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Journal of Virology, February 2006, p. 1574-1583, Vol. 80, No. 3
0022-538X/06/$08.00+0     doi:10.1128/JVI.80.3.1574-1583.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Mass Spectrometric Analyses of Purified Rhesus Monkey Rhadinovirus Reveal 33 Virion-Associated Proteins

Christine M. O'Connor and Dean H. Kedes*

Myles H. Thaler Center for AIDS and Human Retrovirus Research, Box 800734, University of Virginia Health Systems, Charlottesville, Virginia 22908

Received 26 September 2005/ Accepted 14 November 2005


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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.


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INTRODUCTION
 
Kaposi's sarcoma remains the most common AIDS-related malignancy worldwide, and its causative agent is Kaposi's sarcoma-associated herpesvirus (KSHV), a member of the gamma subfamily of herpesviruses (7, 10). Unfortunately, KSHV grows to low titer in culture, making detailed structural and compositional studies of highly purified particles particularly challenging (4, 65). In contrast, rhesus monkey rhadinovirus (RRV), a nonhuman primate gamma-2 herpesvirus with high levels of homology to KSHV, grows to high titer in culture (2, 33, 47, 64). We have previously characterized the capsids of both KSHV and RRV (30, 33, 56, 64). In these studies, we found that the three-dimensional capsid structures and protein homolog compositions were strikingly similar (33, 64). Both KSHV and RRV exhibit structural features similar to those of other herpesviruses, including an icosahedral capsid (30, 56, 62) enclosing the viral genome, a proteinaceous tegument, and an envelope (36, 44, 59) decorated with glycoprotein spikes that facilitate virion-host cell interactions (1, 8, 9, 22, 68). These findings, coupled with the high yields of RRV in culture, have made RRV an attractive model system for the study of KSHV structure and assembly (2, 30, 33, 47, 64).

Alpha- and betaherpesvirus studies provided the basis for early classification schemes for structural components of gammaherpesviruses. However, many structural proteins of the gamma subfamily demonstrate sufficient amino acid sequence divergence from alpha- and betaherpesviruses to suggest that simple extrapolation of structure and potentially function may be invalid. Furthermore, many virion-associated proteins are unique to the gammaherpesviruses and even to individual viral species (2, 67). These groups of proteins likely contribute to those biological and potentially pathological characteristics that distinguish gammaherpesviruses like RRV and KSHV from members of the alpha and beta subfamilies. Therefore, establishing a rigorous system to accurately identify the protein profiles of these virions is critical.

In this study, we sought to develop as thorough an understanding as possible of the proteins comprising the intact RRV virion. We reasoned that a subset of these proteins may play essential roles in the establishment of viral infection, rather than or in addition to their involvement in structure and assembly. Further, the ways that KSHV and RRV rapidly initiate changes to the host cell are most likely distinct from those of other herpesviruses, since each also displays distinct modes of transmission, host cell tropism, balance between latent and lytic replication, and pathogenesis. To this end, we have identified RRV virion-associated proteins utilizing two mass spectrometric (MS) approaches: (i) multidimensional protein identification technology (MudPIT) (23, 24, 60, 61, 63), an en masse approach to tandem MS (MS/MS) that subjects the entire virion to MS analysis, and (ii) MS/MS of a contiguous sets of gel slices excised from polyacrylamide gels containing virion proteins separated by one-dimensional (1D) electrophoresis. Using these methods, we have identified 33 virion-associated proteins comprising 7 capsid-associated proteins, 9 envelope proteins, and 17 putative tegument proteins. (We have chosen in this report to designate most of the proteins that we have assigned to the tegument as "putative tegument proteins", since evidence for their localization is based mainly on homology with other herpesviruses and still requires confirmation by direct experimental observation.) We also found that 6 of these 17 proteins resisted removal by nonionic detergent, suggesting that they may be tightly associated with the underlying capsid.


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MATERIALS AND METHODS
 
Cell culture and production of RRV stocks. Telomerase-immortalized rhesus monkey fibroblasts (RhF) were grown in Dulbecco's modified Eagle's medium (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (Gibco), 110 mg/liter sodium pyruvate, and 500 ng/ml puromycin, as described previously (2, 12, 33). When approximately 90% confluent (8.0 x 104 cells/cm2), the cells were infected with RRV strain 26-95 at a multiplicity of infection of 1.0 for 1 hour (final volume, 0.03 ml/cm2), followed by supplementation with 0.55 ml/cm2 of additional media. The viral supernatants were harvested 8 to 10 days following infection (see below).

Isolation of RRV virions. RRV virions were isolated from the media of infected RhF as described previously (30, 33). In brief, cells and debris were removed from the media of approximately 1.2 x 108 infected cells by low-speed centrifugation after complete cell lysis, and this viral supernatant was then passed through a 0.45-µm filter. The virus was then sedimented from the filtered supernatant by centrifugation for 3 hours at 12,855 x g in a Sorvall SL250T rotor, and the resulting pellet was resuspended overnight at 4°C in a final volume of 1.0 ml of TNE (20 mM Tris, pH 7.5, 100 mM NaCl, 1 mM EDTA). The virus was then purified over a Sepharose CL-4B (Sigma-Aldrich, St. Louis, MO) size exclusion column (Bio-Rad, Hercules, CA), and peak fractions were collected and concentrated by centrifugation in a microcentrifuge at 4°C for 2 h at 16,060 xg. Virus particles were resuspended at 4°C in DNase buffer (10 mM MnCl2, 50 mM Tris HCl [pH 7.5]) for 30 min and then bath sonicated three times for 5 seconds each time on ice. The viral particles were incubated for 30 min at 37°C in the presence of 0.03 U/ml of DNase I (Roche). Where indicated, we added pronase (Calbiochem, La Jolla, CA) to a final concentration of 0.75 µg/µl in the presence of 10 mM CaCl2 for 15 min at 37°C or proteinase K (PK) (150 ng/µl) in the presence of 100 mM Tris-Cl, pH 8.0, 50 mM EDTA, pH 8.0, 600 mM NaCl for 30 min at 37°C. PK was inactivated by the addition of phenylmethylsulfonyl fluoride (5 µg/µl). Following either protease treatment, virions were sedimented through a 20 to 60% sucrose-TNE gradient, and fractions were collected by bottom puncture. Aliquots of each fraction underwent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and viral peaks were identified after the gel was stained with Simply Blue Safe Stain (Invitrogen, Carlsbad, CA) (see below). Sucrose was added to the pooled virus-containing fractions to a final concentration of 60%, and a 20 to 50% sucrose-TNE step gradient was layered above the virion-sucrose mixture. Virions were then "floated" by ultracentrifugation at 43,800 x g for 16 h at 4°C in an SW55Ti. Fractions were collected fromthe top (20-µl fractions), and half of the virion-containing fraction was delipidated for MudPIT analysis.

Delipidation of RRV. Purified virions prepared for MudPIT were delipidated by a modified Folch extraction (15). In brief, contaminating lipids were removed from the purified virus by adding 2.5 volumes of methanol, followed by 0.25 volume of chloroform and 0.5 volume of double-distilled H2O. The sample was then vigorously mixed for 1 minute, and the aqueous and organic layers were separated by centrifugation in a microcentrifuge for 2 min at 16,060 x g. The organic layer was then removed, followed by back extraction with methanol. The protein precipitate was then resuspended in 0.1% SDS and analyzed by MudPIT (see below).

Protein electrophoresis and Western blot analyses. Virion-associated proteins for each fraction were separated by SDS-PAGE on 10% or 12% Bis-Tris gels using the NuPAGE system (Invitrogen). Protein bands were reversibly stained as described above. The gels were then sliced at right angles to the direction of electrophoresis to generate a set of approximately 50 to 60 contiguous slices comprising the entire lane containing the virion proteins. Each slice was then excised for MS/MS (see below).

For Western analyses, proteins first separated by SDS-PAGE were instead transferred to polyvinylidene difluoride (PVDF) membranes for 75 min at 100 V at 4°C. The membranes were then stained briefly with Ponceau S, washed three times for 5 minutes each time with 1x TBST (20 mM Tris base, 150 mM NaCl, 3 mM Tris-Cl, 0.05% Tween) at room temperature, and then blocked in 5% nonfat milk-TBST. Primary antibodies were used at the following concentrations: anti-major capsid protein (MCP) mouse monoclonal antibody (1:100), generously provided by Scott Wong, and anti-open reading frame 52 (ORF52) mouse polyclonal sera (1:300). The secondary antibody, horseradish peroxidase-conjugated sheep anti-mouse, was diluted 1:1,000 (Jackson Immuno Research Laboratories, Inc., West Grove, PA). Western Lightening chemiluminescent reagent (Perkin-Elmer, Boston, MA) was used according to the manufacturer's protocol, and protein bands were visualized using Kodak BioMax XAR film (Fisher Scientific, Hampton, NH).

Detergent-treated virions. Triton X-100 (Fisher Chemicals, Suwanee, GA) was added to column-purified viral particles (a mixture of both) virions and capsids (Fig. 1A) to a final concentration of 2% and incubated overnight at 4°C. The mixture was then briefly bath sonicated at 4°C and sedimented (75,000 x g for 30 min in a Sorvall SW55Ti) through a 35% (wt/vol) sucrose cushion made in 20 mM Tris HCl (pH 8.0), 250 mM NaCl, 1 mM EDTA (modified TNE). The resulting pellet was resuspended, sonicated as described above, and subjected to MudPIT analysis (see below). In parallel experiments, double-gradient-purified virions were also treated with Triton X-100 as described above, although instead of a sucrose cushion, half of the sample containing the Triton-treated virions was treated with PK, as described above for virion purification. These detergent-treated virions were then electrophoresed to separate the capsid-associated proteins, transferred to PVDF membrane, and immunoblotted as described above.



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  		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, x17,000 for panel A and x22,000 for panels B and C.

MudPIT. The sample was dissolved in 1% SDS and slowly diluted with 100 mM ammonium bicarbonate (ambic), pH 8.0, to 0.1% SDS. The sample was reduced with dithiothreitol (DTT) and alkylated with iodoacetamide before addition of 1 µg of trypsin (Promega, Fitchburg Center, WI) for 24 h at room temperature. The sample was desalted and then ion-exchanged (fractions = 1, 2, 5, 10, 25, 50, 100, and 1,000 mM). A portion of each ion-exchange fraction was injected into the mass spectrometer. The liquid chromatography (LC)-MS system consisted of an LTQ ion trap mass spectrometer system (Finnigan, San Jose, CA) with a Protana nanospray ion source (Protana, Inc., Toronto, Ontario, Canada) interfaced with a self-packed 8-cm by 75-µm (inside diameter) Phenomenex Jupiter 10-µm C18 reversed-phase capillary column (Phenomenex, Torrance, CA). The extract was injected in 0.5- to 5-µl fractions, and the peptides were eluted from the column by an acetonitrile-0.1 M acetic acid gradient at a flow rate of 0.25 µl/min. The nanospray ion source was operated at 2.8 kV. The digest was analyzed using the double-play capability of the instrument, acquiring full-scan mass spectra to determine peptide molecular weights and product ion spectra to determine the amino acid sequence in sequential scans. This mode of analysis produces approximately 3,000 collisionally activated dissociation spectra of ions ranging in abundance over several orders of magnitude. Not all collisionally activated dissociation spectra were derived from peptides. The data were analyzed by database searching using the Sequest (NCBI) search algorithm against viral protein and nonredundant databases.

Mass spectrometry. The method of MS determination of tryptic peptides has been described previously (26). Briefly, protein bands were extracted from the gel and destained in 50% methanol overnight. The gel pieces were then dehydrated in acetonitrile, rehydrated in 10 mM DTT in 0.1 M ambic, and reduced at room temperature for 30 min. The DTT solution was removed, and the sample was alkylated in 50 mM iodoacetamide in 0.1 M ambic at room temperature for 30 min. The above dehydration/rehydration steps were repeated, and then the gel pieces were completely dried by vacuum centrifugation. The gel pieces were rehydrated in 20 ng/µl trypsin in 50 mM ambic on ice for 10 minutes. Any excess trypsin solution was removed, and 50 mM ambic was added. The samples were digested overnight at 37°C, and the resultant peptides were extracted from the polyacrylamide in 50% acetonitrile-5% formic acid and evaporated for MS analysis. The LC-MS system, elution of peptides, and mode of analysis were the same as described above. Likewise, the data were again analyzed by database searching using the Sequest search algorithm against viral protein and the nonredundant databases.


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RESULTS
 
To isolate a pure population of virions while preserving their biochemical and structural integrity, we first treated released RRV particles with DNase (to remove any unencapsidated DNA) and then subjected them sequentially to velocity sedimentation and equilibrium centrifugation. Transmission electron microscopy (TEM) both before and after gradient purification (Fig. 1A and B) revealed that the crude viral preparation contained all three (A, B, and C) species of capsids (33), empty virions, partially tegumented virions, and intact virions (Fig. 1A). Following both velocity sedimentation and equilibrium centrifugation, however, the population of particles was comprised largely of mature virions (Fig. 1B), although small amounts of extravirion material were evident (see below).

Analysis of RRV virions by MudPIT. We analyzed the protein contents of our purified samples (Fig. 1B) by the method of MudPIT—a gel-free approach to MS of complex mixtures that subjects the entire sample to both two-dimensional liquid chromatography and MS/MS while it is still in solution (see Materials and Methods). This method, therefore, avoids the potential problems associated with proteins that either enter gels poorly or extract from gel slices inefficiently (see Discussion).

We extracted the lipids from purified virions using a modified Folch extraction (see Materials and Methods) and subjected the complex protein solution to MudPIT analysis. These efforts revealed a total of 29 virus-encoded proteins (Table 1, fourth column). Not surprisingly, we identified four abundant capsid-associated proteins, the major capsid protein (MCP/ORF25), small capsomer-interacting protein (SCIP/ORF65), triplex protein 1 (TRI-1), and TRI-2 (ORF62 and -26, respectively), that we had characterized previously through standard MS/MS of excised bands from purified capsid preparations (33). However, we now also identified the portal protein (PORT/ORF43), the packaging protein (PACK/ORF19), and the viral protease (PRO)/scaffolding (SCAF), encoded by ORF17 (Table 1, fourth column). (Note that our data were unable to distinguish between the potentially coterminal proteins encoded by ORF17 [PRO] and ORF17.5 [SCAF] that, by homology with KSHV, RRV may also possess.) Additionally, we detected 15 putative tegument proteins (Table 1, fourth column). Some of these proteins, such as pORF27, pORF49, and pORF52, for example, are uncharacterized, while several others have only presumed functions based on various degrees of sequence homology to other herpesviral tegument proteins (Table 1, fourth column). The latter group includes pORF27, pORF32, Epstein-Barr virus (EBV) myristoylated protein (MyrP) homolog (ORF38), EBV MyrP binding protein homolog (MyrPBP/ORF33), EBV palmitoylated protein (PalmP) homolog (ORF55), large tegument protein (LTP/ORF64), LTP binding protein (LTPBP/ORF63), and viral phosphoribosylformylglycineamide amidotransferase (vFGARAT/ORF75). We confirmed the presence of several additional RRV proteins that others have characterized in KSHV, including the vIRF-7 binding protein (vIRF-7BP) encoded by ORF45 (67), the KSHV viral protein kinase (vPK) homolog encoded by ORF36 (16), and the thymidine kinase (TK) encoded by ORF21. Finally, we identified seven envelope proteins, gB (ORF8), gH (ORF22), gM (ORF39), gL (ORF47), gN (ORF53), the homolog of the KSHV complement control protein (KCP) encoded by ORF4, and the EBV gp150 homolog encoded by ORF28 (Table 1, fourth column). We then verified these results by repeating the MudPIT analysis four times on four different virion preparations (not shown).


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  		TABLE 1. Virion-associated RRV proteins identified by MudPIT and gel slice MS/MS analysis

Tandem mass spectrometry of virion-associated proteins. To ascertain that the MudPIT approach did not miss lower-abundance virion-associated proteins, we also analyzed our purified samples by a second and somewhat complementary method: MS/MS of excised gel slices. The virion-associated proteins of the particles shown in Fig. 1B were separated by SDS-PAGE (Fig. 2A). We visualized 12 highly abundant virion-associated protein bands (labeled in Fig. 2A) and cut them directly from the gel for traditional MS/MS. Since many additional proteins associated with the virions could have fallen below the level of detection of the stain, we also excised 1-mm slices from the entire remaining portion of the gel lane and subjected each to MS/MS analysis (see Materials and Methods). MS/MS of excised gel slices revealed many of the same proteins identified by MudPIT (Table 1, fifth column). As shown in Table 1 (fourth and fifth columns), the vast majority (29 proteins) of the proteins were observed by both MS/MS and MudPIT approaches. Nevertheless, some proteins, including R8.1 and the EBV epithelial ligand (epiL) homolog (ORF58), were detected only by MS/MS of 1D gel slices and not by MudPIT.



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  		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.

For both MudPIT and MS/MS of gel slices, we included proteins as truly virion associated only after their consistent detection in repeated analyses (Table 1). We excluded proteins that we identified in only one experiment. This excluded group comprised four proteins that we identified only once by either MudPIT or gel slice MS/MS: the small ribonucleotide reductase (RNR-S) encoded by ORF60, the large RNR (RNR-L) encoded by ORF61, SOX encoded by ORF37, and the protein encoded by ORF10 (data not shown). Since detection of these proteins was not reproducible, we omitted them from Table 1. Finally, for each MS analysis, we tabulated the total number of peptides that mapped to each of the virion-associated proteins, as well as the proportion (percent coverage) of each protein accounted for by these peptides. The last two columns of Table 1 show, for example, the results of such calculations from the gel slice MS/MS analysis of a PK-treated sample (see below).

Ensuring specificity: proteolytic treatment of purified RRV virions. Despite the multistep isolation protocol described above, we also detected a number of highly abundant non-virus-encoded proteins among the virions. Although cellular proteins accounted for only 12% of the total peptides we detected by MudPIT, 56% of these mapped to albumin. We hypothesized that our detection of some, if not most, of these proteins, such as albumin, may have resulted from nonspecific sticking to the virion rather than true integration into the particle. An additional confounding problem that can arise in this setting is that such high-abundance proteins can mask the detection of others that may be truly virion associated but present in only one or a few copies per virion (see Discussion).

To address these issues, we treated virions with either pronase or PK, in the absence of detergent, prior to velocity sedimentation and equilibrium centrifugation. (Sequential steps of proteolysis followed by centrifugation were critical to ensuring that subsequent MS did not detect the digested extravirion proteins.) Subsequent analysis by gel slice MS/MS revealed that a number of proteins associated with the purified virions without proteolysis were absent (e.g., albumin peptides) or greatly diminished following these steps. Others, however, were unaffected (see below).

TEMs of these proteolytically treated virions also demonstrated noticeably less extravirion background signal than those without proteolytic digestion (compare Fig. 1B and C). Of note, particles prepared for TEMs underwent an additional concentrating (pelleting) centrifugation step (see Materials and Methods) compared to those portions of the preparation analyzed directly by MudPIT or gel slice MS/MS analysis. This likely accounts for some of the particle distortion we observed after this final concentration step (Fig. 1B and C) that was less apparent at the earliest stages of the preparation (Fig. 1A), though it was difficult to conclude whether the particles subjected to MudPIT or MS/MS sustained similar damage. Despite potential compromise to virion integrity, the proteins remaining were protease resistant (see Discussion).

We separated the virion-associated proteins of these pronase- or PK-treated particles by SDS-PAGE, and the protein banding patterns for the latter are displayed in Fig. 2B (pronase-treated particles are not shown). The numbered protein bands represent the most abundant species (detectable by our staining methods) and were resistant to PK treatment (compare Fig. 2A and B). MS/MS analyses of these bands revealed that they contained peptides that mapped to the following proteins: 1, LTP; 2, MCP; 3, vFGARAT; 4, LTPBP; 5, PORT/TK; 6, PACK; 7, vIRF-7BP; 8, gB; 9, TRI-1; 10, TRI-2; 11, SCIP; and 12, pORF52. To ensure that the PK treatment in the absence of SDS was effective, we treated cytoplasmic and nuclear fractions of RhF under identical conditions and then separated their proteins by SDS-PAGE. Even in the absence of SDS, the vast majority of cellular proteins were sensitive to the PK treatment (not shown).

Proteolytic digestion of the virions not only helped eliminate highly abundant contaminating proteins, such as albumin, but had the serendipitous effect of revealing two previously undetected virion-associated (and proteolysis-resistant) proteins during MS analyses: one encoded by ORF42 and the other by ORF35 (Table 1). We also predicted that viral envelope glycoproteins would be variably susceptible to proteolytic digestion, at least in the regions extending beyond the lipid bilayer. In fact, following proteolytic digestion, MS/MS analyses no longer detected peptides mapping to two glycoproteins, R8.1 and epiL (Table 1), whereas others remained sufficiently intact to allow continued detection, including the KCP homolog, gB, gH, the EBV gp150 homolog, gM, gL, and gN.

MS-identified peptides derive from intact viral proteins rather than proteolytic fragments. To ascertain that we were not merely identifying proteolytic fragments rather than full-length intravirion proteins, we also examined the integrity of the MS-identified proteins (Fig. 3). We predicted that if the majority (or at least a substantial portion) of the viral particles remained intact during the isolation procedure (including proteolytic digestion), the peak of MS-detected tryptic peptides from each intraviral protein should emanate from the region of the gel where the full-length protein would migrate. The MS/MS approach on gel slices allowed us to test this prediction. The peptides of MCP, for example, peaked at approximately 150 kDa (observed molecular mass [MMobs]), coinciding with its predicted molecular mass (MMpre) of 153 kDa (Fig. 3A) (33). The small peak of MCP peptides we observed at the bottom of the gel corresponds to the band containing the dye front (Fig. 2B). We similarly observed breakdown products for several of the other larger-molecular-mass proteins, including vFGARAT, LTP, and LTPBP, but their peaks also correlated well with their respective MMpres (Fig. 3D). The analysis also revealed other proteins, including the EBV MyrPBP homolog (ORF33) and vIRF-7BP (Fig. 3D), in numerous gel slices throughout the gel. Nevertheless, in most cases, the peaks of these proteins were also near their predicted molecular masses. Interestingly, some proteins, such as TRI-2 (Fig. 3A), pORF52 (Fig. 3D), dUTPase, pORF27, and the EBV MyrPBP homolog (Fig. 3A), had some peptides present in excised gel slices that ran at higher molecular masses than predicted for their respective proteins, possibly reflecting the fact that these proteins may be in stable complexes that resist denaturation. The banding patterns of those peptides we identified that map to envelope proteins are shown in Fig. 3B. Although several of the envelope proteins had peptides present in gel slices near their predicted molecular masses (the peaks for gL and the EBV gp150 homolog, as well as the smaller, second peak for the KCP homolog), the remaining envelope proteins had peptides that were revealed in gel slices excised throughout the gel. This aberrant migration may be due to glycosylation (see Discussion).



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  		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 MMobs 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.

A subset of tegument proteins resist detergent treatment. To gain insight into the organization of the tegument, we treated RRV virions with 2% Triton X-100 to remove the envelope and more loosely associated tegument proteins. We then subjected the detergent-treated virions to MudPIT analysis following their purification through sucrose gradients. We observed not only the expected capsid-associated proteins, but also several putative tegument proteins, including the EBV MyrPBP homolog, vIRF-7BP, pORF52, vFGARAT, LTPBP, and LTP (Table 1). Figure 3D depicts the frequencies of peptides mapping to each of these the six detergent-resistant proteins derived from each gel slice after SDS-PAGE.

To corroborate some of our findings with MS and to help localize a subset of the virion-associated proteins to the capsid, tegument, or envelope, we immunoblotted purified virus and detergent-treated particles, with or without PK treatment, using antibodies that recognize several virus-encoded proteins to which antibodies were available: (i) MCP, which comprises the majority of the capsid, including the capsomers (hexons and pentons), as well as the capsid floor; (ii) SCIP, another capsid protein whose homolog in KSHV is located on the distal tips of the hexons but not pentons (25, 30, 62); and (iii) pORF52, which we hypothesize is a tegument protein tightly associated with the capsid and is resistant to detergent treatment.

We found that MCP and SCIP were present in virions regardless of protease treatment, as predicted by the MS/MS results (Fig. 3A, ORF25 and -65). However, in Triton-treated virions that were purified as described above and then treated with or without PK, MCP and SCIP were present only in the absence of PK, since the combination of detergent and proteolytic treatment destroyed the capsid particle, as reflected by the detection of multiple low-molecular-mass breakdown products for MCP (Fig. 4, lane 4) and the absence of signal for SCIP (not shown). pORF52, a gammaherpesvirus-specific protein, was unaffected by PK within the enveloped virion (Fig. 4, lane 2) and remained associated with the particle following detergent treatment (Fig. 3D and Fig. 4, lane 3). These immunoblotting results confirmed our observations with MudPIT analysis of detergent-treated virions and further support the idea that pORF52 is a tegument protein tightly associated with the capsid.



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  		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.


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DISCUSSION
 
A molecular map of primate gammaherpesviruses will help identify not only the proteins involved in structure and assembly, but also those that may play critical roles within the host during entry, establishment of infection, and egress following lytic replication. Toward this end, we isolated pure populations of RRV virions and then identified their virus-encoded protein components using two complementary MS approaches: MudPIT analyses of viral preparations maintained in solution and MS/MS fromcontiguous gel slices following SDS-PAGE. This effort, coupled with parallel analyses in the presence or absence of proteolytic digestion to ensure the highest levels of specificity, resulted in the unequivocal identification of 33 virion-associated proteins, comprising 7 capsid-associated proteins, 17 putatively tegument-associated proteins, and 9 envelope-associated proteins. We also found that detergent-treated virions retained not only the capsid-associated proteins, but also 6 of the 17 putative tegument proteins, suggesting that this subset is more tightly associated with the underlying capsid (Table 1). Further, our results were consistent in multiple analyses of independent viral preparations.

While helping to include only those proteins specifically associated with the virions, the use of proteolytic digestion (with either pronase or PK) had the added benefit of simultaneously increasing the sensitivity of the methods to detect lower-abundance proteins as well. This approach revealed two additional proteins, pORF35 and pORF42 (Table 1), both of which were originally masked by tryptic peptides from highly abundant but nonspecific proteins, such as albumin. Importantly, we avoided the inclusion of these nonspecific peptide fragments by subjecting the treated particles to gradient purification prior to MS analysis. The advantages to this purification step were also critical in eliminating virus-encoded proteins that were also likely nonspecifically adherent to the virion. These included the RNR-S, RNR-L, and SOX, all of which MudPIT analyses detected only once in the absence of this proteolytic step. Our results argue for the importance of such proteolytic steps in rigorously determining the composition of enveloped viruses in general.

Detection of low-abundance RRV-associated proteins by MS analysis. The use of two complementary MS methods to analyze the protein content of RRV is the first reported for any herpesvirus. Further, our MudPIT analyses represent the first data for any gammaherpesvirus by using this technology. MudPIT is significantly more cost-effective than exhaustive MS/MS conducted on an entire gel lane while also avoiding the bias of more traditional MS methods, where visualization of a protein spot or band is crucial. Finally, MudPIT obviates the need for polyacrylamide gels, thereby circumventing the potentially confounding and unpredictable issue that certain proteins are poorly extracted from gel slices prior to the MS analysis. We used MudPIT, therefore, to complement the MS/MS gel slice approach (see below).

Nevertheless, MudPIT, with its simultaneous analyses of complex mixtures of proteins, has its own weakness, namely, a higher probability of missing low-abundance proteins if the mixture also contains highly abundant species. This is especially problematic if this group includes large proteins, such as the MCP of herpesviruses (present in approximately 960 copies/virion). Such large and abundant proteins give rise to multiple peptides during the tryptic preparation step preceding MS analysis. Therefore, to ensure that we attained the most comprehensive molecular map of RRV, we complemented the MudPIT analyses with the more traditional method of 1D gel analysis, followed by MS/MS.

We took an unbiased MS/MS approach by excising contiguous slices from 1D gels. A powerful advantage of using this method was that it allowed us to retroactively determine the observed mass at which each of the proteins (and their resultant MS-detected tryptic peptides) resolved. This enabled us to distinguish proteins that were full length from those that were more likely fragments or, in contrast, potentially multimers (Fig. 3). The "peak" detection of most of the proteins migrated close to their MMpres, although some, such as a subset of envelope proteins (Fig. 3B), migrated at higher masses, likely reflecting the effects of posttranslational modification. None, however, demonstrated peaks significantly below their MMpres, arguing for the effectiveness of the postproteolysis gradient purification step in separating peptides from proteins that may have been nonspecifically adherent to the virions.

Capsid-associated proteins. The MS approaches that we report here detected the capsid-associated proteins that we had previously identified for both KSHV and RRV (30, 33). These include MCP, TRI-1, TRI-2, SCIP, and SCAF/PRO. The greater sensitivity of the present methods, however, also identified PACK and PORT, the first report of the incorporation of these proteins into a gamma-2 herpesvirus. PACK is 52% similar to its KSHV counterpart and 22% similar to herpes simplex virus type 1 (HSV-1) UL25. HSV-1 UL25, capable of binding the viral DNA, is localized to the capsid, where it associates with both VP5 (MCP homolog) and VP19c (TRI-1 homolog). The latter interaction allows its translocation from the cytoplasm to the nucleus during the late stages of HSV-1 infection (34). Whether the RRV PACK has similar functions is currently unknown. PORT gene demonstrates 35% similarity to its counterpart in HSV-1, UL6, which encodes the portal protein described by Newcomb et al. (31). HSV-1 PORT is associated with the HSV-1 virion (37) and is a dodecamer localized to a unique vertex on the capsid (31, 55). Although awaiting direct experimental verification, it is likely that both PACK and PORT genes encode proteins that are members of a group of core component proteins with conserved functions in all herpesviruses.

Gammaherpesvirus-specific proteins within the RRV tegument. The greatest differences in molecular composition among the herpesvirus subfamilies lie within the tegument and envelope layers. We have shown that RRV has 17 putative tegument proteins. Of these, five (pORF27, vIRF7-BP, pORF49, pORF52, and vFGARAT) are gammaherpesvirus specific. The pORF27 and pORF49 homologs in EBV, BDLF2 and BRRF1, respectively, are not well studied, and little is known of their functions. KSHV vIRF-7BP is a tegument protein (65, 67) that interacts with cellular IRF-7, blocking the latter's translocation from the cytoplasm to the nucleus (66). The EBV homolog of RRV pORF52 is BLRF2, and recent data indicate that it is a component of the virion (18, 48), where it interacts with the EBV SCIP homolog, sCP (48). Our data likewise demonstrate inclusion of RRV ORF52 in the virion while also suggesting that it is both highly abundant and tightly associated with the underlying capsid. We are currently investigating this interaction. RRV vFGARAT has significant homology to proteins of other gammaherpesviruses, and several studies have detailed its incorporation into the virions of herpesvirus saimiri, EBV, KSHV, and murine herpesvirus-68 (4, 6, 13, 14, 18, 20, 41-43, 65). The role of RRV vFGARAT in the viral life cycle and whether it encodes a functional homolog of cellular FGARAT remain unknown.

Three novel gammaherpesvirus proteins. We confirmed the presence of several additional proteins within the virion, including the dUTPase and the two uncharacterized proteins pORF23 and pORF42, all of which represent first reports of their inclusion in a gammaherpesvirus. Varnum et al., however, found that the human cytomegalovirus homologs of these three specific proteins are also virion associated (58), and although Johannsen et al. detected trace amounts of the homologs of these proteins in their analysis of purified virions of EBV, they elected to exclude the proteins from their final list of confirmed EBV-associated proteins (18). In contrast, we found the RRV dUTPase and pORF23 consistently in all of our experiments (Table 1). pORF42 was found in both experiments that included proteolytic treatment of virions prior to purification (Table 1). Little is known about the RRV or KSHV pORF23, but homologs in alphaherpesviruses are thought to play a role in intracellular transport of the virus (54). pORF42 is also uncharacterized in gammaherpesviruses, though ultrastructural studies on the HSV-2 homolog demonstrated that it is localized to the tegument (32). The number of peptides that mapped to the RRV dUTPase was consistently high (Fig. 3C). In contrast to other herpesviral dUTPases, the KSHV dUTPase is functional and is expressed during lytic replication (21). The full-length RRV dUTPase is slightly shorter (290 amino acids) but is 53% similar to residues 34 to 318 of KSHV dUTPase. Further, the herpesviral dUTPases are required for efficient viral replication in several systems (19, 40, 50). We have not confirmed whether the RRV dUTPase has catalytic activity.

Conserved herpesviral tegument proteins. Other proteins we identified that likely reside within the tegument include TK, pORF32, MyrP, MyrPBP, vPK, PalmP, LTP, and LTPBP. Our finding that virions of RRV contain pORF32, MyrP, and PalmP represents the first such report for a gamma-2 herpesvirus, though several groups have identified their homologs as virion-associated proteins in other subfamilies, including EBV, a gamma-1 herpesvirus.

A subset of tegument proteins tightly associated with the capsid. We have also identified a subset of tegument proteins that remain associated with viral particles after detergent treatment. These include LTP, LTPBP, MyrPBP, pORF52, vIRF-7BP, and vFGARAT. Furthermore, we detected all six of these proteins in multiple experiments, and each displayed a high number of peptides that mapped to their respective ORFs (Fig. 3D). Since these tegument proteins appear tightly associated with the capsid, they may be some of the first tegument proteins added during assembly and, likewise, may also be critical in processes such as transcytosis during either entry or egress (49). These proteins may also play roles, for example, in tegumentation (28).

RRV envelope proteins. Our MS analyses identified nine envelope proteins encoded by the virus. However, we detected proteins R8.1 and epiL by gel slice MS/MS but not MudPIT analysis. This pattern was reproducible in multiple preparations and may reflect the lower sensitivity of MudPIT compared with the gel slice MS/MS method (see below). Furthermore, R8.1 and epiL were the only predicted glycoproteins that were also sensitive to proteolysis (Table 1). The remaining seven envelope proteins encoded by ORF4 (see below), ORF8, ORF22, ORF28, ORF39, ORF47, and ORF53 remained associated with the particle following proteolytic treatment. This resistance to proteolysis may reflect high levels of glycosylation (3, 5, 11, 17, 27, 29, 35, 38, 39, 45, 46, 53, 57). Based on homology to other herpesviruses, R8.1 (homologous to KSHV K8.1) and epiL are both likely glycoproteins and are also poorly represented in our MS/MS gel slice analyses (Fig. 3D), suggesting (though not proving) their relatively low abundance. A low copy number of epiL molecules per virion may also explain the failure to detect significant amounts of its homolog in EBV, BMRF2, in recent work by others (18).

Incorporation of the complement control protein into the virion envelope. This is the first report of the incorporation of RCP (a homolog of the KSHV complement control protein, KCP), pORF35, and pORF49 into a herpesvirus. While pORF35 and pORF49 encode putative tegument proteins of unknown function, RCP is likely a component of the envelope. The KSHV homolog, KCP, has three isoforms, all of which regulate the complement component 3 (C3) convertase of the complement pathway and contain the domains necessary for complement regulation. Furthermore, all three isoforms retain a transmembrane region (51, 52). Therefore, it is possible that KCP and RCP assist in protecting infected cells from the first wave of the host's innate immune response and the virus from opsinization. Our data were unable to determine whether the virion incorporates a single or multiple potential isoforms of RCP.

We used two complementary MS-based techniques to identify 33 virus-encoded protein components on highly purified RRV. Defining the molecular composition of this primate gammaherpesvirus has laid the foundation for the next stage of this research—to explore bona fide contributions of cellular components to the mature virion and to define the functions of each of these virion proteins, examining their potential roles in initial infection, entry, assembly, and egress.


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ADDENDUM IN PROOF
 
Following submission of this paper, ORF10 was identified as encoding a dUTPase-related protein (A. J. Davison and N. D. Stow, J. Virol. 79:12880-12892, 2005).


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ACKNOWLEDGMENTS
 
This research was supported by the following organizations: NIH (R01 CA88768), the Doris Duke Foundation (20000355), and the Elizabeth Glaser Pediatric AIDS Foundation (28-PG-51381).

We thank Nicholas Sherman at the William Keck Biomedical Mass Spectrometric Laboratory for helpful discussions regarding MS analyses, Nancy Verville for assistance in antibody production, the University of Virginia Cancer Center for support, and Anna Maria Copeland and David Radoff for helpful discussions regarding EM techniques. We also thank Scott Wong at the Oregon National Primate Center, Oregon Health and Science University, for providing us with the ORF25 polyclonal mouse sera and Chris Parsons for critical reading of the manuscript.


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FOOTNOTES
 
* Corresponding author. Mailing address: Myles H. Thaler Center for AIDS and Human Retrovirus Research, Box 800734, University of Virginia Health Systems, Charlottesville, VA 22908. Phone: (434) 243-2758. Fax: (434) 982-1071. E-mail: kedes{at}virginia.edu. Back


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Journal of Virology, February 2006, p. 1574-1583, Vol. 80, No. 3
0022-538X/06/$08.00+0     doi:10.1128/JVI.80.3.1574-1583.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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