808 Rhesus Macaque Rhadinovirus (RRV)-a Model to Decipher Kaposi's Sarcoma-associated Herpes Virus (KSHV) Capsid Structure and Protein Composition C. M. O'Connor*1, X. Yu2, B. Damania3, Z. H. Zhou2, D. H. Kedes1 1Univ of Virginia, Charlottesville; 2Univ of Texas-Houston Med Ctr; and 3Univ of North Carolina, Chapel Hill
Background: One of the closest known phylogenetic relatives to KSHV, the etiologic agent of Kaposi’s sarcoma, is RRV, which grows to high titer and, thus, holds promise as an attractive model to study gamma herpes virus structure and assembly. We have previously isolated KSHV capsids for structural and biochemical analysis. However, extension of this work on KSHV has met with difficulty due to low yields of KSHV in in vitro systems. Further, this same issue has impeded the study of capsid assembly. In contrast to KSHV, RRV displays robust lytic phase growth in cell culture. To determine the utility of RRV as a model for KSHV assembly, we have isolated RRV capsids, determined their structural components, and identified the individual capsid proteins.
Methods: RRV capsids were isolated from the media of immortalized rhesus fibroblasts following de novo infection with RRV, and were separated by density. Capsids were then analyzed for protein content (MS, SDS-PAGE), and by TEM and cryo-EM followed by 3D-reconstructions.
Results: We have isolated 3 (A, B, C) RRV capsid species. The A capsids are empty icosahedral structures, comprised of the major capsid protein (ORF25/MCP), triplex proteins (ORF62/TRI-1, ORF26/TRI-2), and the small capsomer interacting protein (ORF65/SCIP). The B and C capsids contain these same structural proteins, although B capsids also contain the scaffolding protein (ORF17.5/SCAF) found within the capsid, and C capsids contain, instead, the linear viral genome. Intranuclear capsid formation begins 48 hrs post-infection, although capsid and virion release begins 96 hrs post-infection. Such release reaches its peak 6 days post-infection, roughly paralleling the slow kinetics of KSHV release from induced PEL cells. Finally, we have taken advantage of the purity of the capsid populations, and using cryo-EM and computer aided reconstruction, we have resolved the structure of the RRV A capsid to15Å.
Conclusions: Our data indicate that as with other herpesviruses, RRV lytic replication leads to the synthesis of 3 distinct capsid species that resemble those we identified for KSHV. The capsid structure, as determined by high-resolution reconstructions, of RRV and KSHV demonstrate a high degree of similarity. These data, coupled with marked conservation in capsid protein composition and similar kinetics, argue for the use of RRV as a potentially powerful model to study gamma herpes virus structure and assembly.
