Reconstruction of the complete human cytomegalovirus genome in a BAC reveals RL13 to be a potent inhibitor of replication

Stanton, Richard James ORCID: https://orcid.org/0000-0002-6799-1182, Baluchova, Katarina, Dargan, Derrick J., Cunningham, Charles, Sheehy, Orla, Seirafian, Sepher, McSharry, Brian P., Neale, Margaret Lynne, Davies, James Anthony ORCID: https://orcid.org/0000-0003-3569-4500, Tomasec, Peter, Davison, Andrew J. and Wilkinson, Gavin William Grahame ORCID: https://orcid.org/0000-0002-5623-0126 2010. Reconstruction of the complete human cytomegalovirus genome in a BAC reveals RL13 to be a potent inhibitor of replication. Journal of Clinical Investigation 120 (9) , pp. 3191-3208. 10.1172/JCI42955

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Abstract

Human cytomegalovirus (HCMV) in clinical material cannot replicate efficiently in vitro until it has adapted by mutation. Consequently, wild-type HCMV differ fundamentally from the passaged strains used for research. To generate a genetically intact source of HCMV, we cloned strain Merlin into a self-excising BAC. The Merlin BAC clone had mutations in the RL13 gene and UL128 locus that were acquired during limited replication in vitro prior to cloning. The complete wild-type HCMV gene complement was reconstructed by reference to the original clinical sample. Characterization of viruses generated from repaired BACs revealed that RL13 efficiently repressed HCMV replication in multiple cell types; moreover, RL13 mutants rapidly and reproducibly emerged in transfectants. Virus also acquired mutations in genes UL128, UL130, or UL131A, which inhibited virus growth specifically in fibroblast cells in wild-type form. We further report that RL13 encodes a highly glycosylated virion envelope protein and thus has the potential to modulate tropism. To overcome rapid emergence of mutations in genetically intact HCMV, we developed a system in which RL13 and UL131A were conditionally repressed during virus propagation. This technological advance now permits studies to be undertaken with a clonal, characterized HCMV strain containing the complete wild-type gene complement and promises to enhance the clinical relevance of fundamental research on HCMV.

Item Type:	Article
Date Type:	Publication
Status:	Published
Schools:	Medicine Systems Immunity Research Institute (SIURI)
Subjects:	Q Science > QH Natural history > QH426 Genetics Q Science > QR Microbiology
Additional Information:	Pdf uploaded in accordance with publisher's policy at http://www.sherpa.ac.uk/romeo/issn/0021-9738/ (accessed 24/02/2014)
Publisher:	American Society for Clinical Investigation
ISSN:	0021-9738
Date of First Compliant Deposit:	30 March 2016
Last Modified:	11 Oct 2023 20:46
URI:	https://orca.cardiff.ac.uk/id/eprint/28638

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