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Journal of Virology, April 2009, p. 3891-3903, Vol. 83, No. 8
0022-538X/09/$08.00+0     doi:10.1128/JVI.01251-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Human Cytomegalovirus Glycoprotein B Is Required for Virus Entry and Cell-to-Cell Spread but Not for Virion Attachment, Assembly, or Egress{triangledown}

Marisa K. Isaacson and Teresa Compton*

McArdle Laboratory for Cancer Research, University of Wisconsin—Madison Medical School, University of Wisconsin—Madison, Madison, Wisconsin 53706

Received 16 June 2008/ Accepted 23 January 2009

Glycoprotein B (gB) homologs are conserved throughout the family Herpesviridae and appear to serve essential, universal functions, as well as specific functions unique to a particular herpesvirus. Genetic analysis is a powerful tool to analyze protein function, and while it has been possible to generate virus mutants, complementation of essential virus knockouts has been problematic. Human cytomegalovirus (HCMV) gB (UL55) plays an essential role in the replication cycle of the virus. To define the function(s) of gB in HCMV infection, the BAC system was used to generate a recombinant virus in which the UL55 gene was replaced with galK (pAD/Cre{Delta}UL55). UL55 deletions in the viral genome have been made before, demonstrating that UL55 is an essential gene. However, without being able to successfully complement the genetic defect, a phenotypic analysis of the mutant virus was impossible. We generated fibroblasts expressing HCMV gB that complement pAD/Cre{Delta}UL55 and produce infectious virions lacking the UL55 gene but containing wild-type gB on the virion surface ({Delta}UL55-gB HCMV). This is the first successful complementation of an HCMV mutant with a glycoprotein deleted. To characterize {Delta}UL55 infection in the absence of gB, noncomplementing cells were infected with {Delta}UL55-gB virus. All stages of gene expression were detected, and significant amounts of DNase-resistant viral DNA genomes, representing whole intact virions, were released into the infected cell supernatant. Gradient purification of these virions revealed they lacked gB but contained other viral structural proteins. The gB-null virions were able to attach to the cell surface similarly to wild-type gB-containing virions but were defective in virus entry and cell-to-cell spread. Glycoprotein B-null virions do, however, contain infectious DNA, as IE gene expression can be detected in fibroblasts following treatment of attached gB-null virions with a membrane fusion agent, polyethylene glycol. Taken together, our results indicate that gB is required for virus entry and cell-to-cell spread of the virus. However, HCMV gB is not absolutely required for virus attachment or assembly and egress from infected cells.

* Corresponding author. Present address: Novartis Institutes for Biomedical Research, 500 Technology Square, Cambridge, MA 02139. Phone: (617) 871-7470. Fax: (617) 871-5867. E-mail: teresa.compton{at}novartis.com

{triangledown} Published ahead of print on 4 February 2009.

Journal of Virology, April 2009, p. 3891-3903, Vol. 83, No. 8
0022-538X/09/$08.00+0     doi:10.1128/JVI.01251-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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