Genetic stability of bacterial artificial chromosome-derived human cytomegalovirus during culture in vitro

Murrell, I., Wilkie, G. S., Davison, A. J. , Statkute, E., Fielding, C., Tomasec, P., Wilkinson, G. W.G. and Stanton, R. J. (2016) Genetic stability of bacterial artificial chromosome-derived human cytomegalovirus during culture in vitro. Journal of Virology, 90(8), pp. 3929-3943. (doi:10.1128/JVI.02858-15) (PMID:26842472) (PMCID:PMC4810542)

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Abstract

Clinical human cytomegalovirus (HCMV) strains invariably mutate when propagated in vitro. Mutations in gene RL13 are selected in all cell types, whereas in fibroblasts mutants in the UL128 locus (UL128L; genes UL128, UL130, and UL131A) are also selected. In addition, sporadic mutations are selected elsewhere in the genome in all cell types. We sought to investigate conditions under which HCMV can be propagated without incurring genetic defects. Bacterial artificial chromosomes (BACs) provide a stable, genetically-defined source of viral genome. Viruses were generated from BACs containing the genomes of strains TR, TB40, FIX, and Merlin, as well as from Merlin-BAC recombinants containing variant nucleotides in UL128L from TB40-BAC4 or FIX-BAC. Propagation of viruses derived from TR-BAC, TB40-BAC4, and FIX-BAC in either fibroblast or epithelial cells was associated with the generation of defects around the prokaryotic vector, which is retained in the US region of viruses. This was not observed for Merlin-BAC, from which the vector is excised in derived viruses, however propagation in epithelial cells was consistently associated with mutations in the UL/b' region, all impacting on gene UL141. Viruses derived from Merlin-BAC in fibroblasts mutated in UL128L, but this occurred less frequently with recombinants containing UL128L nucleotides from TB40-BAC4 or FIX-BAC. Viruses derived from a Merlin-BAC derivative in which RL13 and UL128L were either mutated or repressed were remarkably stable in fibroblasts. Thus, HCMV containing a wild-type gene complement can be generated in vitro by deriving virus from a self-excising BAC in fibroblasts, and repressing RL13 and UL128L.

Item Type:Articles
Additional Information:This work was supported by the Medical Research Council (grant numbers MC_UU_12014/3, MR/L018373/1, MR/L008734/1), the Wellcome Trust (grant number WT090323), and NRN Ser Cymru.
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Wilkie, Dr Gavin and Davison, Professor Andrew
Authors: Murrell, I., Wilkie, G. S., Davison, A. J., Statkute, E., Fielding, C., Tomasec, P., Wilkinson, G. W.G., and Stanton, R. J.
College/School:College of Medical Veterinary and Life Sciences > Institute of Infection Immunity and Inflammation
Journal Name:Journal of Virology
Publisher:American Society for Microbiology
ISSN:0022-538X
ISSN (Online):1098-5514
Published Online:03 February 2016
Copyright Holders:Copyright © 2016 Murrell et al.
First Published:First published in Journal of Virology 90(8):3929-3943
Publisher Policy:Reproduced under a Creative Commons License

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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
656321Genomics of human cytomegalovirusAndrew DavisonMedical Research Council (MRC)MC_UU_12014/3MVLS III - CENTRE FOR VIRUS RESEARCH