Role of mutations identified in ORFs M27, M36, m139, m141, and m143 in the temperature-sensitive phenotype of murine cytomegalovirus mutant tsm5

A mutant of murine cytomegalovirus (MCMV), tsm5, which is temperature-sensitive for replication in murine embryo fibroblasts at 40 degrees C, failed to replicate to detectable levels in mice. A total of 18 non-synonymous mutations have been identified in tsm5. In a previous study, a mutation (C890Y) identified in the M70 primase gene, when introduced into the wt M70 primase, resulted in a mutant with reduced viral replication at 40 degrees C in vitro and which was severely attenuated in vivo. Five other previously identified mutations may also contribute to the tsm5 phenotype: (1) an A658S mutation in a protein expressed by the M27 ORF; (2) a V54I mutation in M36; (3) a Y565* mutation in m139; (4) a V195M mutation in m141; and (5) an M232I mutation in m143. In the present study, the above-mentioned mutations were introduced individually (M27, M36, m139, m141, m143) or together (M27/M36) into the MCMV K181 (Perth) variant bacterial artificial chromosome (BAC) using RecE/T homologous recombination. Growth in culture revealed that, apart from the double mutant (M27 and M36) and the m139 mutant, the introduced mutations in the above-mentioned genes did not show a temperature-sensitive phenotype in MEF or Raw 264.7 macrophage cells compared to their revertants or the wt virus. In contrast, replication of the M27/M36 double mutant was drastically reduced in MEFs at 40 degrees C and in macrophages at 37 degrees C. Replication of the m139 mutant was reduced in MEF cells at 40 degrees C but not in macrophages. Thus, at least three further mutations contribute to the tsm5 phenotype. J. Med. Virol. 84:912922, 2012. (C) 2012 Wiley Periodicals, Inc.