Terminal nucleotidyl transferase activity of recombinant Flaviviridae RNA-dependent RNA polymerases: implication for viral RNA synthesis.

Recombinant hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) was reported to possess terminal transferase (TNTase) activity, the ability to add nontemplated nucleotides to the 3' end of viral RNAs. However, this TNTase was later purported to be a cellular enzyme copurifying with the HCV RdRp. In this report, we present evidence that TNTase activity is an inherent function of HCV and bovine viral diarrhea virus RdRps highly purified from both prokaryotic and eukaryotic cells. A change of the highly conserved GDD catalytic motif in the HCV RdRp to GAA abolished both RNA synthesis and TNTase activity. Furthermore, the nucleotides added via this TNTase activity are strongly influenced by the sequence near the 3' terminus of the viral template RNA, perhaps accounting for the previous discrepant observations between RdRp preparations. Last, the RdRp TNTase activity was shown to restore the ability to direct initiation of RNA synthesis in vitro on an initiation-defective RNA substrate, thereby implicating this activity in maintaining the integrity of the viral genome termini.

The RNA helicase and nucleotide triphosphatase activities of the bovine viral diarrhea virus NS3 protein are essential for viral replication.

Helicase/nucleoside triphosphatase (NTPase) motifs have been identified in many RNA virus genomes. Similarly, all the members of the Flaviviridae family contain conserved helicase/NTPase motifs in their homologous NS3 proteins. Although this suggests that this activity plays a critical role in the viral life cycle, the precise role of the helicase/NTPase in virus replication or whether it is essential for virus replication is still unknown. To determine the role of the NS3 helicase/NTPase in the viral life cycle, deletion and point mutations in the helicase/NTPase motifs of the bovine viral diarrhea virus (BVDV) (NADL strain) NS3 protein designed to abolish either helicase activity alone (motif II, DEYH to DEYA) or both NTPase and helicase activity (motif I, GKT to GAT and deletion of motif VI) were generated. The C-terminal domain of NS3 (BVDV amino acids 1854 to 2362) of these mutants and wild type was expressed in bacteria, purified, and assayed for RNA helicase and ATPase activity. These mutations behaved as predicted with respect to RNA helicase and NTPase activities in vitro. When engineered back into an infectious cDNA for BVDV (NADL strain), point mutations in either the GKT or DEYH motif or deletion of motif VI yielded RNA transcripts that no longer produced infectious virus upon transfection of EBTr cells. Further analysis indicated that these mutants did not synthesize minus-strand RNA. These findings represent the first report unequivocably demonstrating that helicase activity is essential for minus-strand synthesis.

Pyridinyl imidazoles inhibit the inflammatory phase of delayed type hypersensitivity reactions without affecting T-dependent immune responses.

The effects of pyridinyl imidazoles, specifically SK&F 105809 and its metabolite, on normal T-cell and B-cell mediated immune responses were examined and compared to the fungal macrolide immunosuppressives, cyclosporin A, FK506 and rapamycin and to the corticosteroid, dexamethasone. The orally active prodrug SK&F 105809 [2-(4-methylsulfinylphenyl)-3-(4-pyridyl)-6,7-dihydro-[5H]-pyrrolo [1,2-a] imidazole[ and its metabolite, SK&F 105561 [2-(4-methylthiophenyl)-3-(4-pyridyl)-6,7-dihydro-[5H]-pyrrolo[1,2 -a] imidazole] are dual 5-lipoxygenase (5-LO) and cycloxygenase (CO) inhibitors with potent anti-inflammatory and cytokine (IL-1/TNF) suppressive activities. The anti-inflammatory activity of SK&F 105809 and its metabolite were evaluated in an antigen-specific murine model of delayed type hypersensitivity (DTH) response, where they were found to affect only the inflammatory and not the induction phase of this response. In contrast, these compounds and other pyridinyl imidazoles (SK&F 86002 and its analog, SK&F 104351) exhibited no immunosuppressive activity under conditions where the macrolide rapamycin and the corticosteroid dexamethasone abrogated both the cellular and humoral immune responses. Thus, the ability of pyridinyl imidazoles to attenuate independently the inflammatory components of the disease without causing generalized immunosuppression enhances their profile as candidates for therapy of chronic inflammatory diseases, specifically those mediated by cytokines (e.g. IL-1, TNF) and eicosanoids.

Cytotoxicity assays using cryopreserved target cells pre-labeled with the fluorescent marker europium.

We describe a method for standardizing cytotoxicity assays by the use of cryopreserved, fluorescently labeled target cells. The cells are labeled in batches with the fluorogenic element europium (Eu) and frozen in multiple aliquots. Replicate aliquots can be thawed on different days and used for cytotoxicity assays. Thawed pre-labeled cells from the same batch are killed reproducibly, allowing cytotoxicity assays to be standardized. The target cells need only be thawed, washed, counted for viability and used. The availability of pre-labeled, cryopreserved targets facilitates the assessment of cytotoxic activity of individual donors at different times. The method also simplifies the use of large panels of target cell types. By using this procedure it is not necessary to maintain active cultures of target cells, nor is it necessary to use and dispose of radioisotopes. These pre-labeled target cells can be used in various assays where the result is a destroyed target cell such as cell-mediated cytotoxicity, complement mediated cytotoxicity, or antibody-dependent cellular cytotoxicity.

Identification of the human T-lymphocyte protein-tyrosine kinase by peptide-specific antibodies.

We have recently cloned a cDNA from the human T-cell leukemia, JURKAT, having homology with the src-like family of protein-tyrosine kinases. We have made rabbit polyclonal antibodies against the synthetic peptide CKERPEDRPTFDYLRSVLEDFFTATEGQYQPQP (cys-33-pro) deduced from the carboxy-terminal amino acid sequence predicted by the JURKAT cDNA. In this report, we demonstrate that these antibodies immunoprecipitate the protein-tyrosine kinase activity from solubilized membrane extracts from JURKAT T-leukemia cells and from human peripheral blood T-lymphocytes from normal donors. A 58 kd protein, exhibiting protein-tyrosine kinase activity, was specifically immunoprecipitated in both cases. The antibodies failed to crossreact with pp60c-src from human platelets, but did crossreact with the murine T-lymphocyte protein-tyrosine kinase, pp56T-cell.