Murine gammaherpesvirus 68 (MHV-68

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.Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of KS tumors, an angiogenic skin malignancy, as well primary effusion lymphomas and multicentric Castleman's disease (7,9,40). Malignancies due to KSHV are a major clinical problem for AIDS patients and consequently represent the predominant cancer found in southern Africa. KSHV (or human herpesvirus 8), like the other herpesviruses, assembles icosahedral capsids that display a Tϭ16 shell. For all herpesviruses studied to date the capsids are comprised of six polypeptides (reviewed in references 17, 34, and 42). The capsid shell subunits or capsomeres are composed of the major capsid protein (51). There are 150 hexons of the major capsid protein and 11 pentons, which are located at the vertices of the icosahedral structure. The portal protein occupies the 12th vertex (4, 8, 11) and forms a dodecameric ring structure (48), and it is the portal for DNA entry into ...