TY - JOUR
T1 - Complementation of deletion of the vaccinia virus E3L gene by the Escherichia coli RNase III gene
AU - Shors, Teri
AU - Jacobs, Bertram
N1 - Funding Information:
We thank Donald Court, for providing the RNase III genes and antiserum, Richard Condit for providing antiserum against total vaccinia proteins, and Jim Tartaglia for providing virus stocks of VC-2 and vp1080 and the in vivo recombination vector pMPE3LGPT#748. We also thank Renee Seidler-Wulff for sequencing the recombinant viruses and Cruz Lotz for assistance with preparing the manuscript. This work was supported in part by Public Health Service Grant CA-48654 from the National Cancer Institute. T. Shors was also supported by the ARCs foundation (sponsored by Millie Windrow).
PY - 1997/1/6
Y1 - 1997/1/6
N2 - 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 RK 3 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.
AB - 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 RK 3 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.
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U2 - 10.1006/viro.1996.8319
DO - 10.1006/viro.1996.8319
M3 - Article
C2 - 9007060
AN - SCOPUS:0031555657
SN - 0042-6822
VL - 227
SP - 77
EP - 87
JO - Virology
JF - Virology
IS - 1
ER -