Stability of Murine Cytomegalovirus Genome after In Vitro and In Vivo Passage

Abstract

While large DNA viruses are thought to have low mutation rates, only a small fraction of their genomes have been analyzed at the single-nucleotide level. Here, we defined the genetic stability of murine cytomegalovirus (MCMV) by whole-genome sequencing. Independently assembled sequences of three sister plaques showed only two single-base-pair substitutions after in vitro passage. In vivo-passaged MCMV likewise demonstrated low mutation rates, comparable to those after in vitro passage, indicating high genome stability of MCMV at the single-nucleotide level in the absence of obvious selection pressure.Large DNA viruses, such as herpesviruses, are thought to have low mutation rates as estimated by methods such as analysis of restriction fragment length polymorphisms or function of individual genes (10,16). However, these analyses sample only a small fraction of the genome (11,15). Moreover, in the presence of selective pressure, mutations have been identified in both human and murine cytomegalovirus (HCMV and MCMV, respectively) (5, 7, 17). For example, in HCMV, mutations in UL97 account for ganciclovir (GCV) resistance in up to 25% of immunosuppressed patients infected with HCMV (12, 18). However, whether these resistant mutant strains arise de novo or represent new infection is impossible to ascertain in the clinical setting.Previous studies demonstrated that after in vivo passage, MCMV does acquire de novo mutations. Mutants emerge after passage through mice lacking adaptive immunity but carrying the Cmv1 r allele, which encodes the Ly49H activation receptor on NK cells (7,19). The only known ligand for Ly49H is MCMVencoded open reading frame (ORF) m157. In mice infected with a plaque-purified MCMV clone containing intact m157, all escape viruses had m157 mutations. These mutants carried either single-amino-acid substitutions or premature stop codons and demonstrated increased virulence in naïve Cmv1 r mice (8). In contrast, there were no mutations in the adjacent ORFs, m156 and m158. Taken together, these data indicate that mutations occur in both HCMV and MCMV under selective pressure. However, absent this pressure, their genomewide stability has not been determined at the single-nucleotide level.In the current study, we set out to detail MCMV genomewide sequence changes after in vitro and in vivo passages in the absence of obvious selection pressure. To determine MCMV genome stability in vitro, we subcloned our laboratory stock of Smith strain MCMV (a gift from Herbert Virgin, Washington University, St. Louis, MO) that had been previously passaged in vivo. Our stock was plaque purified twice on NIH-3T12 monolayers. At the second round of plaque purification, three sister plaques were selected and independently amplified in vitro by sequential passages for 21 days in NIH-3T12 cells. Virions were then isolated from the culture medium, and genomic DNA was extracted for shotgun sequencing by the Sanger method. Using the bioinformatics software package Phred/Phrap/Consed (9), we then independently assembled compl...

Limits and patterns of cytomegalovirus genomic diversity in humans

“…The genome-wide average was 2 × 10 −7 mutations per base pair per generation. This estimate was ∼10-fold higher than previous in vitro estimates for herpes simplex virus type 1 (37, 38) but was comparable to in vivo estimates for murine cytomegalovirus (39). Further, there was a weak (Pearson's correlation coefficient r = 0.28, r 2 = 0.083) but highly significant positive correlation between estimated mutation rate and SNP density (Fig.…”

“…The estimates of HCMV genome-wide mutation rates presented here are approximately an order of magnitude higher than those of a closely related herpesvirus, HSV-1, but comparable with murine cytomegalovirus (39). It should be noted, however, that the HCMV estimates may be conservative, given the relatively stringent requirements of calling a de novo mutation, specifically maintaining a sustained frequency above the limits of detection.…”

Virus Progeny of Murine Cytomegalovirus Bacterial Artificial Chromosome pSM3fr Show Reduced Growth in Salivary Glands due to a Fixed Mutation of MCK-2

“…on May 11, 2018 by guest http://jvi.asm.org/ Smith strain sequence are nearly identical, which once again reveals that MCMV genomes are very stable during in vivo and in vitro passage (8).…”

Murine Cytomegalovirus Protein pM79 Is a Key Regulator for Viral Late Transcription

“…MCMV open reading frames (ORFs) were annotated based on the studies by Rawlinson and coworkers (7) and by Cheng and coworkers (38) and updated with details from additional publications whenever possible. Data were converted to gff3 file format and visualized using gBrowse (39).…”