title: Complementation of deletion of the vaccinia virus E3L gene by the Escherichia coli RNase III gene.
abstract: This work investigated whether the Escherichia coli RNase III gene, rnc+, could complement vp1080, a mutant vaccinia virus that is deleted of its E3L gene. Like E3L, rnc+ codes for a dsRNA binding protein that contains an additional nucleolytic activity. Rnc genes were cloned into the eukaryotic expression vector pMTVa-, expressed in COS-1 cells, and shown to be functional. Transient rescue experiments in HeLa cells demonstrated that the cleavage function of the rnc+ gene was necessary for full rescue of vp1080. The rnc 70 gene, which encodes a product deficient in catalytic activity but still capable of binding to dsRNA, rescued vp1080 weakly. The rnc 105 gene, which encodes a product that cannot bind or cleave dsRNA, was unable to rescue vp1080. The rnc genes were also inserted into the E3L locus of vp1080. While recombinants containing the rnc+ gene or the rnc 70 gene regained the IFN resistance phenotype in RK13 cells, full host range of vaccinia virus was only restored in the recombinant containing the rnc+ gene. Thus, the ability of RNase III to process dsRNA appears to be necessary to restore the host range phenotype. The vp-rnc 105 recombinant behaved similarly to vp1080.
authors.affiliation: Department of Microbiology and the Graduate Program in Molecular and Cellular Biology, Arizona State University, Tempe, Arizona, 85287-2701, USA.
authors.forename: T
authors.initials: T
authors.lastname: Shors
authors.forename: B L
authors.initials: BL
authors.lastname: Jacobs
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology; D019556:COS Cells; D004352:Drug Resistance, Microbial / Q000235:genetics; D004722:Endoribonucleases / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D029968:Escherichia coli Proteins; D017353:Gene Deletion; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D006367:HeLa Cells; D006801:Humans; D007372:Interferons / Q000494:pharmacology; D008969:Molecular Sequence Data; D011066:Poly C / Q000378:metabolism; D011069:Poly I / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D043244:Ribonuclease III; D014162:Transfection; D014616:Vaccinia virus / Q000187:drug effects / Q000235:genetics* / Q000502:physiology; D014764:Viral Proteins / Q000235:genetics*; D014779:Virus Replication / Q000235:genetics
chemical_list: D000998:Antiviral Agents; C081374:E3L protein, Vaccinia virus; D029968:Escherichia coli Proteins; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D014764:Viral Proteins; D011069:Poly I; D011066:Poly C; D007372:Interferons; D004722:Endoribonucleases; D043244:Ribonuclease III; C468366:ribonuclease III, E coli