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

Abstract

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.

Mouse Cytomegalovirus M34 Encodes a Non-essential, Nuclear, Early-Late Expressed Protein Required for Efficient Viral Replication

“…Contrary to our expectations based upon previous reports (Baluchova et al, 2008), plaque formation was observed after transfection of the M34-MCMV BAC into CIM cells and a couple of 'blind passages' , in which no CPE or plaque formation was apparent. First plaques were observed after 17 days.…”

“…Previous studies showed that a transposon insertion mutant of M34 (in which the transposon inserted at position 44,827 of the MCMV genome corresponding to codon 582 of M34) as well as a truncation mutant lacking the coding sequence corresponding to the C-terminal amino acids 548-854 of pM34 were replication competent, while "following transfection of NIH/3T3 cells with this BAC construct no virus was recovered, despite many repeated attempts the full M34 knock-out BAC could not be reconstituted" (Baluchova et al, 2008). Intriguingly, the authors stated that "although characteristic virus CPE was evident in mutant BACinfected cells BAC-containing virus of sufficient yield to titrate, grow stocks or passage was not achieved" (Baluchova et al, 2008), suggesting that residual virus replication may have occurred even at that time -a bit like a viable but nonculturable (VBNC) state well-known for certain bacteria.…”

“…Our MS-based analysis showed that pM34 is expressed and that its abundance is significantly reduced upon treatment with MLN4924 (Le- Trilling et al, 2016). Baluchova et al reported that an M34-deficient MCMV could not be reconstituted from a bacterial artificial chromosome (BAC) (Baluchova et al, 2008). This was interpreted as an indication that M34, similar to UL34, is essential for viral replication.…”

“…This was interpreted as an indication that M34, similar to UL34, is essential for viral replication. In contrast, an M34 transposon insertion mutant as well as a truncation mutant, both expressing a C-terminally truncated pM34 form containing the most conserved region, were reported to be in principle replication competent in vitro, albeit with reduced replication efficiency (Baluchova et al, 2008).…”