The Kaposi's sarcoma-associated herpesvirus nuclear egress complex is composed of two proteins, ORF67 and ORF69. In this study, we have recapitulated the KSHV complex by coexpression of these two proteins in insect cells using expression from recombinant baculoviruses. The proteins form a complex at the nuclear membrane as judged by live-cell analysis of protein fusions tagged with green fluorescent protein (GFP) and mCherry. Ultrastructural analysis of infected cells showed that ORF67 expression results in reduplication of the nuclear membrane. When the two proteins are expressed together, numerous virion-size nuclear membrane-derived vesicles were evident at the nuclear margins.

Epstein-Barr virus (EBV), a member of the Gammaherpesvirus family, primarily infects B lymphocytes and is responsible for a number of lymphoproliferative diseases. The molecular genetics of the assembly pathway and high-resolution structural analysis of the capsid have not been determined for this lymphocryptovirus. As a first step in studying EBV capsid assembly, the baculovirus expression vector (BEV) system was used to express the capsid shell proteins BcLF1 (major capsid protein), BORF1 (triplex protein), BDLF1 (triplex protein), and BFRF3 (small capsid protein); the internal scaffold protein, BdRF1; and the maturational protease (BVRF2). Coinfection of insect cells with the six viruses expressing these proteins resulted in the production of closed capsid structures as judged by electron microscopy and sedimentation methods. Therefore, as shown for other herpesviruses, only six proteins are required for EBV capsid assembly. Furthermore, the small capsid protein of EBV (BFRF3), like that of Kaposi's sarcoma-associated herpesvirus, was found to be required for assembly of a stable structure. Localization of the small capsid protein to nuclear assembly sites required both the major capsid (BcLF1) and scaffold proteins (BdRF1) but not the triplex proteins. Mutational analysis of BFRF3 showed that the N-terminal half (amino acids 1 to 88) of this polypeptide is required and sufficient for capsid assembly. A region spanning amino acids 65 to 88 is required for the concentration of BFRF3 at a subnuclear site and the N-terminal 65 amino acids contain the sequences required for interaction with major capsid protein. These studies have identified the multifunctional role of the gammaherpesvirus small capsid proteins. Epstein-Barr virus (EBV) is a gammaherpesvirus that infectsgreater than 90% of the general population and causes several lymphoproliferative diseases. Primary infection in adolescents with EBV results in infectious mononucleosis (46). EBV is also oncogenic and is associated with malignancies such as Burkitt's lymphoma (4), nasopharyngeal carcinoma, and posttransplant lymphoproliferative disease (47).Herpesviruses assemble icosahedral capsid structures in the nuclei of infected cells; six proteins are required for assembly of these structures. The capsid shell is largely composed of multimers of the major capsid protein, which are linked by the triplex structure (trimer of the two triplex proteins) and decorated by the small capsid protein. The internal scaffold protein directs the closure of the shell into an icosahedral structure (reviewed in references 17, 35, and 41). The EBV serine protease is synthesized as a 605-amino-acid polyprotein, which cleaves itself between amino acids Ala235 and Ser236 (release [R] site) and also between amino acids Ala568 and Ser569 (maturation [M] site). The catalytic domain resides in the N-terminal 235 amino acids (13). The six EBV capsid proteins are BcLF1 (major capsid protein), BORF1 (triplex 1), BDLF1 (triplex 2), BdRF1 (scaffold protein), BVRF2 (protease), and BFRF3...