Incorporation of the Kaposi's sarcoma-associated herpesvirus capsid vertex-specific component (CVSC) into self-assembled capsids

•First biochemical studies that examine and discover interactions of the Kaposi's sarcoma-associated herpesvirus (KSHV) capsid vertex-specific component proteins with themselves and with the triplex complex and capsid shell.•Use of an ex-vivo self assembly system to investigate interactions of the KSHV capsid and vertex complex.•New insight into the mode of incorporation of the capsid vertex-specific component specified by ORF19 and ORF32 into the assembled structure.

Self-assembly of herpesvirus capsids can be accomplished in heterologous expression systems provided all six capsid proteins are present. We have demonstrated the assembly of icosahedral Kaposi's sarcoma-associated herpesvirus (KSHV) capsids in insect cells using the baculovirus expression system. Using this self-assembly system we investigated whether we could add additional capsid associated proteins and determine their incorporation into the assembled capsid. We chose the capsid vertex-specific component (CVSC) proteins encoded by open reading frames (ORFs) 19 and 32 to test this. This complex sits on the capsid vertex and is important for capsid maturation in herpesvirus-infected cells. Co-immunoprecipitation assays were used to initially confirm a bi-molecular interaction between ORF19 and ORF32. Both proteins also precipitated the triplex proteins of the capsid shell (ORF26 and ORF62) as well as the major capsid protein (ORF25). Capsid immunoprecipitation assays revealed the incorporation of ORF19 as well as ORF32 into assembled capsids. Similar experiments also showed that the incorporation of each protein occurred independent of the other. These studies reveal biochemically how the KSHV CVSC interacts with the capsid shell.