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.

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.

Cytomegalovirus (CMV) is a widely prevalent herpesvirus that is well tolerated by an immune competent host yet establishes a state of chronic infection. The virus is thought to undergo frequent subclinical episodes of reactivation which leads to an unusually large accumulation of CMV-specific CD8(+) T lymphocytes in the peripheral blood, a phenomenon termed "memory inflation." The high magnitude of the CMV T cell response has been implicated in impaired immunity to heterologous pathogens such as EBV, influenza and West Nile virus. Here, using murine CMV (MCMV), we show that memory inflation of virus-specific CD8(+) T cells is avoided if mice are infected with a replication defective virus called temperature-sensitive mutant 5 (tsm5), which carries an attenuating mutation within the DNA primase gene. Mice infected with tsm5 do generate primary T cell responses towards viral proteins but these do not amass to skew the memory repertoire of CD8(+) T cells. Therefore, attenuation of the virus replication machinery may be valuable in future CMV vaccine designs because the virus remains immunogenic but does not contribute to CMV associated T cell immune senescence.

Identification of mutations in mutants derived chemically is a difficult and relatively random process. NimbleGen Comparative Genome Sequencing (CGS) was assessed as an inexpensive, rapid method of identifying mutations in the temperature-sensitive mutant tsm5 of the K181 (Birmingham) variant of murine cytomegalovirus (MCMV). This genome resequencing approach requires an established genome sequence as a reference. Comparison of tsm5 and the K181 (Birmingham) variant with the published K181 (Perth) MCMV genomic sequence revealed a total of 10 synonymous and 15 non-synonymous SNPs in tsm5 and 14 of the latter were confirmed by sequencing. Thus, while CGS cannot be relied upon to identify correctly all mutations it was helpful for identifying a large number of mutations for further investigation that could contribute to the ts phenotype of tsm5.

A mutation (C890Y) introduced into the M70 primase gene of murine cytomegalovirus (MCMV) resulted in reduced viral replication in murine embryo fibroblasts at 40°C and the mutant was severely attenuated in vivo. The attenuated replication of the M70 mutant was also observed in Raw 264.7 macrophages at 37°C, demonstrating that the mutation produced a defective rather than an unstable protein possibly reducing the amount of functional protein under different environmental conditions. Many synonymous mutations were introduced into this ORF by changing codon preferences that should reduce the efficiency of gene translation, but not change protein sequence or structure. Two Bacterial Artificial Chromosome (BAC) constructs were produced with 155 codons (at the distal third of the M70 gene) changed to MCMV less preferred codons and with either cysteine (BAC70(155Cys) ) or tyrosine (BAC70(155Tyr) ) at residue 890. Upon transfection of these BACs into NIH 3T3 cells, only BAC70(155Cys) produced virus and this mutant Mt70(155Cys) replicated similarly to its revertant and the wt MCMV K181 (Perth) variant. A metagenomic analysis of the protein structure of the primase using PredictProtein showed that the change from cysteine (M70Cys) to tyrosine (M70Tyr) has a marked effect on protein structure. However, when the cysteine residue was replaced by serine (M70Ser) or methionine (M70Met), which produced mutant viruses with a wild-type phenotype, the predicted structure was similar to the wild-type structure. J. Med. Virol. 88:1613-1621, 2016. © 2016 Wiley Periodicals, Inc.