Complementation of deletion of the vaccinia virus E3L gene by the Escherichia coli RNase III gene

This work investigated whether the Escherichia coli RNase III gene, mc+, could complement vp1080, a mutant vaccinia virus that is deleted of its E3L gene. Like E3L Mcf 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 mc+ gene was necessary for full rescue of vp1080. The me 70 gene, which encodes a product deficient in catalytic activity but still capable of binding to dsRNA, rescued vp1080 weakly. The me 105 gene, which encodes a product that cannot bind or cleave dsRNA, was unable to rescue vp1080. The me genes were also inserted into the E3L locus of vp1080. While recombinants containing the mcf gene or the me 70 gene regained the IFN resistance phenotype in RK(13) cells, full host range of vaccinia Virus was only restored in the recombinant containing the mc+ gene. Thus, the ability of RNase III to process dsRNA appears to be necessary to restore the host range phenotype. The vp-me 105 recombinant behaved similarly to vp1080. (C) 1997 Academic Press