Chronic hepatitis C virus (HCV) infections can be cured only in a fraction of patients treated with alpha interferon (IFN-␣) and ribavirin combination therapy. The mechanism of the IFN-␣ response against HCV is not understood, but evidence for a role for viral nonstructural protein 5A (NS5A) in IFN resistance has been provided. To elucidate the mechanism by which NS5A and possibly other viral proteins inhibit the cellular antiviral program, we have constructed a subgenomic replicon from a known infectious HCV clone and demonstrated that it has an approximately 1,000-fold-higher transduction efficiency than previously used subgenomes. We found that IFN-␣ reduced replication of HCV subgenomic replicons approximately 10-fold. The estimated half-life of viral RNA in the presence of the cytokine was about 12 h. HCV replication was sensitive to IFN-␣ independently of whether the replicon expressed an NS5A protein associated with sensitivity or resistance to the cytokine. Furthermore, our results indicated that HCV replicons can persist in Huh7 cells in the presence of high concentrations of IFN-␣. Finally, under our conditions, selection for IFN-␣-resistant variants did not occur.Hepatitis C virus (HCV) causes persistent infection in approximately 80% of infected adults and variable and severe liver disease in an estimated 70% of those who cannot clear the virus (1). HCV is an enveloped, positive-stranded RNA virus encoding a polyprotein that is proteolytically processed into 10 polypeptides. Four of them are enzymes: cysteine and serine proteases, an ATP-dependent helicase, and an RNA-directed RNA polymerase (17). While these enzymes are used as potential targets for virus-specific antiviral therapies, their genes exhibit high variability among the different HCV genotypes, and, most likely, drug-resistant variants will evolve during antiviral therapy. Currently available combination therapy with alpha interferon (IFN-␣) and ribavirin is effective in less than 50% of treated patients. Although the mechanism controlling the IFN response in patients is likely to be complex, there is evidence that nonstructural (NS) protein 5A (NS5A) evolves to confer resistance against IFN-␣ during antiviral therapy (5, 6). This resistance is believed to be a consequence of a specific interaction between NS5A and protein kinase R (PKR), an important mediator of the antiviral program induced by IFN-␣.Unfortunately, efforts to investigate the molecular mechanisms responsible for IFN resistance were hampered by the lack of tissue culture systems permissive for the replication and production of infectious HCV from available cDNA clones. Recently, Lohmann and colleagues (13) reported that a subgenomic replicon containing a neomycin phosphotransferase gene (neoR) in lieu of the viral structural genes replicated in Huh7 cells (see Fig. 1A). However, from the low frequency with which Huh7 cells supported replication, it is possible that the selected isolate is defective and requires genetic changes for efficient genome synthesis. This possibili...

The entire genome of human hepatitis B virus (HBV) occurring in Latvia was sequenced. This sequence, which is 3182 nucleotides long. was compared with the other previously published HBV genomes and was shown to share maximum homology with HBV subtype ayw DNA. The coordinates of 4 main open reading frames as well as hairpin structures are very well conserved in the two genomes. The distribution of nucleotide substitutions among different HBV genomes suggests that the open reading frames P and X can fultil a coding function. On the basis of primary stucture comparison for hepadnaviral DNAs several evolutionary conclusions can be drawn. Hepatitis B virusDNA sequence

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Cationic liposomes are known to facilitate efficient transfection of animal cells with DNA and even some viruses. As reported here, we have been able to use such a commercially available formulation (Lipofectamine) and introduce human hepatitis delta virus (HDV) into lines of cultured cells and demonstrate replication of the HDV genome both by immunofluorescence and by Northern (RNA) analysis. As much as 10%/ of the human hepatoma cell line Huh7 was transfected with HDV. Also transfected were the baby hamster kidney cell line BHK-21 and the Morris rat hepatoma line 7777. Two initial applications of HDV transfection have been made. (i) The ribonucleoprotein structure of HDV was isolated from disrupted virions and demonstrated as being sufficient to transfect Huh7 cells. In contrast, naked HDV RNA was not sufficient. (ii) From a study of cells transfected with HDV particles, it was found that, even after as long as 7 weeks and the associated replication of the transfected cells, the HDV RNA genome was still replicating. Apparently, HDV, in the absence of helper virus and in the absence of virus assembly, can maintain persistent replication and expression of the HDV genome. Transfection was also achieved with woodchuck hepatitis virus introduced into Huh7 cells. In on August 3, 2020 by guest http://jvi.asm.org/ Downloaded from 5248 BICHKO ET AL.