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Journal of Virology, July 2008, p. 7201-7211, Vol. 82, No. 14
0022-538X/08/$08.00+0     doi:10.1128/JVI.00423-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Small Capsid Protein pORF65 Is Essential for Assembly of Kaposi's Sarcoma-Associated Herpesvirus Capsids{triangledown}

Edward M. Perkins,3 Daniel Anacker,1 Aaron Davis,1,{dagger} Vishwam Sankar,1 Richard F. Ambinder,2 and Prashant Desai1*

Viral Oncology Program,1 Division of Hematologic Malignancies, The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins,2 Integrated Imaging Center, Department of Biology, Johns Hopkins University, Baltimore, Maryland3

Received 26 February 2008/ Accepted 1 May 2008

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent for KS tumors, multicentric Castleman's disease, and primary effusion lymphomas. Like other herpesvirus capsids, the KSHV capsid is an icosahedral structure composed of six proteins. The capsid shell is made up of the major capsid protein, two triplex proteins, and the small capsid protein. The scaffold protein and the protease occupy the internal space. The assembly of KSHV capsids is thought to occur in a manner similar to that determined for herpes simplex virus type 1 (HSV-1). Our goal was to assemble KSHV capsids in insect cells using the baculovirus expression vector system. Six KSHV capsid open reading frames were cloned and the proteins expressed in Sf9 cells: pORF25 (major capsid protein), pORF62 (triplex 1), pORF26 (triplex 2), pORF17 (protease), pORF17.5 (scaffold protein), and also pORF65 (small capsid protein). When insect cells were coinfected with these baculoviruses, angular capsids that contained internal core structures were readily observed by conventional electron microscopy of the infected cells. Capsids were also readily isolated from infected cells by using rate velocity sedimentation. With immuno-electron microscopy methods, these capsids were seen to be reactive to antisera to pORF65 as well as to KSHV-positive human sera, indicating the correct conformation of pORF65 in these capsids. When either virus expressing the triplex proteins was omitted from the coinfection, capsids did not assemble; similar to observations made in HSV-1-infected cells. If the virus expressing the scaffold protein was excluded, large open shells that did not attain icosahedral structure were seen in the nuclei of infected cells. The presence of pORF65 was required for capsid assembly, in that capsids did not form if this protein was absent as judged by both by ultrastructural analysis of infected cells and rate velocity sedimentation experiments. Thus, a novel outcome of this study is the finding that the small capsid protein of KSHV, like the major capsid and triplex proteins, is essential for capsid shell assembly.

* Corresponding author. Mailing address: Viral Oncology Program, 3M07 CRB, 1650 Orleans St., Johns Hopkins University, Baltimore, MD 21117. Phone: (410) 614-1581. Fax: (410) 955-0840. E-mail: pdesai{at}jhmi.edu

{triangledown} Published ahead of print on 7 May 2008.

{dagger} Present address: Johns Hopkins Bloomberg School of Public Health, Baltimore, MD.

Journal of Virology, July 2008, p. 7201-7211, Vol. 82, No. 14
0022-538X/08/$08.00+0     doi:10.1128/JVI.00423-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Henson, B. W., Perkins, E. M., Cothran, J. E., Desai, P. (2009). Self-Assembly of Epstein-Barr Virus Capsids. J. Virol. 83: 3877-3890 [Abstract] [Full Text]  


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