Mass spectrometric analyses of purified rhesus monkey rhadinovirus reveal 33 virion-associated proteins

Abstract

The repertoire of proteins that comprise intact gammaherpesviruses, including the human pathogen Kaposi's sarcoma-associated herpesvirus (KSHV), is likely to have critical functions not only in viral structure and assembly but also in the early stages of infection and evasion of the host's rapidly deployed antiviral defenses. To develop a better understanding of these proteins, we analyzed the composition of rhesus monkey rhadinovirus (RRV), a close phylogenetic relative of KSHV. Unlike KSHV, RRV replicates to high titer in cell culture and thus serves as an effective model for studying primate gammaherpesvirus structure and virion proteomics. We employed two complementary mass spectrometric approaches and found that RRV contains at least 33 distinct virally encoded proteins. We have assigned 7 of these proteins to the capsid, 17 to the tegument, and 9 to the envelope. Of the five gammaherpesvirus-specific tegument proteins, three have no known function. We also found three proteins not previously associated with a purified herpesvirus and an additional seven that represent new findings for a member of the gamma-2 herpesviruses. Detergent extraction resulted in particles that contained six distinct tegument proteins in addition to the expected capsid structural proteins, suggesting that this subset of tegument components may interact more directly with or with higher affinity for the underlying capsid and, in turn, may play a role in assembly or transport of viral or subviral particles during entry or egress.

FIG. 1.

TEMs of RRV virions at various stages of purification. (A) Initial concentration of the virus from the medium prior to gradient purification reveals a mixture of viral particles, including A, B, and C capsids; partially tegumented virions; empty virions; and mature virions. White chevron, A capsid; white arrow, B capsid; white arrowhead, C capsid; black arrowhead, partially tegumented virion; black arrow, empty virion; black chevron, virion. (B) Following velocity sedimentation and equilibrium centrifugation, the population of particles is comprised mostly of mature virions, although there is considerable background debris. (C) PK-treated virions that are then gradient purified are a purer population of particles, with reduced background debris. Original magnification, ×17,000 for panel A and ×22,000 for panels B and C.

FIG. 2.

Simply Blue-stained SDS-PAGE analysis of purified RRV virions before and after proteolytic treatment. Following gradient purification, samples were separated on a 10% Bis-Tris gel, and bands were excised from the well to the bottom of the gel (see Materials and Methods). (A) Virion-associated proteins following sequential velocity and equilibrium gradients. (B) Virions treated first with PK and then purified through velocity and equilibrium gradients. The gel in panel B was electrophoresed for a shorter time. The numbered visible bands represent those that were excised individually and that were present both before (A) and after (B) PK treatment (see the text). Migrations of mass markers (kDa) are indicated at the sides of both lanes. In panel B, “d” indicates the dyefront.

FIG. 3.

MS/MS-identified peptides labeled by their corresponding ORFs and displayed according to their original positions after SDS-PAGE. The frequencies of peptides identified by MS/MS analysis within each sequential gel slice were plotted against MM_obs for (A) capsid proteins, (B) envelope proteins, (C) putative tegument proteins that are sensitive to detergent treatment, and (D) tegument proteins that resist detergent treatment. The numbers above some bars indicate the frequencies of peptides detected that were greater than the upper limit of the scale.

FIG. 4.

Western blot analyses of virion-associated proteins. Purified virus (+PK) was treated with Triton X-100 in the presence or absence of PK. In all cases, particle-associated proteins were separated by SDS-PAGE, transferred to PVDF, and immunoblotted for MCP or ORF52.