Hepatitis C virus (HCV) is one of the most important Flaviviridae infections in humans and is responsible for the second most common cause of viral hepatitis. Presently, nearly 2% of the U.S. population, and an estimated 170 million people worldwide, are HCV carriers (2). The only approved therapy for chronic hepatitis C is alpha interferon (IFN-␣), either alone or in combination with ribavirin. Anemia is the most common adverse effect associated with ribavirin treatment, and neuropsychiatric adverse effects of IFN-␣ lead to premature cessation of therapy in 10 to 20% of patients (9, 13).As additional treatment options are urgently needed, there is an ongoing search for more potent antiviral compounds with fewer adverse effects. However, the search for improved antiviral agents is hampered by the limited and cumbersome propagation of HCV in vitro (4). Therefore, surrogate models such as the HCV RNA replicon that replicates in the human hepatoma cell line Huh7 have been developed (6,29). Improved versions of these HCV replicons contain adaptive mutations (25), and their use has facilitated the evaluation of candidate anti-HCV drugs.Bovine viral diarrhea virus (BVDV) is one of the best characterized members of the Flaviviridae family and has one of the largest RNA genomes (12.5 kb) in this family (8). This virus has the remarkable property of existing as noncytopathic and cytopathic (cpBVDV) biotypes, with cpBVDV strains showing insertions or viral genome rearrangements at the junction site between nonstructural protein 2 (NS2) and NS3 (32). BVDV may provide a surrogate model for HCV, both for the molecular study of viral proteins (33) and for the evaluation of antiviral compounds (3,7,47).In the search for therapeutic agents, any element that is essential for viral (or replicon) RNA replication may be considered a drug discovery target. Such elements can be either viral proteins (NS2-NS3 protease, NS3-NS4A serine proteinase, NS3 RNA helicase, or RNA-dependent RNA polymerase [3,24,34,36] [18,31,41,43]). Current knowledge of the human genome, combined with array technology and pathogen infection models, will likely lead to more defined host-pathogen-related targets for future drug design (17, 23). Today, however, the most successful classes of antiviral compounds with clinical utility in combat against other human viral pathogens (human immunodeficiency virus type 1 [HIV-1], hepatitis B virus [HBV], herpes simplex virus [HSV], and cytomegalovirus) are the protease, the nonnucleoside analogue, and the nucleoside analogue inhibitors. As the latter class of compounds is crucial in controlling herpesvirus, HIV-1, and HBV infections, it is likely and anticipated that

It is well known that hepatitis B virus infections can be transient or chronic, but the basis for this dichotomy is not known. To gain insight into the mechanism responsible for the clearance of hepadnavirus infections, we have performed a molecular and histologic analysis of liver tissues obtained from transiently infected woodchucks during the critical phase of the recovery period. We found as expected that clearance from transient infections occurred subsequent to the appearance of CD4 ؉ and CD8 ؉ T cells and the production of interferon gamma and tumor necrosis factor alpha in the infected liver. These events were accompanied by a significant increase in apoptosis and regeneration of hepatocytes. Surprisingly, however, accumulation of virus-free hepatocytes was delayed for several weeks following this initial influx of lymphocytes. In addition, we observed that chronically infected animals can exhibit levels of T-cell accumulation, cytokine expression, and apoptosis that are comparable with those observed during the initial phase of transient infections. Our results are most consistent with a model for recovery predicting replacement of infected hepatocytes with regenerated cells, which by unknown mechanisms remain protected from reinfection in animals that can be cured.Human hepatitis B virus (HBV), as well as the related woodchuck hepatitis virus (WHV), can cause transient or chronic infections in its native host (11,24,27). The molecular basis for the dichotomy of disease outcomes is not known. As in humans, in woodchucks chronic, lifelong WHV infections generally occur when virus is transmitted during or soon after birth. Infection of adults leads to transient infections in over 90% of cases. Experiments with woodchucks have shown that clearance of infections can occur within a few weeks even when nearly all hepatocytes in the liver have been infected (14, 20). Thus, a major question concerns the molecular mechanism responsible for the regulation of clearance of virus from infected hepatocytes.Clearance from infections with noncytopathic viruses, such as hepadnaviruses, requires the elimination of infected cells by cytotoxic T lymphocytes (CTLs) and the production of neutralizing antibodies directed against one or several viral proteins (13). A role for T cells in the recovery from natural hepadnavirus infections has been demonstrated through treatment with cyclosporin A, a known suppressor of T-cell function, which prevents recovery from otherwise transient WHV infections in adult woodchucks (4). It also appears that the number of CTLs present in the peripheral blood of chronically infected patients is approximately 10 to 100 times lower than that in the blood of patients with transient infections (23), suggesting that a critical number of reactive CTLs are required for recovery. In this scenario all infected hepatocytes would have to be killed by CTLs and replaced by uninfected cells. In order to sustain sufficient liver function, the rate of cell death should not exceed the rate of cell replacement ov...

) is a potent inhibitor of hepatitis C virus (HCV) RNA replication in an HCV replicon assay. The 5-triphosphate of PSI-6130 is a competitive inhibitor of the HCV RNA-dependent RNA polymerase (RdRp) and acts as a nonobligate chain terminator. Recently, it has been shown that the metabolism of PSI-6130 also results in the formation of the 5-triphosphate of the uridine congener, ␤-D-2-deoxy-2-fluoro-2-C-methyluridine (PSI-6206; RO2433). Here we show that the formation of the 5-triphosphate of RO2433 (RO2433-TP) requires the deamination of PSI-6130 monophosphate and that RO2433 monophosphate is subsequently phosphorylated to the corresponding di-and triphosphates by cellular UMP-CMP kinase and nucleoside diphosphate kinase, respectively. RO2433-TP is a potent inhibitor of the HCV RdRp; however, both enzymatic and cell-based assays show that PSI-6130 triphosphate is a more potent inhibitor of the HCV RdRp than RO2433-TP.Hepatitis C virus (HCV), a member of the Flaviviridae family of viruses, is one of the major causes of liver disease. Nearly 2% of the U.S. population and an estimated 170 million people worldwide are believed to be infected with HCV (2, 20). Approximately 80% of infected individuals develop a chronic infection, and long-term chronic HCV infection can lead to liver cirrhosis and hepatocellular carcinoma (7,20,24). The current standard of care is a combination of pegylated interferon alpha and ribavirin (2,6,8,21), which produces viral response rates in approximately 50% of patients infected with genotype 1 virus. Due to the adverse effects associated with both interferon and ribavirin therapy and the lack of an optimal sustained viral response in the majority of patients infected with HCV, there is a need for more potent anti-HCV compounds with fewer adverse effects.The HCV RNA-dependent RNA polymerase (RdRp; the NS5B protein) is essential for virus RNA replication and therefore represents an attractive target for therapy (3,11,14,15,25). Since nucleoside analogs form the cornerstone of therapy against human immunodeficiency virus, hepatitis B virus, and herpesviruses, such an approach to the treatment of HCV infection should prove equally effective. Recently, several nucleoside analogs with modifications at either the 2Ј or the 4Ј position have demonstrated good activity against HCV in vitro and in vivo (4, 12, 18).The discovery and development of nucleoside analogs require an understanding of the pathways and enzymes involved in the anabolism of an analog to the active triphosphate form. We have shown that ␤-D-2Ј-deoxy-2Ј-fluoro-2Ј-C-methylcytidine (PSI-6130) is a potent and selective inhibitor of HCV RNA synthesis in an HCV replicon assay (5,19,22). Recently, we reported that PSI-6130 is anabolized to the 5Ј-triphosphate by enzymes involved in the deoxycytidine salvage pathway and that 2Ј-fluoro-2Ј-C-methylcytidine 5Ј-triphosphate (PSI-6130-TP) is an inhibitor of the HCV RdRp (19). In vitro metabolism studies have now shown that in addition to the formation of PSI-6130-TP, the 5Ј-triphosphate of th...

) is a potent specific inhibitor of hepatitis C virus (HCV) RNA synthesis in Huh-7 replicon cells. To inhibit the HCV NS5B RNA polymerase, PSI-6130 must be phosphorylated to the 5-triphosphate form. The phosphorylation of PSI-6130 and inhibition of HCV NS5B were investigated. The phosphorylation of PSI-6130 by recombinant human 2-deoxycytidine kinase (dCK) and uridine-cytidine kinase 1 (UCK-1) was measured by using a coupled spectrophotometric reaction. PSI-6130 was shown to be a substrate for purified dCK, with a K m of 81 M and a k cat of 0.007 s ؊1 , but was not a substrate for UCK-1. PSI-6130 monophosphate (PSI-6130-MP) was efficiently phosphorylated to the diphosphate and subsequently to the triphosphate by recombinant human UMP-CMP kinase and nucleoside diphosphate kinase, respectively. The inhibition of wild-type and mutated (S282T) HCV NS5B RNA polymerases was studied. The steady-state inhibition constant (K i ) for PSI-6130 triphosphate (PSI-6130-TP) with the wild-type enzyme was 4.3 M. Similar results were obtained with 2-C-methyladenosine triphosphate (K i ‫؍‬ 1.5 M) and 2-C-methylcytidine triphosphate (K i ‫؍‬ 1.6 M). NS5B with the S282T mutation, which is known to confer resistance to 2-C-methyladenosine, was inhibited by PSI-6130-TP as efficiently as the wild type. Incorporation of PSI-6130-MP into RNA catalyzed by purified NS5B RNA polymerase resulted in chain termination.Hepatitis C virus (HCV) is an RNA virus which possesses a single-stranded positive-sense RNA as the viral genome. This viral RNA plays important roles during viral replication, as it serves as an mRNA for viral protein synthesis, a template for RNA replication, and a nascent RNA genome for a newly formed virus (17). HCV NS5B RNA-dependent RNA polymerase is a key enzyme in viral RNA replication. This enzyme, which does not require a primer for initiation of RNA synthesis, catalyzes de novo RNA synthesis (8, 11). Nucleoside analogs have been used to treat viral infections, such as herpes simplex virus, human immunodeficiency virus, and hepatitis B virus infections (5,6,21). These drugs are designed to inhibit viral polymerases by a process called chain termination, in which DNA synthesis is quenched by incorporating the triphosphate forms of these drugs, which lack the 3Ј-hydroxyl group on the sugar moiety. In order for nucleoside analogs to inhibit a viral polymerase, they must be transported into the cell and converted to the active 5Ј-triphosphate form by cellular kinases. 2Ј-C-Methylnucleosides have been investigated as anti-HCV agents targeting HCV NS5B RNA polymerase (2, 20). 2Ј-C-Methyladenosine (2Ј-C-Me-A) and 2Ј-C-methylguanosine (2Ј-C-Me-G) showed potent anti-HCV activities in a cell-based replicon assay, and their triphosphate forms inhibited replicase and NS5B RNA polymerase in vitro (20). In addition, 2Ј-C-Me-A exhibited significant activity against HCV in a cell culture system which involves complete HCV replication and which produces infectious HCV (16). A resistant replicon has been selected by passage of HCV in the...

The antiviral activity of 2'-C-MeC against strains of two different NoV genogroups and the low EC(50) suggest that this nucleoside analogue may be effective against the more prevalent GII NoVs. In the absence of a vaccine, antiviral agents could be an effective intervention to control the spread of human NoV in populations at a high risk for NoV disease.