The helicase domain of hepatitis C virus NS3 (genotype 1b) was expressed in Escherichia coli and purified to homogeneity. The purified protein catalyzed the hydrolysis of nucleoside triphosphates (NTP) and the unwinding of duplex RNA in the presence of divalent metal ion. The enzyme was not selective for the NTP substrate. For example, UTP and acyclovir triphosphate were hydrolyzed efficiently by the enzyme. The rate of NTP hydrolysis was stimulated up to 27-fold by oligomeric nucleic acids (NA). Furthermore, NA bound to the enzyme with concomitant quenching of the intrinsic protein fluorescence. The dissociation constants of the enzyme for selected NA in the absence of NTP were between 10 and 35 M at pH 7.0 and 25°C. ؊1 ), respectively. These data were consistent with a random kinetic mechanism. Hepatitis C virus (HCV)1 is the principle agent responsible for non-A, non-B hepatitis (1, 2). Approximately 1% of the human population is infected with HCV. Standard interferon therapies are effective for some patients, but the majority do not clear the virus, resulting in relapse (3). Consequently, an urgent medical need for an effective antiviral agent exists.Development of an effective therapeutic agent has been hindered by the lack of reliable cell culture systems for propagating HCV. Consequently, efforts to characterize potential therapeutic agents have relied on surrogate expression systems and the techniques of molecular biology. For example, the NS3 protein of hepatitis C virus has several enzymatic activities necessary for viral replication that make it an attractive antiviral target. The N-terminal 20 kDa of NS3 is a serine proteinase that cleaves the HCV-encoded polyprotein at a minimum of four specific sites (4). The C-terminal 50 kDa of NS3 has NTPase (5) and RNA helicase activity (6). A detailed understanding of either of these enzymatic activities could facilitate identification of potent antiviral agents. Consequently, we have initiated a program to characterize the interaction of the HCV helicase domain with nucleoside triphosphates (NTP) and polymeric nucleic acids (NA). HCV helicase catalyzes the three reactions shown in Scheme I.dsNA ϩ NTP 3 ssNA ϩ NDP ϩ P i (c) SCHEME I. Reactions catalyzed by HCV helicase where NA is single stranded (ss) or double stranded (ds) polynucleic acid and NTP is a nucleoside triphosphate.The physiologically relevant reaction for viral replication is probably unwinding of double-stranded NA (reaction c). The kinetic studies presented herein, which represent the first comprehensive kinetic analysis of a RNA helicase, have focused on the NTPase activities of the HCV helicase in the presence or absence of NA (reactions a and b in Scheme I). We have chosen to work with HCV genotype 1b, which is a major subtype found in both the Japanese and American populations (7). In summary, we found that 1) the NTPase activity was nonselective for the nucleobase and sugar of the NTP substrate, 2) the NTPase activity was stimulated up to 25-fold by selected NA in a reaction that was rela...
1592U89, (-)-(1S,4R)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclo pentene-1-methanol, is a carbocyclic nucleoside with a unique biological profile giving potent, selective anti-human immunodeficiency virus (HIV) activity. 1592U89 was selected after evaluation of a wide variety of analogs containing a cyclopentene substitution for the 2'-deoxyriboside of natural deoxynucleosides, optimizing in vitro anti-HIV potency, oral bioavailability, and central nervous system (CNS) penetration. 1592U89 was equivalent in potency to 3'-azido-3'-deoxythymidine (AZT) in human peripheral blood lymphocyte (PBL) cultures against clinical isolates of HIV type 1 (HIV-1) from antiretroviral drug-naive patients (average 50% inhibitory concentration [IC50], 0.26 microM for 1592U89 and 0.23 microM for AZT). 1592U89 showed minimal cross-resistance (approximately twofold) with AZT and other approved HIV reverse transcriptase (RT) inhibitors. 1592U89 was synergistic in combination with AZT, the nonnucleoside RT inhibitor nevirapine, and the protease inhibitor 141W94 in MT4 cells against HIV-1 (IIIB). 1592U89 was anabolized intracellularly to its 5'-monophosphate in CD4+ CEM cells and in PBLs, but the di- and triphosphates of 1592U89 were not detected. The only triphosphate found in cells incubated with 1592U89 was that of the guanine analog (-)-carbovir (CBV). However, the in vivo pharmacokinetic, distribution, and toxicological profiles of 1592U89 were distinct from and improved over those of CBV, probably because CBV itself was not appreciably formed from 1592U89 in cells or animals (<2%). The 5'-triphosphate of CBV was a potent, selective inhibitor of HIV-1 RT, with Ki values for DNA polymerases (alpha, beta, gamma, and epsilon which were 90-, 2,900-, 1,200-, and 1,900-fold greater, respectively, than for RT (Ki, 21 nM). 1592U89 was relatively nontoxic to human bone marrow progenitors erythroid burst-forming unit and granulocyte-macrophage CFU (IC50s, 110 microM) and human leukemic and liver tumor cell lines. 1592U89 had excellent oral bioavailability (105% in the rat) and penetrated the CNS (rat brain and monkey cerebrospinal fluid) as well as AZT. Having demonstrated an excellent preclinical profile, 1592U89 has progressed to clinical evaluation in HIV-infected patients.
The recently developed hepatitis C virus (HCV) subgenomic replicon system was utilized to evaluate the efficacy of several known antiviral agents. Cell lines that persistently maintained a genotype 1b replicon were selected. The replicon resident in each cell line had acquired adaptive mutations in the NS5A region that increased colony-forming efficiency, and some replicons had acquired NS3 mutations that alone did not enhance colony-forming efficiency but were synergistic with NS5A mutations. A replicon constructed from the infectious clone of the HCV-1 strain (genotype 1a) was not capable of inducing colony formation even after the introduction of adaptive mutations identified in the genotype 1b replicon. Alpha interferon (IFN-␣), IFN-␥, and ribavirin exhibited antiviral activity, while double-stranded RNA (dsRNA) and tumor necrosis factor alpha did not. Analysis of transcript levels for a series of genes stimulated by IFN (ISGs) or dsRNA following treatment with IFN-␣, IFN-␥, and dsRNA revealed that both IFNs increased ISG transcript levels, but that some aspect of the dsRNA response pathway was defective in Huh7 cells and replicon cell lines in comparison to primary chimpanzee and tamarin hepatocytes. The colony-forming efficiency of the replicon was reduced or eliminated following replication in the presence of ribavirin, implicating the induction of error-prone replication. The potential role of error-prone replication in the synergy observed between IFN-␣ and ribavirin in attaining sustained viral clearance is discussed. These studies reveal characteristics of Huh7 cells that may contribute to their unique capacity to support HCV RNA synthesis and demonstrate the utility of the replicon system for mechanistic studies on antiviral agents.Chronic hepatitis C virus (HCV) infections are one of the leading causes of liver disease worldwide (2). The prevalence of HCV infections is 1 to 2%, although certain geographical regions, age groups, and ethnic groups have much higher rates of infection (3). Although symptoms may be mild for decades, 20% of persistently infected individuals may eventually develop serious liver disease including cirrhosis and liver cancer (2). HCV infection is the leading cause for liver transplantation in the United States (12). Although the initial use of interferon (IFN) for treatment of chronic infections yielded marginal results, the current therapeutic regimen of pegylated alpha 2b IFN (IFN-␣2b) and ribavirin provides substantially improved rates of sustained viral clearance of 42 and 82% for genotype 1 and genotype 2 and 3, respectively (45). Treatment of acute infections with standard IFN therapy without ribavirin is highly efficacious and approaches 100% sustained viral clearance (33). Nonetheless, a great need exists for improved antiviral agents, since many patients still do not benefit from IFN therapy, and IFN therapy is associated with undesirable side effects. The lack of a suitable tissue culture system has previously hampered the development of antiviral agents, but the re...
Hepatitis B viruses establish a chronic, productive, and noncytopathic infection of hepatocytes. Viral products are produced by transcription from multiple copies (5-50) of covalently closed circular (ccc) viral DNA. This cccDNA does not replicate, but can be replaced by DNA precursors that are synthesized in the cytoplasm. The present study was carried out to determine if long-term treatment with an inhibitor of viral DNA synthesis would lead to loss of virus products, including cccDNA, from the liver of woodchucks chronically infected with woodchuck hepatitis virus. Viral DNA synthesis was inhibited with the nucleoside analog, lamivudine (2'-deoxy-3'-thiacytidine). Lamivudine treatment produced a slow but progressive decline in viral titers in serum, to about 0.3% or less of the initial level. However, even after maintenance of drug therapy for 3-12 months, > 95% of the hepatocytes in most animals were still infected. Significant declines in the percentage of infected hepatocytes and of intrahepatic cccDNA levels were observed in only three woodchucks, two in the group receiving lamivudine and one in the placebo control group. Moreover, virus titers eventually rose in woodchucks receiving lamivudine, suggesting that drug-resistant viruses began to spread through the liver starting at least as early as 9-12 months of treatment. Three types of mutation that may be associated with drug resistance were found at this time, in a region upstream of the YMDD motif in the active site of the viral reverse transcriptase. The YMDD motif itself remained unchanged. Not unexpectedly, the lamivudine therapy did not have a impact on development of liver cancer.
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