Previous mass spectrometry studies have characterized the viral and host protein content of several herpesvirus virions, including HSV-1 (83
), Kaposi's sarcoma-associated herpesvirus (KSHV) (8
), murine gammaherpesvirus 68 (MHV68) (9
), alcelaphine herpesvirus 1 (AlHV-1) (33
), EBV (56
), murine cytomegalovirus (MCMV) (61
), rhesus monkey rhadinovirus (RRV) (107
), and HCMV (6
). Additionally, Michael et al. utilized stable isotope labeling with amino acids in cell culture (SILAC) in combination with mass spectrometry to quantitatively analyze changes in the incorporation of select virion components in different mutant PRV strains. However, the proteins identified in this study did not represent all of the virion constituents that were identified by additional approaches, especially viral envelope proteins (see Tables S1 and S3 in the supplemental material) (99
). Therefore, in this current study, we performed a comprehensive analysis of the protein content of PRV, including both viral and host proteins.
To produce purified virions suitable for the high sensitivity of mass spectrometry, we utilized an isolation approach previously used to purify HSV-1 virions (83
). This yielded a purified sample of extracellular virions as confirmed by negative-stain electron microscopy and Western blot analysis ( and ). We then analyzed the protein content of virions using two complementary mass spectrometric approaches. The first approach involved in-gel digestion and analysis on a LTQ Orbitrap XL mass spectrometer configured with a MALDI ionization source. Luo et al. recently demonstrated that this configuration is suitable for rapid and sensitive detection of proteins present within a complex sample (84
). Since sample loss is commonly associated with in-gel digestion strategies, we confirmed and extended our results using a second proteomic approach involving in-solution digestion by FASP and nLC-MS/MS analyses (138
). While we reproducibly identified 35 known viral components using both approaches, the in-solution digestion approach enabled the identification of an additional 12 viral proteins associated with PRV virions (). Therefore, we concluded that this approach is suitable for characterizing samples with the composition and complexity of extracellular virions.
To discriminate between virion components and potential contaminating proteins, we performed our analysis on proteinase K-treated virions as well as samples isolated from mock-infected cells. Protease treatment has been previously utilized to eliminate contaminants present on the surface of KSHV (8
) and RRV (107
) virions and may be a useful tool for future proteomic studies of other enveloped viral particles or membranous organelles. Analysis of proteinase K-treated PRV virions by mass spectrometry and Western blotting confirmed that capsid and tegument proteins were mostly resistant to digestion. Surprisingly, following proteinase K treatment of virions, we noticed a striking reduction of the high-molecular-weight band in the gel region that contains pUL36 (). While Zhu and colleagues found evidence that open reading frame 64 (ORF64), the KSHV orthologue of PRV pUL36, is completely sensitive to protease treatment in KSHV virions (144
), our data suggest that this is not the case for PRV pUL36. Our Western blot analyses and mass spectrometry data confirm that pUL36 is a tegument protein, a conclusion that is consistent with previous studies on the localization and function of pUL36 in virions (10
Furthermore, several viral glycoproteins with domains that are exposed on the outer virion envelope were sensitive to protease digestion (e.g., gE, gI, gD, gH, and gL), and the protein sequence coverage obtained by mass spectrometry analysis was in agreement with prior knowledge regarding their domain topology. Unexpectedly, gB and gC, which have topologies and membrane orientations similar to those of gE, gI, gH, and gD, did not display decreased peptide sequence coverage after proteinase K treatment (). We hypothesize that this is because gB and gC are major components of the virion envelope (50
), and the sensitivity and sequence-based analysis of the FASP-mass spectrometry approach detected partially digested gB and gC proteins. For readily detected proteins or those with few protease cleavage sites, sequence coverage will not always provide the most discriminating metric for quantitative analysis of protease sensitivity. Indeed, the proteinase K-dependent increase in unique semi-tryptic peptides for gB and gC is supported by our Western blot analysis that confirmed the efficient cleavage of these proteins by proteinase K ( and ).
Overall, we determined by mass spectrometry that PRV virions contain a total of 47 viral proteins ( and ). This list includes 40 of the 42 previously known viral virion proteins and seven novel viral proteins ( and ). One of the novel virion components, RSp40/ICP22, is an orthologue of HSV-1 ICP22, a protein implicated in multiple aspects of infection. Early in infection, ICP22 induces the formation of discrete nuclear foci containing cellular chaperone proteins known as VICE domains (7
). These domains are thought to play an important role in nuclear alterations and protein quality control necessary for viral infection. Additionally, ICP22 has been implicated in modulating viral gene expression (1
) and ensuring proper virion morphology (110
). However, ICP22 was undetectable in HSV-1 virions (83
). Further work is necessary to determine if PRV RSp40/ICP22 is functionally homologous to HSV-1 ICP22. Incorporation of this protein into PRV virions is consistent with a potential role in the early stages of infection. This may be relevant to a successful infection since PRV, unlike HSV-1, does not express RSp40/ICP22 with immediate-early kinetics (113
Fig. 8. Schematic summary of previously known and novel viral virion components. Using mass spectrometry, a total of 47 viral proteins were detected in PRV virions including 40 (of the 42) previously identified viral virion proteins (white) and seven novel viral (more ...)
In addition to identifying viral virion components, we detected phosphorylated peptides from pUL26, pUL36, pUL46, and pUL48. Posttranslational modifications of virion proteins may serve a variety of functions during viral infection, such as modulation of viral particle assembly, egress, or entry (14
). However, key modifications, such as phosphorylation, can be challenging to detect using mass spectrometry and often require further enrichment (21
). Our ability to detect these modifications without enrichment suggests that they are abundant within virions.
Phosphorylation of PRV pUL26, pUL36, pUL46, and pUL48 within virions is, to our knowledge, a novel finding. Phosphorylation of the HSV-1 orthologues of three of these proteins, pUL36, pUL46, and pUL48, has been previously described following virion entry, but the specific sites at which these proteins are modified have not been defined (90
). For pUL48, a phosphorylation site has been predicted at Ser375, which is within a consensus casein kinase II (CKII) site near the C terminus. Mutation of this site to alanine disrupts pUL48 transactivation function (109
). While the single phosphorylation we detected on PRV pUL48 could not be unambiguously localized to a particular residue, the phosphopeptide we detected contains the same CKII site, which is conserved in PRV and HSV-1 (109
). Additionally, the phosphorylation sites within pUL36 (Ser2726 and Ser2969) are within a proline-rich domain near the C terminus. Interestingly, a PRV strain with a large deletion that encompasses this site displays decreased neurovirulence in infected mice (10
). Further analyses will determine if this phosphorylation is critical for pUL36 function. For example, this modification may have a role during viral entry or assembly by facilitating nucleocapsid binding (79
), nuclear pore docking (25
), or recruitment of molecular motor proteins (114
). Interestingly, while Morrison et al. have shown that phosphorylation of the HSV-1 orthologues of these proteins occurs only after entry (105
), our findings show that these proteins are already phosphorylated in extracellular virions. It is tempting to speculate that phosphorylation may serve to activate viral proteins for specific functions during the early stages of infection.
In addition to viral proteins, herpesvirus virions have been previously shown to contain cellular proteins (8
). However, incorporation of host proteins into PRV virions has been poorly characterized. In this study we identified a total of 48 host proteins that were present in both untreated and proteinase K-treated PRV virions but absent in samples from mock-infected cells (). This number of host proteins was comparable to that previously detected by mass spectrometry in other herpesvirus virions, such as 49 in HSV-1 (83
), 71 in HCMV (133
), and 43 in EBV (56
). Among the host proteins we detected, many have well-characterized functions and participate in a variety of biological pathways, such as cellular signaling, membrane organization and trafficking, RNA processing, and metabolism. Comparison of the host proteins we detected in PRV virions with those detected in other herpesvirus virions by mass spectrometry revealed several interesting similarities. Notably, both PRV and HSV-1 virions share four small GTPase family members (), while none was detected in HCMV. Additionally, the virions of PRV, HSV-1, and HCMV, but not EBV, contain members of the 14-3-3 family of signaling proteins (56
). While these similarities and differences may be due to experimental techniques, the comparison may offer insights into the underlying mechanisms of herpesvirus assembly and the role of host proteins during infection.
In addition to their functions during PRV infection, the mechanisms by which host proteins are localized to virions remain unclear. Several studies have shown that incorporation of certain cellular proteins into virions may be targeted, such as MAP kinase (88
). An additional example is the incorporation of cellular actin, which is increased in the absence of the tegument proteins VP22, pUS3, and pUL47 (30
). Alternatively, host proteins may be included in virions nonspecifically, and their presence could reflect proteins that are highly abundant within the cell or enriched at sites of viral assembly. Although little is known about the copy numbers of host proteins in mock- and PRV-infected PK15 cells, several studies have investigated the expression of host genes in a variety of PRV- and HSV-1-infected cell types and tissues (115
). Interestingly, the mRNA expression levels corresponding to 9 of the 48 cellular proteins we detected were decreased more than 3-fold during PRV infection of rat embryonic fibroblasts (115
). In contrast, gene expression of only one of the proteins we detected (thioredoxin) was increased (115
). These data do not directly address the relationship between protein abundance and virion incorporation; however, they suggest that the majority of the host proteins were detected in PRV virions despite their unchanged or decreased expression levels during infection. Further targeted studies involving dominant negative proteins or knockdown approaches are necessary to determine the functional roles that these proteins may have during infection.
In summary, we describe the first comprehensive analysis of PRV virion composition. Future analysis of the host and viral protein content of virions will enhance our understanding of the involvement of these proteins in the processes of viral assembly, egress, entry, pathogenesis, and spread.