Re-Analysis of the Widely Used Recombinant Murine Cytomegalovirus MCMV-m157luc Derived from the Bacmid pSM3fr Confirms Its Hybrid Nature

Abstract

Murine cytomegalovirus (MCMV), and, in particular, recombinant virus derived from MCMV-bacmid pSM3fr, is widely used as the small animal infection model for human cytomegalovirus (HCMV). We sequenced the complete genomes of MCMV strains and recombinants for quality control. However, we noticed deviances from the deposited reference sequences of MCMV-bacmid pSM3fr. This prompted us to re-analyze pSM3fr and reannotate the reference sequence, as well as that for the commonly used MCMV-m157luc reporter virus. A correct reference sequence for this frequently used pSM3fr, containing a repaired version of m129 (MCK-2) and the luciferase gene instead of ORF m157, was constructed. The new reference also contains the original bacmid sequence, and it has a hybrid origin from MCMV strains Smith and K181.

Keywords: MCMV; MCMV bacmid reference; MCMV-m157lucMCK-2 repair; pSM3fr.

Figure 1

Genome alignment of the MCMV strains Smith and K181. The reference sequences for the MCMV strains Smith (OP429142) and K181 (AM88612) from GenBank were aligned and sequence conservation is displayed. Black bars indicate lower conservation. The genome areas of m02-m06 as well as m145-m159 are enlarged to demonstrate high sequence diversity in these respective areas between the two strains.

Figure 2

Comparison of ORFs m150 and m151 between MCMV strains Smith and K181. (A) Mapping of original pSM3fr reads against MCMV strain Smith and pSM3fr-MCK-2fl reference sequence and variant detection. Colored vertical lines indicate multiple nucleotide polymorphisms. (B) Section of an alignment of pSM3fr-MCK-2fl sequence of ORFs m151 and m152 as well as the respective regions of database reference sequences for MCMV strains Smith (OP429142, GU305914) and K181 (AM88612). Alignment was constructed using the Jukes–Cantor model. Black bars indicate lower conservation. (C) Maximum-likelihood tree generation with 1000 bootstrap repeats from the alignment of (B); numbers at bifurcation give percent of trees with the respective branching. Scale bars indicate the phylogenetic distance in percent of number of substitutions/changes per nucleotide.

Figure 3

Schematic representation of corrected reference sequences. (A) Mapping showing the bacmid-specific sequences within the references. (B) The corrected pSM3fr reference sequence, with the frameshift mutation in m129 and strain-K181-derived sequences in m151-m158. (C) The newly generated reference sequence for the repaired and modified pSM3fr-m157luc bacmid, containing the repaired version of m129 and a luciferase gene cassette instead of m157.

Figure 4

Mapping of reads obtained from low-passage MCMV viruses (m157-luc). Displayed are the mapped reads from two different viral stocks. The genome area spanning the bacmid components, which are flanked by the repetitive elements for recombination, is shown. In both cases, gaps (without specific read mapping or with strongly reduced specific read mapping) can be observed.