Carl Laxton

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 microM) with similar potency compared with 2'-C-methylcytidine (IC(50) = 1.13 microM). 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.

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.