Cytomegalovirus destruction of focal adhesions revealed in a high-throughput Western blot analysis of cellular protein expression

Stanton, Richard James ORCID: https://orcid.org/0000-0002-6799-1182, McSharry, Brian Patrick, Rickards, Carole Ruth, Wang, Edward Chung Yern ORCID: https://orcid.org/0000-0002-2243-4964, Tomasec, Peter and Wilkinson, Gavin William Grahame ORCID: https://orcid.org/0000-0002-5623-0126 2007. Cytomegalovirus destruction of focal adhesions revealed in a high-throughput Western blot analysis of cellular protein expression. Journal of Virology 81 (15) , pp. 7860-7872. 10.1128/JVI.02247-06

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Abstract

Human cytomegalovirus (HCMV) systematically manages the expression of cellular functions, rather than exerting the global shutoff of host cell protein synthesis commonly observed with other herpesviruses during the lytic cycle. While microarray technology has provided remarkable insights into viral control of the cellular transcriptome, HCMV is known to encode multiple mechanisms for posttranscriptional and posttranslation regulation of cellular gene expression. High-throughput Western blotting (BD Biosciences Powerblot technology) with 1,009 characterized antibodies was therefore used to analyze and compare the effects of infection with attenuated high-passage strain AD169 and virulent low-passage strain Toledo at 72 hpi across gels run in triplicate for each sample. Six hundred ninety-four proteins gave a positive signal in the screen, of which 68 from strain AD169 and 71 from strain Toledo were defined as being either positively or negatively regulated by infection with the highest level of confidence (BD parameters). In follow-up analyses, a subset of proteins was selected on the basis of the magnitude of the observed effect or their potential to contribute to defense against immune recognition. In analyses performed at 24, 72, and 144 hpi, connexin 43 was efficiently downregulated during HCMV infection, implying a breakdown of intercellular communication. Mitosis-associated protein Eg-5 was found to be differentially upregulated in the AD169 and Toledo strains of HCMV. Focal adhesions link the actin cytoskeleton to the extracellular matrix and have key roles in initiating signaling pathways and substrate adhesion and regulating cell migration. HCMV suppressed expression of the focal-adhesion-associated proteins Hic-5, paxillin, and -actinin. Focal adhesions were clearly disrupted in HCMV-infected fibroblasts, with their associated intracellular and extracellular proteins being dispersed. Powerblot shows potential for rapid screening of the cellular proteome during HCMV infection.

Item Type:	Article
Date Type:	Publication
Status:	Published
Schools:	Medicine Systems Immunity Research Institute (SIURI)
Additional Information:	Pdf uploaded in accordance with publisher's policy at http://www.sherpa.ac.uk/romeo/issn/0022-538X/ (accessed 25/02/2014)
Publisher:	American Society for Microbiology
ISSN:	0022538X
Date of First Compliant Deposit:	30 March 2016
Last Modified:	01 Nov 2023 07:38
URI:	https://orca.cardiff.ac.uk/id/eprint/258

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