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J. Virol. doi:10.1128/JVI.00423-08
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Assembly of Kaposi's sarcoma-associated herpesvirus (KSHV) capsids in insect cells using recombinant baculoviruses: the small capsid protein is essential for assembly

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

^* To whom correspondence should be addressed. Email: pdesai{at}jhmi.edu.

	Abstract

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 (BEV) 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) and pORF17.5 (scaffold protein) and also pORF65 (small capsid protein). When insect cells were co-infected with these viruses 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 using rate velocity sedimentation. Using immuno-EM methods these capsids were 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 co-infection, 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.