Preliminary characterization of murine cytomegaloviruses with insertional and deletional mutations in the M34 open reading frame.

A viable virus could not be recovered from a mutant murine cytomegalovirus (MCMV) BAC in which the M34 ORF had been deleted (BACDeltaM34). In contrast, an M34 mutant virus (RcM34), in which the M34 ORF was interrupted by transposon insertion at nt 44,827 of the Smith MCMV BAC, was attenuated in replication both in tissue culture and in SCID mice. Similarly, mutant virus Rc3'DeltaM34, in which the 3'-end was deleted from nt 44,724 to nt 45,647, produced similar replication kinetics in tissue culture to RcM34 while BAC5'DeltaM34, in which the 5'-end from nt 43,083 up to nt 44,896 was deleted, was non-viable like BACDeltaM34. A transcript analysis of wt and RcM34 virus-infected cells showed that a truncated transcript encoding a putative protein of 624 amino acids was produced by RcM34, of which the amino terminal 582 amino acids would be identical to the predicted wt 854 amino acids product. Recent, re-annotations of the MCMV genome have identified three putative M34 overlapping ORFs (m33.1, m34.1, and m34.2) that may be interrupted in the above mutants. All three were transcribed in RcM34 virus-infected cells confirming that the RcM34 virus phenotype was probably due to interruption of the M34 ORF. These results suggest that M34, like human CMV UL34, is an essential gene.