Complementation of Deletion of the Vaccinia Virus E3L Gene by theEscherichia coliRNase III Gene

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This work investigated whether theEscherichia coliRNase 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.Rncgenes 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 thernc+ gene was necessary for full rescue of vp1080. Thernc70 gene, which encodes a product deficient in catalytic activity but still capable of binding to dsRNA, rescued vp1080 weakly. Thernc105 gene, which encodes a product that cannot bind or cleave dsRNA, was unable to rescue vp1080. Therncgenes were also inserted into the E3L locus of vp1080. While recombinants containing thernc+ gene or thernc70 gene regained the IFN resistance phenotype in RK13cells, full host range of vaccinia virus was only restored in the recombinant containing thernc+ 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.