Patatin is a family of glycoproteins that accounts for 30-40% of the total soluble protein in potato (Solanum tuberosum) tubers. This protein has been reported not only to serve as a storage protein, but also to exhibit enzymic activity. By using a baculovirus system to express protein from the patatin cDNA clone pGM01, it was unambiguously shown that the patatin coded by this DNA has lipid acyl hydrolase and acyltransferase activities. The enzyme is active with phospholipids, monoacylglycerols and p-nitrophenyl esters, moderately active with galactolipids, but is apparently inactive with di- and tri-acylglycerols.
GB virus B (GBV-B) is the closest relative of hepatitis C virus (HCV)and is an attractive surrogate model for HCV antiviral studies. GBV-B induces an acute, resolving hepatitis in tamarins. Utilizing primary cultures of tamarin hepatocytes, we have previously developed a tissue culture system that exhibits high levels of GBV-B replication. In this report, we have extended the utility of this system for testing antiviral compounds. Treatment with human interferon provided only a marginal antiviral effect, while poly(I-C) yielded >3 and 4 log units of reduction of cell-associated and secreted viral RNA, respectively. Interestingly, treatment of GBV-B-infected hepatocytes with ribavirin resulted in an approximately 4-log decrease in viral RNA levels. Guanosine blocked the antiviral effect of ribavirin, suggesting that inhibition of IMP dehydrogenase (IMPDH) and reduction of intracellular GTP levels were essential for the antiviral effect. However, mycophenolic acid, another IMPDH inhibitor, had no antiviral effect. Virions harvested from ribavirin-treated cultures exhibited a dramatically reduced specific infectivity. These data suggest that incorporation of ribavirin triphosphate induces error-prone replication with concomitant reduction in infectivity and that reduction of GTP pools may be required for incorporation of ribavirin triphosphate. In contrast to the in vitro studies, no significant reduction in viremia was observed in vivo following treatment of tamarins with ribavirin during acute infection with GBV-B. These findings are consistent with the observation that ribavirin monotherapy for HCV infection decreases liver disease without a significant reduction in viremia. Our data suggest that nucleoside analogues that induce error-prone replication could be an attractive approach for the treatment of HCV infection if administered at sufficient levels to result in efficient incorporation by the viral polymerase.
Hepatitis E virus (HEV) is an unclassified, plus-strand RNA virus whose genome contains three open reading frames (ORFs). ORF1, the 5' proximal ORF of HEV, encodes nonstructural proteins involved in RNA replication which share homology with the products of the corresponding ORF of members of the alphavirus-like superfamily of plus-strand RNA viruses. Among animal virus members of this superfamily (the alphavirus and rubivirus genera of the family Togaviridae), the product of this ORF is a nonstructural polyprotein (NSP) that is cleaved by a papain-like cysteine protease (PCP) within the NSP. To determine if the NSP of HEV is similarly processed, ORF1 was introduced into a plasmid vector which allowed for expression both in vitro using a coupled transcription/translation system and in vivo using a vaccinia virus-driven transient expression system. A recombinant vaccinia virus expressing ORF1 was also constructed. Both in vitro and in vivo expression under standard conditions yielded only the full-length 185 kDa polyprotein. Addition of co-factors in vitro, such as divalent cations and microsomes which have been shown to activate other viral proteases, failed to change this expression pattern. However, in vivo following extended incubations (24-36 hours), two potential processing products of 107 kDa and 78 kDa were observed. N- and C-terminus-specific immunoprecipitation and deletion mutagenesis were used to determine that the order of these products within the NSP is NH2-78 kDa-107 kDa-COOH. However, site-specific mutagenesis of Cys483, predicted by computer alignment to be one member of the catalytic dyad of a PCP within the NSP, failed to abolish this cleavage. Additionally, sequence alignment across HEV strains revealed that the other member of the proposed catalytic dyad of this PCP, His590, was not conserved. Thus, the cleavage of the NSP observed following prolonged in vivo expression was not mediated by this protease and it is doubtful that a functional PCP exists within the NSP. Attempts to detect NSP expression and processing in HEV-infected primary monkey hepatocytes were not successful and therefore this proteolytic cleavage could not be authenticated. Overall, the results of this study indicate that either the HEV NSP is not processed or that it is cleaved at one site by a virally-encoded protease novel among alpha-like superfamily viruses or a cellular protease.
GB virus-B (GBV-B)causes an acute hepatitis in tamarins characterized by increased alanine transaminase levels that quickly return to normal as the virus is cleared. Phylogenetically, GBV-B is the closest relative to hepatitis C virus (HCV), and thus GBV-B infection of tamarins represents a powerful surrogate model system for the study of HCV. In this study, the course of infection of GBV-B in tamarins was followed using a real-time 5 exonuclease (TaqMan) reverse transcription-PCR assay to determine the level of GBV-B in the serum. Peak viremia levels exceeded 10 9 genome equivalents/ml, followed by viral clearance within 14 to 16 weeks. Rechallenge of animals that had cleared infection resulted in viremia that was limited to 1 week, suggestive of a strong protective immune response. A robust tissue culture system for GBV-B was developed using primary cultures of tamarin hepatocytes. Hepatocytes obtained from a GBV-B-infected animal maintained high levels of cellassociated viral RNA and virion secretion for 42 days of culture. In vitro infection of normal hepatocytes resulted in rapid amplification of cell-associated viral RNA and secretion of up to 10 7 genome equivalents/ml of culture supernatant. In addition, infection could be monitored by immunofluorescence staining for GBV-B nonstructural NS3 protein. This model system overcomes many of the current obstacles to HCV research, including low levels of viral replication, lack of a small primate animal model, and lack of a reproducible tissue culture system. Hepatitis C virus (HCV) is a major worldwide health problem, with an estimated 2% of the population chronically infected with this virus. Chronic HCV infection can cause significant liver disease and cirrhosis of the liver and, in some patients, lead eventually to liver cancer. The current animal model for the study of HCV is the chimpanzee. However, this model system suffers from the limited availability of chimpanzees and the high cost associated with conducting studies on large nonhuman primates. A smaller, less expensive model system would be desirable.One alternative model is the hepatitis GB virus-B (GBV-B) in Saguinus species (tamarins). The GB agents, GBV-A, -B, and -C, are members of Flaviviridae (21); GBV-B is the virus most closely related to HCV (22). GBV-A and GBV-B were isolated from tamarins inoculated with a blood sample from a surgeon (GB) suffering from acute hepatitis (10). Although GBV-A and GBV-B were isolated from a tamarin inoculated with human serum believed to contain a human hepatitis virus, GBV-A and GBV-B are considered tamarin viruses. While GBV-A has been isolated from a number of tamarins (5), GBV-B has been isolated only once, and its origins are unclear. A third GB agent, GBV-C (27), also known as hepatitis G virus (19), was isolated from human serum samples in attempts to isolate new hepatitis viruses; however its association with hepatitis is tenuous (1).GBV-A causes no recognized disease in tamarins, while GBV-B causes an acute, self-limited hepatitis, as evidenced by a r...
Hepadnavirus polymerases initiate reverse transcription in a protein-primed reaction that involves the covalent linkage of the first deoxyribonucleotide to the polymerase polypeptide. Analysis of the initial steps in this reaction as well as certain details of genome replication has been hampered by the difficulties encountered in the expression of functional hepadnavirus polymerases in heterologous systems. We have expressed human hepatitis B virus (HBV) polymerase (pol) in insect cells, using the recombinant baculovirus system. Analysis of immunoaffinity-purified pol indicated that (i) a portion of pol had initiated minus-strand DNA synthesis within infected insect cells; (ii) the pol mRNA appeared to be the template for reverse transcription; (iii) the products were small (100 to 500 nucleotides); (iv) only minus-strand DNA was synthesized; (v) the products were covalently bound to protein; and (vi) the 5 end of the minus-strand DNA mapped to DR1 by primer extension. The purified pol was also active in an in vitro polymerase assay. Analyses suggested that a different fraction of pol was active in the in vitro assays. Incubation of pol with labeled deoxyribonucleotide triphosphates resulted in the labeling of the pol polypeptide in a reaction that appeared to represent in vitro nucleotide priming. In vitro nucleotide priming was confirmed by the appearance of 32 P-labeled phosphotyrosine on pol following in vitro reactions with 32 P-labeled deoxyribonucleotide triphosphates. The ability to purify significant quantities of HBV pol will facilitate functional and physical analysis of this enzyme as well as the search for novel inhibitors of HBV replication.
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