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

Abstract

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.

Lessons from studies with murine cytomegalovirus that could lead to a safe live attenuated vaccine for human cytomegalovirus

“…These non-synonymous mutations were introduced individually and in combination into the MCMV K181 (Perth) variant virus using bacterial artificial chromosome (BAC) RecE/T homologous recombination, and showed that M56 and M98 individually did not contribute to the ts phenotype, but the M70 mutation alone and in combination with M56 and/or M98 rendered the virus ts , unable to replicate in mice and highly defective in DNA synthesis [ 18 ]. Experiments suggested that the M70 mutation produced a defective rather than an unstable protein, as replication was attenuated in Raw 264.7 macrophages at 37 °C, possibly reducing the amount of functional protein under different environmental conditions [ 19 ]. M70 is homologous to HCMV UL70 and a multiple alignment of herpesvirus UL70 primase homologues showed that the cysteine mutated in tsm5 is conserved in all cytomegaloviruses, MCMV (K181, Smith), HCMV (human herpes virus 5, strain Merlin), monkey cytomegaloviruses (chimpanzee, rhesus), but not other herpesviruses [herpes simplex virus (HSV-1), human herpes viruses 3, 4, 6, 7 and 8 (HHV-3, HHV-4, HHV-6, HHV-7, HHV-8)] [ 18, 20 ].…”

“…Unfortunately, this region of the primase appears to be particularly important for its function, as the tyrosine mutation had a marked effect on the protein structure and was, thus, very unstable and readily reverted to methionine or serine. A metagenomic analysis showed that these reversions produced a protein with a structure similar to that of the wt and the resultant viruses had a wt phenotype [ 19 ].…”

“…Several strategies were examined for changing the codon sequence of the MCMV M70 ORF to those used less frequently by the mouse [ 19 ]. Initially, all codons that were not already used least frequently were changed to the least frequently used mouse codons.…”