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

Abstract

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.

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

“…In mouse tissues or at 40°C in vitro more dysfunctional primase is produced resulting in reduced DNA synthesis and virus replication. That virus reverted the tyrosine mutation to cysteine or methionine during virus passage at 37°C supports this [Timoshenko et al, ].…”

“…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, ].…”

“…Mutant tsm 5 is defective in DNA synthesis and a major mutation contributing to this is a nucleotide change (C to T) at position 99,286 in the primase ORF (M70; C890Y). When introduced into the wt K181 (Perth) variant using Bacterial Artificial Chromosome (BAC) technology to create mutant virus Mt70 890Y , this mutation reduced peak titres of infectious virus in MEF cells by ∼0.5 log 10 pfu/ml at 37°C and by >4 log 10 pfu/ml at 40°C [Timoshenko et al, ]. Similarly, DNA synthesis was reduced ∼2‐fold and 100–1,000‐fold at 37 and 40°C, respectively [Timoshenko et al, ].…”

“…When introduced into the wt K181 (Perth) variant using Bacterial Artificial Chromosome (BAC) technology to create mutant virus Mt70 890Y , this mutation reduced peak titres of infectious virus in MEF cells by ∼0.5 log 10 pfu/ml at 37°C and by >4 log 10 pfu/ml at 40°C [Timoshenko et al, ]. Similarly, DNA synthesis was reduced ∼2‐fold and 100–1,000‐fold at 37 and 40°C, respectively [Timoshenko et al, ]. This defect was also evident in immunocompromised mice at 37°C [Timoshenko et al, ].…”

“…Similarly, DNA synthesis was reduced ∼2‐fold and 100–1,000‐fold at 37 and 40°C, respectively [Timoshenko et al, ]. This defect was also evident in immunocompromised mice at 37°C [Timoshenko et al, ].…”