Expression of functional parvoviral NS1 from recombinant vaccinia virus: Effects of mutations in the nucleotide-binding motif

摘要: The gene encoding the major replicative protein, NS1, of minute virus mice (MVM) was transferred into a recombinant vaccinia vector in place thymidine kinase gene. NS1 placed under control bacteriophage T7 promoter and expressed cells coinfected with another virus, vTF7-3, which encodes RNA polymerase. Expression further enhanced by presence 5' untranslated region, derived from encephalomyocarditis allows efficient cap-independent translation. This system used to produce analyze wild-type two mutant forms NS1K405R NS1K405M, highly conserved lysine codon located putative purine triphosphate binding site changed arginine methionine, respectively. Full-length efficiently both human mouse infected each three viruses, case rapidly translocated nucleus. Wild-type this way biologically active. It able trans-activate an MVM P38 host chromosomal site, whereas showed no significant activity assay, it capable resolving palindromic junction fragments cloned multimeric form DNA molecules. These substrates, representing genomic left-end:left-end right-end:right-end fusions, were resolved synthesis-dependent vitro reaction supplemented nuclear extracts containing NS1. Neither polypeptide had any detectable assay.