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. J. Med. Virol. 84:912–922, 2012. © 2012 Wiley Periodicals, Inc.

Further studies on the role of the residue 890 cysteine to tyrosine mutation in the M70 primase ORF of the temperature‐sensitive mutant (tsm5) of murine cytomegalovirus

“…The MCMV variants, K181 (Birmingham) and K181 (Perth), and the mutant tsm 5 and their production and titration in primary mouse embryo fibroblasts (MEFs) have been described previously [Sammons and Sweet, ; Timoshenko et al, ]. The Raw 264.7 macrophage cell line (ECACC 91062702) was cultured and inoculated as previously [Al‐Ali et al, ].…”

“…The PCR reaction was carried out as previously, products sequenced and sequences aligned as described [Al‐Ali et al, ].…”

“…A two‐step Red‐mediated homologous recombination was performed to completely or partially replace the M70 gene sequence of the wt K181 (Perth) strain with the synthetic M70 ORF using BAC technology as described previously [Timoshenko et al, ; Al‐Ali et al, ].…”

“…PCR products were digested with Dpn I (NEB) to remove any residual template DNA. 100–200 ng in 1–2 μl of the PCR product was added to DH10B Escherichia coli cells containing the MCMV BAC and electroporation performed as previously [Al‐Ali et al, ]. Recombinants were selected on their chloramphenicol and kanamycin resistance and streptomycin sensitivity and integration of the rpsL‐neo cassette confirmed by PCR.…”

“…In the second step, the RpsL‐neo cassette was replaced with either the cysteine or the tyrosine version of the synthetic M70 gene (obtained by Bam HI/ Hin dIII digestion of the pPCR‐Script Amp SK(+) plasmid) to yield the mutated BACs, BAC70 403Cys and BAC70 403Tyr . Recombinants were identified as above and suitable colonies selected, PCR screened and virus generated as described previously [Al‐Ali et al, ].…”