De novo infection with rhesus monkey rhadinovirus leads to the accumulation of multiple intranuclear capsid species during lytic replication but favors the release of genome-containing virions

Abstract

Rhesus monkey rhadinovirus (RRV) is one of the closest phylogenetic relatives to the human pathogen Kaposi's sarcoma-associated herpesvirus (KSHV), yet it has the distinct experimental advantage of entering efficiently into lytic replication and growing to high titers in culture. RRV therefore holds promise as a potentially attractive model with which to study gammaherpesvirus structure and assembly. We have isolated RRV capsids, determined their molecular composition, and identified the genes encoding five of the main capsid structural proteins. Our data indicate that, as with other herpesviruses, lytic infection with RRV leads to the synthesis of three distinct intranuclear capsid species. However, in contrast to the inefficiency of KSHV maturation following reactivation from latently infected B-cell lines (K. Nealon, W. W. Newcomb, T. R. Pray, C. S. Craik, J. C. Brown, and D. H. Kedes, J. Virol. 75:2866-2878, 2001), de novo infection of immortalized rhesus fibroblasts with RRV results in the release of high levels of infectious virions with genome-containing C capsids at their center. Together, our findings argue for the use of RRV as a powerful model with which to study the structure and assembly of gammaherpesviruses and, specifically, the human rhadinovirus,KSHV.

FIG. 1.

Maturation of RRV particles. (A) TEM of intranuclear RRV capsids 6 days postinfection. Three morphologically distinct capsid species are evident. The inset displays the dashed boxed area at a greater magnification with type A, B, and C capsids indicated. (B) Released RRV particles collected from the supernatant of infected RhF. Virions contain a capsid with an inner density surrounded by a tegument layer (t) and an envelope layer with protrusions consistent with glycoproteins (g). A capsid (c) lacking these two outer layers is also present in this micrograph.

FIG. 2.

Separation of RRV capsids by velocity sedimentation. Purified capsids were separated by velocity sedimentation on a 20 to 50% sucrose gradient (see reference 38). The three light-scattering bands in the gradient are labeled A, B, and C capsids on the basis of a similar banding pattern previously observed for KSHV.

FIG. 3.

Thin-section TEM of RRV capsids. (A) Mixture of three morphologically distinct capsids prior to separation. A capsids (thick arrow) are empty icosahedral structures, B capsids (arrowhead) contain an inner ring-like structure likely composed of SCAF (see text), and C capsids (thin arrow) contain a dense core consistent with encapsidated viral DNA. Velocity sedimentation separated the mixed capsid population into distinct populations of A, B, and C capsids (B to D, respectively).

FIG. 4.

Protein composition of RRV capsids. (A) SDS-PAGE of capsid fractions from a 20 to 50% sucrose gradient (Fig. 2). Fractions 3, 5, and 9 contain mainly A, B, and C capsids, respectively (see the corresponding micrographs, Fig. 3B to D). Fraction 4 is a mixture of both A and B capsids. (B) Expanded view of fractions 3, 5, and 9. M, molecular mass standards (kilodaltons). The arrow and arrowheads to the right indicate the five capsid-associated proteins migrating with the following apparent molecular masses: 1, 134 kDa; 2, 41 kDa; 3, 37 kDa; 4, 34 kDa; 5, 16 kDa. The values on the left of each panel are molecular sizes in kilodaltons.

FIG. 5.

Mass spectrometric analysis of protein bands 1 to 5 in Fig. 4B. Peptides identified by tryptic digestion (shaded) are shown within the sequence of the protein (black) they identified after screening of protein sequence databases. The protein bands are as follows: 1, MCP; 2, TRI-1; 3, SCAF; 4, TRI-2; 5, SCIP. Note that the translational start site and transcript of ORF17.5 (which encodes SCAF) is predicted from its homolog in KSHV and other herpesviruses (see text).

FIG. 6.

RRV C capsids cosediment with encapsidated DNA and, in contrast to KSHV, represent the most abundant species that arise during lytic replication. (A) RRV capsids were separated by velocity sedimentation and relative amounts of encapsidated RRV-specific DNA (solid squares) in each fraction were measured by Southern dot blot analysis. The RRV DNA peaks in the fractions correlating with the most rapidly sedimenting core-filled capsid species (Fig. 3D). For direct comparison, the MCP profile (open diamonds) determined by densitometry (Fig. 4, band 1) from a second aliquot of each fraction is also shown. A, B, and C indicate the fractions containing the purest populations of A, B, and C capsids (determined by TEM and SDS-PAGE), respectively. (B) In similar experiments (38), KSHV-specific DNA (solid squares) also cosediments with the KSHV C capsids. However, with the human virus, C capsids are the least-abundant species. The peak in KSHV MCP (open diamonds) correlates, instead, with the A and B capsids.