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

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 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 ...

CryoEM and mutagenesis reveal that the smallest capsid protein cements and stabilizes Kaposi's sarcoma-associated herpesvirus capsid

“…Therefore, in contrast to the suggested essential role of SCP in the in vitro assembly studies, KSHV SCP is not essential for capsid assembly in vivo, but rather dramatically enhances the efficiency of capsid assembly. It is also possible that capsids were also assembled in the absence of SCP in previous in vitro studies but might have eluded detection as a result of their scarcity (16,38). Our structure and in vivo mutagenesis studies support the notion that SCP is nonessential and plays a secondary or auxiliary role.…”

“…The aforementioned cross-linking of hexon MCPs by SCPs suggests that SCP may function by strengthening the integrity of hexons in the capsid. In this regard, previous studies with recombinant capsid proteins in a baculovirus expression system have suggested an essential role of SCP in KSHV and EBV in vitro capsid assembly (16,17), and the "essential" region of SCP was further mapped to the N-terminal half by mutagenesis scan with the in vitro capsid assembly system (17,38). To assess whether SCP is also essential for in vivo viral assembly and to better understand its mechanism of action, we performed conventional transmission EM of mutant virus-infected cells and cryoEM of purified viruses bearing SCP-null mutation or SCP C-terminal truncation.…”

“…The SCP of HSV-1 was dispensable for capsid assembly (13) and virus propagation (15). However, SCPs of KSHV and EpsteinBarr virus (EBV), the two known human pathogens of the gammaherpesvirus subfamily, have been demonstrated to be vital for capsid assembly in vitro (16,17) and important for virus propagation (18). How SCP of tumor herpesviruses affects capsid assembly is poorly understood because of the lack of highresolution capsid structures of these viruses.…”

Proteomic Characterization of Bovine Herpesvirus 4 Extracellular Virions

“…Although its homologues are nonessential in Alphaherpesvirinae and Betaherpesvirinae, it has recently been shown to be required for KSHV capsid formation (43). On the basis of full occupancy, it should be the second most abundant capsid protein.…”

Interactions of the Kaposi's Sarcoma-Associated Herpesvirus Nuclear Egress Complex: ORF69 Is a Potent Factor for Remodeling Cellular Membranes

“…Spodoptera frugiperda (Sf9 and Sf21) cells were grown in Grace's insect cell medium, supplemented with 10% fetal calf serum (Gibco-Invitrogen). Sf9 and Sf21 cells were propagated as described by Perkins et al (37) and Okoye et al (38). Baculoviruses were made using the BAC-to-BAC system from Invitrogen.…”