rates still remains the majo...

Replication of hepatitis C virus (HCV) RNA in virus-infected cells is believed to be catalyzed by viral replicase complexes (RCs), which may consist of various virally encoded nonstructural proteins and host factors. In this study, we characterized the RC activity of a crude membrane fraction isolated from HCV subgenomic replicon cells. The RC preparation was able to use endogenous replicon RNA as a template to synthesize both single-stranded (ss) and double-stranded (ds) RNA products. Divalent cations (Mg 2؉ and Mn 2؉ ) showed different effects on RNA synthesis. Mg 2؉ ions stimulated the synthesis of ss RNA but had little effect on the synthesis of ds RNA. In contrast, Mn 2؉ ions enhanced primarily the synthesis of ds RNA. Interestingly, ss RNA could be synthesized under certain conditions in the absence of ds RNA, and vice versa, suggesting that the ss and ds RNA were derived either from different forms of replicative intermediates or from different RCs. Pulse-chase analysis showed that radioactivity incorporated into the ss RNA was chased into the ds RNA and other larger RNA species. This observation indicated that the newly synthesized ss RNA could serve as a template for a further round of RNA synthesis. Finally, 3 deoxyribonucleoside triphosphates were able to inhibit RNA synthesis in this cell-free system, presumably through chain termination, with 3 dGTP having the highest potency. Establishment of the replicase assay will facilitate the identification and evaluation of potential inhibitors that would act against the entire RC of HCV.Hepatitis C virus (HCV) is the major cause of non-A, non-B transfusion-associated hepatitis and accounts for a significant proportion of viral hepatitis cases worldwide (9, 18). Although HCV infection is resolved in some subjects, up to 80% of infected individuals develop chronic HCV infection. HCV is a positive-sense RNA virus belonging to the Flaviviridae family (5). The life cycle of HCV consists of several important processes that occur primarily in the cytoplasm of the host cells. Among these important processes, RNA replication is a key step in the viral proliferation cycle and is thus an important target for antiviral development. The genome of HCV consists of approximately 9,600 bases, with a single open reading frame encoding a polyprotein of around 3,010 amino acids. The open reading frame is flanked by 5Ј and 3Ј untranslated regions, which are important for translation and RNA replication. The polyprotein is cleaved both co-and posttranslationally by cellular and virally encoded proteases into at least 10 different structural and nonstructural proteins (1, 9, 18).The NS5B protein of HCV has been shown to possess RNAdependent RNA polymerase activity in vitro. However, recombinant NS5B alone lacks the strict template specificity required for RNA synthesis (2, 6-8, 11, 13-15, 21, 27, 28). The lack of template specificity is presumably due to the absence of other viral or host factors that constitute the viral replicase complexes (RCs) in vivo. It is believed that RCs...

Hepatitis C virus (HCV) nonstructural protein 5B (NS5B) is an RNA-dependent RNA polymerase (RdRp) essential for virus replication. Several consensus sequence motifs have been identified in NS5B, some of which have been shown to be critical for its enzymatic activity. A unique beta-hairpin structure located between amino acids 443 and 454 in the thumb subdomain has also been shown to play an important role in ensuring terminal initiation of RNA synthesis in vitro. However, the importance of these sequence and structural elements in viral RNA replication in infected cells has not been established, mainly due to the lack of a reliable cell culture system for HCV. In this study, we investigated the effect of several single amino acid substitutions and beta-hairpin truncations in NS5B on viral RNA replication by using the subgenomic replicon cell culture system. A strong correlation between in vitro polymerase activity and viral RNA replication was observed with most of the substitutions. Interestingly, truncations of the beta-hairpin (by four and eight amino acid residues, respectively), which did not reduce the in vitro enzymatic activity, completely abolished the ability of the replicon RNA to replicate in Huh-7 cells, demonstrating its essential role in viral RNA replication. Furthermore, a conservative substitution in motif D, from an arginine residue (AMTR(345)), which is conserved among all HCV isolates, to a lysine residue, resulted in significant improvements in both transient RNA replication and colony formation efficiencies. This result also correlates with a previous observation that the enzymatic activity of NS5B increased by about 50% when the same NS5B substitution was introduced (V. Lohmann, F. Korner, U. Herian, and R. Bartenschlager, J. Virol. 1997, 71, 8416-8428).

We describe the development of a selectable, bi-cistronic subgenomic replicon for bovine viral diarrhea virus (BVDV) in Huh-7 cells, similar to that established for hepatitis C virus (HCV). The selection marker and reporter (Luc-Ubi-Neo) in the BVDV replicon was fused with the amino-terminal protease N pro , and expression of the nonstructural proteins (NS3 to NS5B) was driven by an encephalomyocarditis virus internal ribosome entry site. This BVDV replicon allows us to compare RNA replication of these two related viruses in a similar cellular background and to identify antiviral molecules specific for HCV RNA replication. The BVDV replicon showed similar sensitivity as the HCV replicon to interferons (alpha, beta, and gamma) and 2-␤-C-methyl ribonucleoside inhibitors. Known nonnucleoside inhibitor molecules specific for either HCV or BVDV can be easily distinguished by using the parallel replicon systems. The HCV replicon has been shown to block, via the NS3/4A serine protease, Sendai virus-induced activation of interferon regulatory factor 3 (IRF-3), a key antiviral signaling molecule. Similar suppression of IRF-3-mediated responses was also observed with the Huh-7-BVDV replicon but was independent of NS3/4A protease activity. Instead, the amino-terminal cysteine protease N pro of BVDV appears to be, at least partly, responsible for suppressing IRF-3 activation induced by Sendai virus infection. This result suggests that different viruses, including those closely related, may have developed unique mechanisms for evading host antiviral responses. The parallel BVDV and HCV replicon systems provide robust counterscreens to distinguish viral specificity of small-molecule inhibitors of viral replication and to study the interactions of the viral replication machinery with the host cell innate immune system. Hepatitis C virus (HCV) and bovine viral diarrhea virus (BVDV) are both members of the Flaviviridae family and share many molecular and virological similarities (20, 27). They both have single-strand RNA genomes that replicate via negativestrand intermediates and produce a single polyprotein that is cleaved into individual proteins by a combination of host and viral proteases. Although HCV is a hepacivirus and BVDV is a pestivirus, there is a low degree of sequence homology between their respective nonstructural proteins and both RNAdependent RNA polymerases are structurally very similar (9).HCV is a major etiological agent for viral hepatitis. The World Health Organization estimates that 170 million people are chronically infected with HCV worldwide, and of those, 4 million are in the United States. Within 10 to 20 years of infection, 20 to 30% of chronic carriers develop cirrhosis, making HCV infection one of the major reasons for liver transplantation. Current therapies are limited to interferon treatment, either alone or in combination with ribavirin (23, 38), but patient response for certain genotypes is still unsatisfactory. This unmet medical need has created an urgent demand for the development of new dr...

Mutations in the Fas (apo-1, CD95) gene result in autoimmune lymphoproliferative syndrome (ALPS).