Role of mutations identified in ORFs M56 (terminase), M70 (primase) and M98 (endonuclease) in the temperature-sensitive phenotype of murine cytomegalovirus mutant tsm5

Twenty-six non-synonymous and synonymous mutations have been identified in the temperature-sensitive (ts) mutant (tsm5) of the K181 (Birmingham) variant of murine cytomegalovirus that is deficient in DNA synthesis, processing and packaging at the non-permissive temperature and produces undetectable levels of infectious virus in mice. Non-synonymous mutations identified in the M70 (primase), M56 (terminase) and M98 (nuclease) ORFs were introduced individually and in combination into the K181 (Perth) variant using BAC technology to examine their role in the ts phenotype. The M56 (G439R) and M98 (P324S) mutations had no evident role in the ts phenotype. However, the C890Y M70 mutation alone and in combination with the M56 and/or M98 mutations rendered the virus ts, unable to replicate in mice and highly defective in DNA synthesis. Reversion of the tyrosine mutation to cysteine or introduction of C890M (experimentally) or C890S (naturally) restored the wt phenotype.