title: Role of mutations identified in ORFs M27, M36, m139, m141, and m143 in the temperature-sensitive phenotype of murine cytomegalovirus mutant tsm5.
abstract: A mutant of murine cytomegalovirus (MCMV), tsm5, which is temperature-sensitive for replication in murine embryo fibroblasts at 40°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°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°C and in macrophages at 37°C. Replication of the m139 mutant was reduced in MEF cells at 40°C but not in macrophages. Thus, at least three further mutations contribute to the tsm5 phenotype.
authors.affiliation: School of Biosciences, University of Birmingham, Edgbaston, Birmingham, UK.
authors.forename: Abdulaziz
authors.initials: A
authors.lastname: Al-Ali
authors.forename: Olga
authors.initials: O
authors.lastname: Timoshenko
authors.forename: Brian A B
authors.initials: BA
authors.lastname: Martin
authors.forename: Clive
authors.initials: C
authors.lastname: Sweet
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics; D005347:Fibroblasts / Q000821:virology; D008297:Male; D051379:Mice; D018146:Muromegalovirus / Q000235:genetics* / Q000254:growth & development* / Q000528:radiation effects; D020125:Mutation, Missense; D016366:Open Reading Frames; D010641:Phenotype; D017422:Sequence Analysis, DNA; D013696:Temperature; D014779:Virus Replication / Q000528:radiation effects*
chemical_list: D004279:DNA, Viral