We have produced a cell line which lacks the protein tyrosine kinase JAK1 and is completely defective in interferon response. Complementation of this mutant with JAK1 restored the response, establishing the requirement for JAK1 in both the interferon-alpha/beta and -gamma signal transduction pathways. The reciprocal interdependence between JAK1 and Tyk2 activities in the interferon-alpha pathway, and between JAK1 and JAK2 in the interferon-gamma pathway, may reflect a requirement for these kinases in the correct assembly of interferon receptor complexes.

Mutants in complementation group U3, completely defective in the response of all genes tested to interferons (IFNs) alpha and gamma, do not express the 91 and 84 kDa polypeptide components of interferon‐stimulated gene factor 3 (ISGF3), a transcription factor known to play a primary role in the IFN‐alpha response pathway. The 91 and 84 kDa polypeptides are products of a single gene. They result from differential splicing and differ only in a 38 amino acid extension at the C‐terminus of the 91 kDa polypeptide. Complementation of U3 mutants with cDNA constructs expressing the 91 kDa product at levels comparable to those observed in induced wild‐type cells completely restored the response to both IFN‐alpha and ‐gamma and the ability to form ISGF3. Complementation with the 84 kDa component similarly restored the ability to form ISGF3 and, albeit to a lower level, the IFN‐alpha response of all genes tested so far. It failed, however, to restore the IFN‐gamma response of any gene analysed. The precise nature of the DNA motifs and combination of factors required for the transcriptional response of all genes inducible by IFN‐alpha and ‐gamma remains to be established. The results presented here, however, emphasize the apparent general requirement of the 91 kDa polypeptide in the primary transcriptional response to both types of IFN.

Hepatitis C virus (HCV) polymerase activity is essential for HCV replication. Targeted screening of nucleoside analogs identified R1479 (4-azidocytidine) as a specific inhibitor of HCV replication in the HCV subgenomic replicon system (IC 50 ‫؍‬ 1.28 M) with similar potency compared with 2-C-methylcytidine (IC 50 ‫؍‬ 1.13 M). R1479 showed no effect on cell viability or proliferation of HCV replicon or Huh-7 cells at concentrations up to 2 mM. HCV replicon RNA could be fully cleared from replicon cells after prolonged incubation with R1479. The corresponding 5-triphosphate derivative (R1479-TP) is a potent inhibitor of native HCV replicase isolated from replicon cells and of recombinant HCV polymerase (NS5B)-mediated RNA synthesis activity. R1479-TP inhibited RNA synthesis as a CTP-competitive inhibitor with a K i of 40 nM. On an HCV RNA-derived template substrate (complementary internal ribosome entry site), R1479-TP showed similar potency of NS5B inhibition compared with 3-dCTP. R1479-TP was incorporated into nascent RNA by HCV polymerase and reduced further elongation with similar efficiency compared with 3-dCTP under the reaction conditions. The S282T point mutation in the coding sequence of NS5B confers resistance to inhibition by 2-C-MeATP and other 2-methyl-nucleotides. In contrast, the S282T mutation did not confer cross-resistance to R1479. Hepatitis C virus (HCV)2 infection is a major cause of chronic liver disease, cirrhosis, and hepatocellular carcinoma and is currently the leading cause of liver transplantation (1, 2). Viral genome sequence analysis established six HCV genotype classes (HCV genotypes 1-6), with genotypes 1-3 being the most prevalent in the United States, Europe, and Japan. Current treatment options available to HCV-infected persons are limited, and sustained virological response rates are particularly low for HCV genotype 1-infected patients. Only ϳ50% of individuals infected with HCV genotype 1 with serum viral titers of Ͼ2 ϫ 10 6 copies/ml achieved sustained virological response rates when treated with a combination of pegylated interferon-␣ and ribavirin (3, 4). Response rates are even lower in persons with HIV co-infection or cirrhosis and also decrease with age (1, 5-7). Urgently required improvements in anti-HCV therapy will depend on the development of novel therapeutic approaches, especially in difficult to treat populations.HCV is an enveloped (ϩ)-strand RNA virus that enters host cells via receptor-mediated endocytosis and replicates in the host cell cytoplasm. A membrane-associated replicase complex containing HCV genome-encoded nonstructural proteins and HCV genomic RNA in a tight complex is responsible for the formation of viral RNA for packaging into new virus particles during the HCV replication process. The viral NS5B protein contains the HCV polymerase active site within the replicase complex, an RNA-dependent RNA polymerase. The concept of polymerase inhibition to attain antiviral efficacy has been successfully established in other viral infections (human immunodefi...

Expression of the E1A oncogene of adenovirus type 5 inhibits the response of interferon (IFN)-inducible constructs to Type I (alpha,beta) and II (gamma) IFNs in transient transfection assays. In human cell lines stably expressing E1A mRNA and protein acquisition of an antiviral state and the induction of a number of genes in response to alpha- and gamma-IFNs is inhibited. A short IFN-stimulable response element (ISRE) present in the 5' flanking region of a number of genes mediates induction by alpha- and gamma-IFNs. In cells expressing E1A there is a substantial reduction in the levels of the ISRE-binding factors E and M, inducible by alpha-IFN, and of factor G, inducible by gamma-IFN. In E1A-expressing cells the E alpha subunit of factor E is activated normally in response to alpha-IFN; the defect is in the production or activation of the E gamma subunit. The inhibitory activity of E1A is lost upon deletion of the CR1 domain. The induction of HLA class II genes by gamma-IFN, which involves a different DNA response element(s), and of beta-IFN mRNA in response to double-stranded RNA are also inhibited by E1A. An essential component(s) of a number of signalling pathways must, therefore, be subject, directly or indirectly, to inhibition by E1A.

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