ANALYSIS OF A TEMPERATURE-SENSITIVE VACCINIA VIRUS MUTANT IN THE VIRAL MESSENGER-RNA CAPPING ENZYME ISOLATED BY CLUSTERED CHARGE-TO-ALANINEMUTAGENESIS AND TRANSIENT DOMINANT SELECTION

We have previously reported the successful development of a targeted g enetic method for the creation of temperature-sensitive vaccinia virus mutants [9. E. Hassett and R. C. Condit (1994) Proc. Natl. Acad. Sci. USA 91, 4554-4558]. This method has now been applied to the large sub unit of the multifunctional vaccinia virus capping enzyme, encoded by gene DIR. Ten clustered charge-to-alanine mutations were created in a cloned copy of D1R. Four of these mutations were successfully transfer red into the viral genome using transient dominant selection, and each of these four mutations yielded viruses with plaque phenotypes differ ent from that of wild-type virus. Two of the mutant viruses, 516 and 7 93, were temperature sensitive in a plaque assay. Mutant 793 was also temperature sensitive in a one-step growth experiment. Phenotypic char acterization of the 793 virus under bath permissive and nonpermissive conditions revealed nearly normal patterns of viral protein and mRNA s ynthesis. Under nonpermissive conditions the 793 virus was defective i n telomere resolution and blocked at an intermediate stage of viral mo rphogenesis. In vitro assays of various capping enzyme activities reve aled that in permeabilized virions, enzyme guanylylate intermediate fo rmation was reduced and methyltransferase activity was thermolabile, w hile in solubilized virion extracts enzyme guanylylate activity was re duced and both guanylyltransferase and methyltransferase activities we re absent Thus, the 793 mutation affects at least two separate enzymat ic activities of the capping enzyme, guanylyltransferase and methyltra nsferase, and when incorporated into the virus genome, the mutation yi elds a virus that is temperature sensitive for growth, telomere resolu tion, and Virion morphogenesis. (C) 1997 Academic Press.