Structural and proteomic analyses of the gammaherpesviruses KSHV and RRV

Rhesus monkey rhadinovirus (RRV) is one of the closest phylogenetic relatives to the human pathogen, Kaposi's sarcoma-associated herpesvirus (KSHV)---a gammaherpesvirus and the etiologic agent of three malignancies associated with immunosuppression. Unlike KSHV, RRV displays robust lytic phase growth and replicates to high titer and, thus, holds promise as an effective model for studying primate gammaherpesvirus structure, assembly and virion proteomics---the major goals of this thesis. Our data indicate that RRV lytic replication leads to the synthesis of three (A, B and C) distinct capsid species that resemble those we identified previously for KSHV. In contrast to patterns of capsid production during KSHV replication, RRV replication results in high proportions of the genome-filled C capsids, providing the first potential explanation for the differences in replication efficiency between the two viruses. We have also shown that the protein composition of RRV and KSHV capsids are highly conserved and, in parallel collaborative work with the Zhou laboratory at the University of Texas, Houston, have determined the three-dimensional structures of the three RRV capsid species, employing cryoelectron microscopy. We resolved the empty "A" capsid to approximately 8A, the highest resolution for any herpesvirus. Comparisons of these reconstructions to those of KSHV A capsids demonstrated remarkable structural conservation between these two primate gammaherpesviruses but both differed significantly from the capsids of alpha- and betaherpesviruses. In the last portion of the thesis, we analyzed the protein content and structure of purified RRV, employing two complementary mass spectrometric approaches. We found that RRV contains at least 33 distinct virally encoded and three host-encoded proteins. We have tentatively assigned seven of the virally encoded proteins to the capsid, 17 to the tegument and nine to the envelope. We found six novel proteins not previously reported as virion-associated (three encoded by the virus and three by the cell). Seven of the 33 virally encoded, virion-associated proteins represent novel findings for a gamma-2-herpesvirus. Detergent-treated RRV revealed not only the expected capsid structural proteins, but also six distinct tegument proteins, suggesting that these components may interact more directly with and with higher affinity for the underlying capsid.