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Journal of Virology, March 2009, p. 2480-2490, Vol. 83, No. 6
0022-538X/09/$08.00+0     doi:10.1128/JVI.01818-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Major Tegument Protein pp65 of Human Cytomegalovirus Is Required for the Incorporation of pUL69 and pUL97 into the Virus Particle and for Viral Growth in Macrophages{triangledown}

Meike Chevillotte,1 Sandra Landwehr,1,2 Leonhard Linta,1 Giada Frascaroli,1 Anke Lüske,1 Christopher Buser,1,{dagger} Thomas Mertens,* and Jens von Einem1

Institute of Virology, University Hospital Ulm,1 Electron Microscopy Facility, University Ulm, D-89081 Ulm, Germany2

Received 29 August 2008/ Accepted 17 December 2008

The tegument protein pp65 of human cytomegalovirus (HCMV) represents the major component of mature virus particles. Nevertheless, deletion of pp65 has been shown to have no effects on virus replication and morphogenesis in fibroblasts in vitro. We have studied the HCMV virion composition in the absence of pp65 and viral growth of a pp65 stop mutant in different cell types, including monocyte-derived macrophages. Two stop codons at amino acids 11 and 12 of pp65 were introduced by bacterial artificial chromosome mutagenesis into the endotheliotropic strain TB40/E. Clear changes of the tegument composition could be observed in purified mutant virus particles, where the amount of tegument protein pUL25 was drastically reduced. In addition, pUL69 and the virally encoded protein kinase UL97 were undetectable in the pp65 stop mutant. Expression of pUL69 in infected cells was unaltered while pUL25 accumulated in the absence of pp65, thus demonstrating that only incorporation into virus particles is dependent on pp65. Coimmunoprecipitation experiments using lysates of infected cells revealed an interaction between pUL69 and pp65. This interaction was verified in pull-down experiments using transfected cells, which showed that pp65 and pUL69 do not require the presence of other viral proteins for their interaction. We conclude that pp65 is required for the incorporation of other viral proteins into the virus particle and thus is involved in the protein-protein interaction network leading to normal tegument formation. When studying growth kinetics of the pp65 stop mutant in different cell types, we found a severe impairment of viral growth in monocyte-derived macrophages, showing for the first time a strong cell-specific role of pp65 in viral growth.

* Corresponding author. Mailing address: Institute of Virology, University Hospital Ulm, Ulm 89081, Germany. Phone: 49 (0)731 500 65100. Fax: 49 (0)731 500 65102. E-mail: thomas.mertens{at}uniklinik-ulm.de

{triangledown} Published ahead of print on 30 December 2008.

{dagger} Present address: Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3202.

Journal of Virology, March 2009, p. 2480-2490, Vol. 83, No. 6
0022-538X/09/$08.00+0     doi:10.1128/JVI.01818-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Chevillotte, M., Schubert, A., Mertens, T., von Einem, J. (2009). Fluorescence-Based Assay for Phenotypic Characterization of Human Cytomegalovirus Polymerase Mutations Regarding Drug Susceptibility and Viral Replicative Fitness. Antimicrob. Agents Chemother. 53: 3752-3761 [Abstract] [Full Text]  


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