Free access
Editor's Pick
Research Article
1 December 2022

Proteomic Comparison of Three Wild-Type Pseudorabies Virus Strains and the Attenuated Bartha Strain Reveals Reduced Incorporation of Several Tegument Proteins in Bartha Virions


Pseudorabies virus (PRV) is a member of the alphaherpesvirus subfamily and the causative agent of Aujeszky’s disease in pigs. Driven by the large economic losses associated with PRV infection, several vaccines and vaccine programs have been developed. To this day, the attenuated Bartha strain, generated by serial passaging, represents the golden standard for PRV vaccination. However, a proteomic comparison of the Bartha virion to wild-type (WT) PRV virions is lacking. Here, we present a comprehensive mass spectrometry-based proteome comparison of the attenuated Bartha strain and three commonly used WT PRV strains: Becker, Kaplan, and NIA3. We report the detection of 40 structural and 14 presumed nonstructural proteins through a combination of data-dependent and data-independent acquisition. Interstrain comparisons revealed that packaging of the capsid and most envelope proteins is largely comparable in-between all four strains, except for the envelope protein pUL56, which is less abundant in Bartha virions. However, distinct differences were noted for several tegument proteins. Most strikingly, we noted a severely reduced incorporation of the tegument proteins IE180, VP11/12, pUS3, VP22, pUL41, pUS1, and pUL40 in Bartha virions. Moreover, and likely as a consequence, we also observed that Bartha virions are on average smaller and more icosahedral compared to WT virions. Finally, we detected at least 28 host proteins that were previously described in PRV virions and noticed considerable strain-specific differences with regard to host proteins, arguing that the potential role of packaged host proteins in PRV replication and spread should be further explored.
IMPORTANCE The pseudorabies virus (PRV) vaccine strain Bartha—an attenuated strain created by serial passaging—represents an exceptional success story in alphaherpesvirus vaccination. Here, we used mass spectrometry to analyze the Bartha virion composition in comparison to three established WT PRV strains. Many viral tegument proteins that are considered nonessential for viral morphogenesis were drastically less abundant in Bartha virions compared to WT virions. Interestingly, many of the proteins that are less incorporated in Bartha participate in immune evasion strategies of alphaherpesviruses. In addition, we observed a reduced size and more icosahedral morphology of the Bartha virions compared to WT PRV. Given that the Bartha vaccine strain elicits potent immune responses, our findings here suggest that differences in protein packaging may contribute to its immunogenicity. Further exploration of these observations could aid the development of efficacious vaccines against other alphaherpesvirus vaccines such as HSV-1/2 or EHV-1.


The alphaherpesviruses represent a large subfamily of enveloped double-stranded DNA (dsDNA) viruses (1). Important members include herpes simplex virus 1 and 2 (HSV-1/2) and varicella zoster virus (VZV) in humans, bovine herpesvirus 1 (BoHV-1) in cattle, equine herpesvirus 1 (EHV-1) in horses, and the suid herpesvirus 1 or pseudorabies virus (PRV) in pigs (1). PRV is the causative agent of Aujeszky’s disease in pigs, which may involve respiratory, neurological and reproductive symptoms (2). Given its high homology to other alphaherpesviruses, PRV is often employed as a model organism to study alphaherpesvirus biology and interactions with the host (3). The infectious particle (or virion) of PRV, like that of all herpesviruses, is characterized by a dsDNA genome which is enclosed by a capsid (4, 5). The capsid is a highly organized, icosahedral structure (T = 16) consisting of 150 VP5 (pUL19) hexamers, 11 VP5 pentamers, and 1 pUL6 dodecamer (4), which is decorated with triplexes of VP19C (pUL38) and VP23 (pUL18), capped by VP26 (pUL35) and the heterodimer pUL17 and pUL25 (4). The capsid resides in a proteinaceous layer called the tegument (6) that is composed of both viral and cellular proteins. It has an icosahedrally organized inner layer where viral tegument proteins, such as VP1/2 (pUL36), pUL37, pUS3, and VP13/14 (pUL47), closely interact with the capsid (68) and an outer layer that is less organized and contains several viral proteins, such as VP16 (pUL48), VP11/12 (pUL46), VP22 (pUL49), pUL41, pUL16, pUL21, pUL11, pUL51, and pUL7, but also host proteins such as actin (6, 916). Finally, the tegument is enclosed by a membrane envelope containing several transmembrane (glyco)proteins, such as glycoprotein B (gB), gC, gD, gE, gH, gM, gH, gI, gK, pUS9, pUL56, and pUL43 (3, 1724). The assembly of PRV virions starts in the nucleus of the infected cell by incorporation of the newly formed genome into the capsid, creating the nucleocapsid, which then migrates to the cytoplasm (25). Here, the nucleocapsid is partly tegumented and docks onto tegument proteins that have accumulated at the trans-Golgi network (TGN) by interactions with the cytoplasmic domains of viral envelope (glyco)proteins (26). This docking induces budding of the particle into the TGN, creating the mature virion, which is then transported to the plasma membrane and released in the extracellular medium.
Scientific research on PRV is usually performed with well-established strains, with the Becker, Kaplan, and NIA3 wild-type (WT) strains arguably representing the most commonly used WT PRV strains (2729). The Bartha strain, on the other hand, is an attenuated PRV strain that was created by serial passaging in cell cultures and represents one of the most widely used vaccine strains against PRV worldwide (30, 31). Bartha PRV is also used as a vector vaccine for other pathogens (32) and as a retrograde neuronal tracer to elucidate neuronal interactions (33). The Bartha genome is characterized by a large deletion in the unique short (US) region, affecting the genes US7 (gI), US8 (gE), US9, and US2, but also displays a myriad of other mutations, affecting genes such as UL21, gM, gC, and gH (3439).
Viral entry represents the first step of viral infection of a host cell and the composition of the virion is thus crucial for the initial interaction between the virus and the host. A previous proteome characterization of purified Becker virions by Kramer et al. (40) has identified 47 viral proteins of which 40 were established before as being structural virion components, while 7 were earlier presumed to be nonstructural. These authors also reported 48 host proteins putatively incorporated in the virion. For Bartha PRV, a previous proteome characterization performed by Yu et al. (41) identified 34 viral and 25 host proteins in the extracellular virions of a Bartha-derived mutant (expressing an mRFP-labeled VP26 and an enhanced green fluorescent protein [EGFP]-labeled gM protein). In addition, a study using stable isotope labeling by amino acids in cell culture (SILAC) revealed that Bartha PRV virions incorporate less VP11/12, VP22, and pUS3 compared to WT Kaplan virions because of mutations in the UL21 gene (42). Nevertheless, a comprehensive proteome comparison of virions of different WT PRV strains and the Bartha PRV strain has not been done thus far.
In the present study, we compared virion preparations of three of the most commonly used WT PRV strains—i.e., Becker, Kaplan, and NIA3—and the attenuated Bartha vaccine strain using mass spectrometry-based proteomics. We identified a total of 37 established viral structural proteins as well as 6 presumed nonstructural viral proteins by data-dependent acquisition (DDA) (4345). By using the more sensitive data-independent acquisition (DIA) (4648), we additionally found 3 established structural and 8 presumed nonstructural viral proteins. Comparison of the PRV strains revealed that capsid and most envelope proteins are incorporated to near-identical amounts in all strains. Nonetheless, distinct interstrain differences, particularly when comparing Bartha to the WT strains, were observed in virion incorporation of several established tegument proteins (VP11/12, IE180, pUS3, VP22, and pUL41), two envelope proteins (pUL43 and pUL56) and at least three presumed nonstructural proteins (pUS1, pUL40, and pUL50). Probably as a result of the reduced incorporation of tegument proteins, we observed that Bartha virions are smaller in size and display a more icosahedral morphology compared to WT virions. Finally, we also detected the presence of at least 28 host proteins that were previously reported to be packaged in PRV virions (40, 41) and noticed considerable strain-specific differences in the accumulation of several host proteins, arguing that the potential role of cellular proteins incorporated in virions should be further explored.


Isolation of PRV virions.

To generate and collect virions for mass spectrometry, ST cells were infected with either Bartha, Becker, Kaplan, or NIA3 PRV strains at a multiplicity of infection (MOI) of 10, and supernatants were harvested at 24 h postinfection (hpi). To confirm efficient virus replication in ST cells, infected cells were harvested and analyzed by Western blotting. Figure 1A demonstrates expression of the major viral capsid protein VP5 in the infected cells for all PRV strains. Moreover, as expected due to the large US deletion in the Bartha genome, Bartha-infected cells did not express gE. Extracellular virions were collected from supernatants of infected cells by ultracentrifugation through a Ficol-400 cushion, as described before to isolate mass spectrometry (MS)-grade PRV or HSV-1 virions. Based on these prior studies, it is reasonable to assume that these preparations are devoid of noninfectious light particles (L-particles) (40, 49). Virus preparations were subsequently subjected to Western blot analysis, revealing similar quantities of virions based on VP5 levels and again confirming the absence of gE in Bartha virions. Tubulin was not detected in the purified virions, indicating that any possible contamination of cellular material is minimal or nonexistent (Fig. 1B).
FIG 1 Purity assessment of virions by Western blotting and Coomassie blue staining. Infected cell (A) or virus particle (B) lysates were separated by SDS-PAGE and analyzed by Western blotting with antibodies against VP5, gE, and tubulin. (C) Virion lysates were separated by SDS-PAGE and visualized by Coomassie blue before extraction and processing for mass spectrometry. Several abundant proteins were annotated based on previous observations (50).
For MS analysis, virus preparations were lysed, separated by SDS-PAGE, and visualized by Coomassie blue staining (Fig. 1C). Distinct bands were noted for all four virus preparations similar to what has been observed by others, making it possible to distinguish several abundant viral proteins with a high degree of certainty (see arrows in Fig. 1C) (40, 50). For each replicate, all the proteins of the entire lane were then in-gel digested, extracted from the gels, and processed for MS analysis.

Proteomic analysis of extracellular virions.

Since we aimed to compare the proteome of the four viral strains in this study, we needed to correct the peptide data set for strain-specific differences, i.e., point mutations. Therefore, we deployed a custom analysis leveraging the Progenesis QIP match-between-runs label-free quantification strategy. More specifically, all runs were aligned in the MS1 space, and all fragmentation spectra acquired on a specific peptide over the experiment were aligned to a single “precursor feature” in a single project. This way, nonidentical peptides between strains can be discarded by serially performing the peptide annotation against every strain, creating a data set with only those peptides that are conserved in all four strains. Unfortunately, this approach also inadvertently discarded all the peptides of pUS2, gE, gI, and pUS9 due to the absence of the corresponding protein sequences in the Bartha proteome. Therefore, another Progenesis QIP project was created for which the peptides were not annotated against the Bartha strain, allowing the identification of pUS2, gE, gI, and pUS9 in the WT strains.
In most proteome analyses, at least two unique peptides are required to claim protein identification. Although the aforementioned filtering step sacrificed several nonconserved viral peptides, this only resulted in a minor reduction in unique peptides. In fact, pUL34 was the only protein that fell below the identification threshold due to this filtering process. Using this approach, we identified a total of 37 structural viral proteins (Table 1) with at least two unique peptides by data-dependent acquisition (DDA). In addition, we detected six proteins that were presumed to be nonstructural (Table 2). Notably, these six proteins were also observed in Becker virions in a proteomic study by Kramer et al. (40), whereas 3 of these proteins were observed in Bartha virions in the proteomic study by Yu et al. (41). The only established structural proteins that were not identified in our study were gL, gK, gG, and EP0 (Table 1).
TABLE 1 Viral structural proteins present or absent in PRV virion preparations as determined by DDA analysis
Viral proteinCommon nameVirion localizationMass (kDa)No. of conserved peptidesNo. of unique conserved peptidesDetected by:
Kramer et al. (40)Yu et al. (41)
 pUL6 Capsid701918++
 pUL17 Capsid641717++
 pUL25 Capsid573333++
 pUL43 Envelope3844+a+
 pUS9c Envelope1122+a
 pUL7 Tegument291010+a+
 pUL11 Tegument722+a
 pUL16 Tegument352222++
 pUL21 Tegument552121++
 pUL37 Tegument985555++
 pUL51 Tegument2555++
 pUS2c Tegument281818+
Not detected or uncertain       
Only detected by Kramer et al. by using the nLC-ESI workflow (40).
Only detected by Kramer et al. by Western blotting (40).
Proteins were identified using the Progenesis QIP project not annotated to the Bartha strain.
TABLE 2 Presumed nonstructural viral proteins detected in PRV virion preparations by DDA analysis
Viral proteinCommon nameMass (kDa)No. of conserved peptidesNo. of unique conserved peptidesDetected by:
Kramer et al. (40)Yu et al. (41)
pUL20 1766+
pUL32 5233+a+
Only detected by Kramer et al. by using the nLC-ESI workflow (40).
To increase the sensitivity of the analysis, we reanalyzed all the samples in the same randomized order, this time operating the MS instrument in data-independent acquisition (DIA) mode (SWATH). As recently shown, using predicted spectral libraries increases the depth of the detectable proteome (47). Here, predicted libraries were used to search for peptides using the neural network-based DIA-NN search engine, and peptides were considered detected upon a minimum posterior error probability value below 0.01 (51). This approach allowed us to confirm the presence of the structural proteins EP0, gL, and gK. However, for gG, detection remained uncertain as only 1 unique peptide was detected (Table 3). Moreover, using this SWATH data set, we additionally identified the presumed nonstructural proteins pUL2, pUL5, pUL8, pUL14, pUL23, pUL29, pUL31, and pUL34 (Table 3). Similar unique peptides were found for all four strains for most of these proteins, except for pUL29, which had less peptides detected in Becker and Kaplan virions. Curiously, we identified both the proteins pUL31 and pUL34, albeit in very small amounts. Both proteins are thought to serve as markers for immature, primary virions (52), yet pUL34 was previously also detected in mature PRV virions, while both pUL31 and pUL34 have been found in HSV-1 virions (40, 53, 54).
TABLE 3 Viral proteins detected in virion preparations by SWATH acquisition (DIA) that were not detected by DDA
Viral proteinCommon nameVirion localizationMass (kDa)No. of unique peptides detected in each straincDetected by:
BeKaN3BaKramer et al. (40)Yu et al. (41)
pUL2 Uncertain333454
pUL5 Uncertain923222
pUL8 Uncertain711111118+a+
pUL14 Uncertain183221
pUL31 Uncertain302122
pUL34 Uncertain284624+b
Only detected by Kramer et al. by using the nLC-ESI workflow (40).
Only detected by Kramer et al. by Western blotting (40).
Be, Becker; Ka, Kaplan; N3, NIA3; Ba, Bartha.

Differential proteome analysis identifies reduced incorporation of tegument proteins in Bartha virions.

We next performed a differential proteome analysis of Becker, Kaplan, NIA3, and Bartha virions. Therefore, the viral proteins for each strain were quantified based on the data set of conserved peptides in the DDA background (Tables 1 and 2) and protein abundances were normalized to the major capsid protein VP5 (4). Figure 2 shows the normalized protein abundance organized by virion localization for each of the PRV strains. Of note, the normalized abundance of gE, gI, pUS2, and pUS9 (which are absent in Bartha) were superimposed based on the approach mentioned in the previous section. It should also be pointed out that these normalized abundances do not allow strong claims about the absolute abundance of individual proteins in virions because of potential differences in detectability of individual peptides. It is therefore not strictly possible to compare abundance of different proteins within the same virus preparation. However, since the same peptides were used to quantify viral proteins in each strain, it is possible to compare relative abundance of individual proteins in-between strains. Even though label-free liquid chromatography-mass spectrometry (LC-MS) analysis does not allow for absolute quantification, VP5 was by far the most abundantly detected protein (Fig. 2), which is in line with its dominant abundance in virions as the major capsid protein and correlates with the strong VP5 signal observed in the Coomassie blue staining in Fig. 1C. Similarly, gB and tegument proteins VP13/14 (pUL47), VP22 (pUL49), and VP1/2 (pUL36), which are known to be present in high copy numbers in the viral particle, also stand out as highly abundant in the proteome analysis (Fig. 2). At the other end of the spectrum, pUL6, of which only 12 copies are present in each capsid (55), shows the lowest abundance of all capsid proteins (Fig. 2). When comparing the different strains, all of the capsid proteins depict a near-identical virion abundance across strains, which is in accordance with the stringent position of these proteins in the icosahedral capsid structure (4). Similar as observed for the capsid proteins, all envelope proteins display fairly similar distributions among the four PRV strains, with the obvious exception of gE, gI, and pUS9, which are absent in Bartha as the corresponding genes are (partially) deleted from its genome. The same holds true for the tegument protein pUS2, which is also absent in Bartha virions. Interestingly, tegument proteins VP11/12 (pUL46) and VP22 are both drastically less incorporated in Bartha virions, which corresponds with previous observations by Western blotting and mass spectrometry (42, 56). Remarkably, the tegument protein IE180 that represents the only immediate early protein of PRV is almost completely absent in Bartha virions, even though it shows substantial and well-conserved abundance in all the WT strains. This virtual lack of IE180 in Bartha virions is also noticeable on the Coomassie blue staining, as shown in Fig. 1C.
FIG 2 Normalized abundance of viral proteins in virions of the four tested PRV strains. Viral proteins for each strain were quantified based on a data set of conserved peptides and normalized to the major capsid protein VP5. Error bars represent the standard deviations for the different replicates (n = 6 for Kaplan, NIA3, and Bartha; n = 5 for Becker).
To better evaluate the observed differences in-between strains, a data set was created showing the normalized protein abundance of each strain set relative to the Kaplan strain (Fig. 3). This further supports the notion that all capsid proteins have a highly similar abundance in all the virus strains (Fig. 3, upper graph). However, for the tegument proteins, we noticed that several proteins were prone to high interstrain variation, whereas others showed very little interstrain variation. Moreover, of those proteins that show the highest variance, almost all were drastically reduced in Bartha (Fig. 3, middle graph). Indeed, the analysis not only pointed out that the relative amounts of the VP11/12, pUS3, VP22, and IE180 proteins are severely compromised in Bartha virions, in line with the analysis in Fig. 2 and published literature (42, 56), but also that pUL41 shows a reduced abundance in Bartha virions compared to all the WT strains (Fig. 3, middle graph). This demonstrates that the number of tegument proteins that display reduced incorporation in Bartha virus particles stretches substantially beyond the previously described pUS3, VP11/12, and VP22 proteins (50, 56). Becker and NIA3 virions, on the other hand, show an overall increased incorporation of these tegument proteins compared to Kaplan virions. Of interest, many of the tegument proteins that have a highly similar abundance in between PRV strains (VP13/14, VP1/2, pUL37, VP16, pUL21, and pUL16) have been described as inner tegument proteins or closely associated with the capsid, whereas the more variable tegument proteins mainly represent outer tegument proteins (6). The envelope proteins on the other hand show a fairly similar incorporation in-between strains, with the exception of pUL43 and pUL56 (Fig. 3, lower left graph). Whereas the former is substantially more abundant in Becker and NIA3 virions, the latter is distinctly less abundant in Bartha virions. Of interest, gM and gN, which form a disulfide-linked heterodimeric complex (57), appear to show the least variability with regard to virion incorporation. Lastly, with regard to the presumed nonstructural proteins, pUL50 displays an almost uniquely high abundance in Becker virions (Fig. 3, lower right graph). In conclusion, despite overall strong similarities in viral protein incorporation in virions of different WT PRV strains, we observed some drastic strain-specific differences with regard to the incorporation of selected viral proteins into the PRV virion. Most notably, we found that Bartha PRV not only shows reduced (and in some cases virtually absent) virion incorporation of pUS3, VP22, and VP11/12 but also of additional tegument proteins (IE180 and pUL41) and an envelope protein (pUL56).
FIG 3 Normalized protein abundance in virions for each strain set relative to the normalized abundance in Kaplan virions. The average normalized abundance of each viral protein was set relative to that in Kaplan virions. Viral proteins were categorized according to their virion localization and displayed in a descending order of maximum fold change between strains. Error bars represent propagated standard deviations (n = 6 for Kaplan, NIA3, and Bartha; n = 5 for Becker).
Proper protein quantification based on peptide measurements is a challenging aspect of proteomics. Therefore, to further confirm our observations and apply statistical analysis, viral peptide measurements were statistically analyzed in six pairwise comparisons using MSqRob and represented as volcano plots (Fig. 4) (58). Note that gE, gI, pUS9, and pUS2 were not included in the pairwise comparison, since these proteins are not present in the Bartha strain, and superimposition of the measurements would falsify the statistical analysis. Clearly, the reduced incorporation of the proteins IE180, VP11/12, pUS3, VP22, pUL41, and pUL56 in Bartha virions compared to any of the WT strains is apparent. In addition, also the presumed nonstructural proteins pUS1 and pUL40 were less incorporated in Bartha virions compared to the WT virions. This was statistically confirmed, as -log10 q values above 1.3 are considered statistically significant (58, 59). Similarly, this analysis confirms the increased incorporation of pUL50 in Becker virions. (Fig. 4).
FIG 4 Volcano plots of the six pairwise comparisons of the four PRV strains. Log2-fold changes are represented in function of the –log10 q value of each pairwise comparison. Significantly different proteins are marked with a filled circle and have a –log(q) value of >1.3 (marked by the dashed line); nonstatistically significant different proteins are marked with an open circle.

The reduced incorporation of IE180 in Bartha virions is largely independent of pUL21 or an altered intracellular localization of IE180.

The observation that Bartha PRV incorporates less pUS3, VP11/12, and VP22 in virions has been linked to mutations in the UL21 gene of Bartha, which encodes the pUL21 tegument protein (42). Indeed, the Bartha-derived strain Ba43/25aB4, in which the mutated UL21 locus as well as the large US deletion in the Bartha genome were repaired, shows a substantially increased incorporation of pUS3, VP11/12, and VP22 in its virions which was not observed when only the US region was repaired (42). Therefore, we checked whether the virion incorporation of IE180 was restored in virions of the Ba43/25aB4 strain. Western blot analysis revealed, as expected, equal amounts of the major capsid protein VP5 and the tegument protein VP16 in all virus preparations and an absence of gE in Bartha, but not in the Bartha partial-rescue strain Ba43/25aB4 in which the US deletion and UL21 mutation of Bartha are repaired (Fig. 5A). Nevertheless, we only noticed a marginal increase in IE180 protein levels in Ba43/25aB4 virions compared to Bartha virions (Fig. 5A). Interestingly, the incorporation of pUS3 in Ba43/25aB4 virions was increased compared to Bartha, as expected (42), yet remained substantially lower compared to WT strains. Hence, although repair of the US region and the UL21 locus in the Bartha genome partially restored packaging of pUS3 and, to a lesser extent, IE180, the current data indicate that additional mechanisms are involved in the packaging defect of Bartha.
FIG 5 Western blot analysis of purified virions. ST cells were infected (MOI of 10) with the indicated PRV strains, and virions were purified at 24 hpi (A) or at 12 or 24 hpi (B). Virion lysates were fractionated and analyzed by Western blotting. The membranes were incubated with antibodies directed against VP5, IE180, pUS3, VP16, and/or gE.
Comparison of WT PRV and Bartha virions was done at 24 hpi. Previous research showed that PRV virions are assembled and/or released earlier in cells infected with PRV Bartha compared to cells infected with WT PRV (60). Hence, to assess whether or not the reduced incorporation of pUS3 and IE180 in Bartha virions may be time dependent, we analyzed Bartha virions harvested at 12 hpi. Figure 5B shows that also at 12 hpi, Bartha virions contain almost no IE180 and substantially reduced amounts of pUS3, similar to the observations at 24 hpi. This indicates that also early during infection, the incorporation of tegument proteins is altered in Bartha-infected cells.
An alternative explanation for the largely pUL21-independent reduced incorporation of IE180 in Bartha virions could be an altered subcellular distribution of IE180 in Bartha-infected cells versus WT PRV-infected cells, leading to reduced virion incorporation. Indeed, for HSV, it was shown that ICP4 (the IE180 ortholog in HSV) needs to interact with ICP27 (pUL54) to induce its cytoplasmic translocation and subsequent virion incorporation (61). However, fractionation assays revealed similar amounts of IE180 in the cytoplasm and nucleus of Bartha- versus Kaplan-infected cells at different time points post inoculation (Fig. 6). As a control for successful fractionation, the nuclear marker histone H3 was nearly undetectable in the cytoplasmic fraction, whereas no tubulin could be detected in the nuclear fractions (Fig. 6). In line with this, immunofluorescence assays did not show obvious differences in subcellular IE180 localization in Bartha- versus Kaplan-infected ST cells (data not shown).
FIG 6 Cell fractionation assays demonstrate similar subcellular distribution of IE180 in Bartha- versus Kaplan-infected cells. ST cells were infected with Bartha or Kaplan PRV (MOI of 10) and lysed at the indicated time points. Nuclear and cytoplasmic fractions were separated and analyzed by Western blotting with antibodies directed against IE180, histone H3, and tubulin.

Bartha virions show morphological differences compared to WT PRV virions.

It is remarkable that several abundant tegument proteins, often proteins that make part of the outer tegument layer, display a substantially reduced incorporation in Bartha virions, seemingly without compensation by other viral proteins (Fig. 3). We therefore wondered whether this difference would correlate with a reduced size of Bartha virions compared to that of WT virions. Figure 7A shows that the average particle area of Bartha virions is indeed significantly smaller compared to that of WT virions (respectively, 16,507 ± 1,521 nm2 and 17,141 ± 1,634 nm2). Moreover, we also observed that the average circularity of Bartha virions is slightly but significantly decreased (Fig. 7B) (respectively, 0.935 ± 0.016 and 0.944 ± 0.021). A possible explanation for these observations could be that the reduced abundance of tegument proteins results in an overall compacted virion. In line with this, Bartha virions often display an icosahedral shape that more closely matches the icosahedral shape of the capsid, compared to the rounder, more flexible shape of WT virions (Fig. 7C). We hypothesize that, as a consequence of the reduced incorporation of tegument proteins, the rigid icosahedral shape of the capsid has a greater influence on the overall shape of the membrane envelope of Bartha virions. This appears to be in line with the observation that the inner tegument layer in HSV-1 exhibits an icosahedral structure (62).
FIG 7 Bartha virions are morphologically different from WT PRV virions. Bartha or Kaplan virions were analyzed by TEM. The area (A) and circularity (B) of Kaplan and Bartha virions were calculated. (C) Representative pictures are shown for virions of both strains. Scale bar, 50 nm.

Analysis of host proteins in virion preparations.

A total of 128 host proteins with at least two unique peptides associated with the PRV virion preparations were identified in the DDA analysis (Table 4). Unfortunately, the current experimental setup does not allow a clear distinction between proteins that are truly packaged inside the virions or proteins that are associated with the virions. In other studies, proteinase K treatment was used to remove proteins that were not incorporated into PRV virions (40, 41). Upon comparison with these reports assessing packaged host proteins, we identified a total of 28 host proteins in our PRV virion preparations that were discovered in at least one of these studies (Table 4). We next investigated how the DIA approach (SWATH) could increase the detection sensitivity of the host proteins. Similar as for the viral proteins, predicted libraries were used to search for peptides using the neural network-based DIA-NN search engine, and peptides were considered detected upon a minimum posterior error probability value below 0.01 (57). By these standards, we identified 678 host proteins with at least two unique peptides in at least one of the virus strain preparations. (see Data Set S1).
TABLE 4 Host proteins detected by DDA in the virion preparations of the present study
Protein classificationIdentifieraProtein nameIdentified as a virion componentb by:Difference between strainsc
Kramer et al. (40)Yu et al. (41)
Small GTPasesARL1Arl1  Yes
RB11ARab11a  Yes
RRASR-Ras  Yes
MetabolismACSL4Acyl-CoA synthetase long-chain family member 4+  
ENOAAlpha-enolase  Yes
AMPNAminopeptidase N   
CBPDCarboxypeptidase D  Yes
SERAd-3-Phosphoglycerate dehydrogenase  Yes
FASFatty Acid Synthase   
G3PTGlyceraldehyde-3-phosphate dehydrogenase  Yes
ENPP6Glycerophosphocholine cholinephosphodiesterase   
LDHAl-Lactate dehydrogenase A  Yes
LDHBl-Lactate dehydrogenase B chain  Yes
NDKBNucleoside diphosphate kinase B  Yes
PRDX6Peroxiredoxin-6  Yes
PGK1Phosphoglycerate kinase 1  Yes
PARP6Poly[ADP-ribose] polymerase 6   
PSAPuromycin-sensitive aminopeptidase   
AL1A1Retinal dehydrogenase 1  Yes
TRY1Serine protease 1  Yes
TERATransitional endoplasmic reticulum ATPase   
TPISTriosephosphate isomerase  Yes
ChaperonesF10A1Hsc70-interacting protein  Yes
HS90BHeat shock protein HSP 90-beta  Yes
HS90AHeat shock protein HSP 90-alpha  Yes
HSPB1Heat shock protein beta-1+ Yes
HSP7CHeat shock cognate 71 kDa protein  Yes
HS71BHeat shock 70 kDa protein 1B  Yes
BIPEndoplasmic reticulum chaperone BiP   
Membrane trafficking and organizationANXA1Annexin A1 + 
ANX13Annexin A13   
ANXA2Annexin A2   
ANXA8Annexin A8+  
AP1B1AP-1 complex subunit beta-1  Yes
AP1G1AP-1 complex subunit gamma-1   
CLH1Clathrin heavy chain 1  Yes
EHD2EH domain-containing protein 2   
GDIBRab GDP dissociation inhibitor beta   
CytoskeletonACTBActin, cytoplasmic 1   
PLAKJunction plakoglobin   
K1C10Keratin, type I cytoskeletal 10   
K1C17Keratin, type I cytoskeletal 17   
K1C9Keratin, type I cytoskeletal 9   
KRHB1Keratin, type II cuticular Hb1   
KRHB5Keratin, type II cuticular Hb5   
K2C1Keratin, type II cytoskeletal 1   
K22EKeratin, type II cytoskeletal 2   
K2C5keratin, type II cytoskeletal 5   
LASP1LIM and SH3 domain protein 1  Yes
SRBS2Sorbin and SH3 domain-containing protein 2  Yes
TBA1BTubulin alpha-1B  Yes
VIMEVimentin  Yes
RNA bindingDDX3XATP-dependent RNA helicase DDX3X++ 
ELAV1ELAV-like protein 1  Yes
EF1A1Elongation factor 1-alpha 1  Yes
EF2KEukaryotic elongation factor 2   
IF4A1Eukaryotic initiation factor 4A-I++ 
IF4HEukaryotic translation initiation factor 4H+ Yes
IF5A1Eukaryotic translation initiation factor 5A-1++ 
HNRPKHeterogeneous nuclear ribonucleoprotein K+  
ROA2Heterogeneous nuclear ribonucleoproteins A2/B1++Yes
PCBP1Poly(rC)-binding protein 1+ Yes
SMD3Small nuclear ribonucleoprotein Sm D3  Yes
SND1Staphylococcal nuclease domain-containing protein 1  Yes
YTHD3YTH N6-methyladenosine RNA binding protein 3   
Signaling1433B14-3-3 protein beta/alpha++Yes
1433E14-3-3 protein epsilon++Yes
1433T14-3-3 protein theta++Yes
1433Z14-3-3 protein zeta + 
CSK22Casein kinase II subunit alpha’   
CSK23Casein kinase II subunit alpha 3+ Yes
ES8L2Epidermal growth factor receptor kinase substrate 8-like protein 2   
GRB2Growth factor receptor-bound protein 2+ Yes
GNAI2Guanine nucleotide-binding protein G(i) subunit alpha-2++ 
MK03Mitogen-activated protein kinase 3  Yes
PDLI5PDZ and LIM domain protein 5   
PDLI7PDZ and LIM domain protein 7   
IASPPRelA-associated inhibitor   
MARK2Serine/threonine-protein kinase MARK2  Yes
PP1ASerine/threonine-protein phosphatase PP1-alpha+ Yes
Cell adhesionCRKAdapter molecule crk  Yes
ITA2Integrin alpha-2 precursor   
ITA3integrin alpha-3  Yes
ITB1Integrin beta-1+ Yes
MAGI1Membrane-associated guanylate kinase, WW and PDZ domain-containing protein 1  Yes
VINEXVinexin  Yes
Carbohydrate bindingLEG1Galectin-1  Yes
Ion transferCLIC1Chloride intracellular channel protein 1+  
AT1A1Sodium/potassium-transporting ATPase subunit alpha-1  Yes
HistonesH13Histone H1.3-like protein   
H2AZHistone H2A type 2-A-like   
H2B3BHistone H2B type 3-B   
H4Histone H4  Yes
ANXA6Annexin A6   
HBAHemoglobin A   
HBBHemoglobin B   
IGF1RInsulin-like growth factor 1 receptor  Yes
ISG15ISG15 ubiquitin-like modifier   
LYSCLysozyme C   
PPIAPeptidyl-prolyl cis-trans isomerase A   
FKB1APeptidyl-prolyl cis-trans isomerase FKBP1A   
PDIA1Protein disulfide-isomerase  Yes
RS27AUbiquitin-40S ribosomal protein S27a  Yes
Protein identifiers are represented as their human homologue UniProt entry names (e.g., ARL1_HUMAN).
Host proteins detected in virion preparations in the present study identified in proteinase K-treated virions by Kramer et al. (40) or Yu et al. (41).
Host proteins that showed a statistically significant difference between the studied viral strains.
As a subsequent step, we wanted to explore whether the acquisition of host proteins in viral particles exhibits a certain strain specificity. Therefore, it was investigated which host proteins showed a statistically significant difference between strains (indicated in Table 4). The 54 host proteins that did show significant differences were then compared to the VP5-normalized abundance in Kaplan (Fig. 8). However, given that most of these host peptide measurements reside in virion preparations near the limit of detection of the DDA acquisition, interpretation of these relative abundances should be done with caution. Due to these low abundances, quantification and comparison of several host proteins were more susceptible to batch effects compared to the analyses of viral proteins, resulting in several large error bars in Fig. 8 and making it difficult to draw robust conclusions. Even so, it appears that, on average, fewer host proteins were detected in Becker virion preparations. Furthermore, specific host proteins appeared to show a strain-specific abundance. In particular, virion preparations of the Bartha strain display reduced abundance of the mitogen activated kinase 3 (MK03 or ERK1), the protein phosphatase 1 catalytic subunit α (PP1A), the adapter molecule crk (CRK), and the casein kinase II subunit α (CSK23), whereas an increased abundance of the carboxypeptidase D (CBPD) and the growth factor receptor-bound protein 2 (GRB2) was observed (Fig. 8). However, additional research is required to further clarify the suspected differences and elucidate whether and how these may affect virus biology.
FIG 8 Normalized abundances of host proteins that display significant differences in abundance in virion preparations between viral strains, set relative to the respective normalized abundance of the Kaplan strain. Error bars represent propagated standard deviations determined on the replicate values (n = 6 for NIA3, Kaplan, and Bartha; n = 5 for Becker). Protein annotations are represented as their human homologue UniProt entry names (e.g., PP1A_HUMAN).


In the current manuscript, the virion proteomes of four PRV strains commonly used in research—Becker, Kaplan, NIA3, and Bartha—were analyzed using LC-MS-based proteomics. The former three strains represent established WT strains (2729), whereas Bartha represents the most widely used attenuated vaccine strain (30). As a first approach, viral proteins were identified by DDA analysis. As such, a total of 43 viral proteins were detected, of which 37 represented established structural proteins and 6 were previously presumed nonstructural viral proteins. In addition, three established structural and eight presumed nonstructural viral proteins were detected by a more sensitive DIA analysis. Our study also confirmed previous findings that Bartha virions incorporate less pUS3, VP11/12, and VP22 (42, 56). Although the relative abundance of capsid proteins and most envelope proteins show very little variation in-between PRV strains, this is not the case for several tegument proteins, often those that make part of the outer tegument layer. Strikingly, we noted that the Bartha strain incorporates almost no detectable amounts of the tegument proteins IE180 and VP11/12 and drastically reduced amounts of the tegument proteins pUS3, pUL41, VP22, pUL40, and pUS1 and the envelope protein pUL56. Although the mutated UL21 locus in the Bartha genome has been identified as an important regulator for the reduced packaging of VP11/12, VP22, and pUS3, our current data suggest the existence of at least one additional, pUL21-independent mechanism that contributes to this phenotype. Curiously, transmission electron microscopy (TEM) analysis indicates that this reduced packaging may affect the size and morphology of Bartha virions, which we observed to be significantly smaller and more icosahedral in shape compared to WT virions. Furthermore, we detected 28 host proteins in virion preparations that were also reported as packaged in virions by the earlier proteomic characterization of PRV virions (40, 41). Lastly, even though many host proteins were detected in similar quantities in-between strains, we also observed some strain-specific differences which should be further explored in follow-up research to assess whether and how these may affect viral replication fitness.
The strong correspondence of our observations with those of two other independent studies (40, 41), in particular the Kramer et al. paper (40), is remarkable. Indeed, only a few structural viral proteins were not mutually found. Differences in comparison to the study by Kramer et al. (40) were gG, pUL13 and possibly pUL56 (the former one absent in our study, the latter two absent in the other study). Nonetheless, we did detect one unique peptide of gG in our analyses and pUL13 was detected in virions by Western blotting in the other study (40). It is therefore reasonable to assume that these proteins reside in virions at the limit of detection. Of note, the PRV gene UL56 (ORF1), encoding the pUL56 protein, has an upstream in-frame start codon, creating the additional gene ORF1.2 (63). Even though Kramer et al. did not identify pUL56, they did detect pORF1.2 (40), whereas the two peptides discovered in the present study could be annotated to both proteins, preventing unambiguous identification of either or both proteins. With regard to the Bartha virions analyzed by Yu et al. (41), it is noteworthy that the authors could not detect IE180 in their Bartha virion preparations, in line with our current comparative proteome analysis. For the presumed nonstructural proteins that were detected in virion preparations, we discovered seven more in addition to those that were already identified in at least one of these two previous studies (40, 41). It has to be noted, however, that the novel presumed nonstructural proteins in virion preparations were only identified upon inclusion of recent advances in MS analysis by using predicted fragment intensities and retention times, allowing us to increase the depth of the detectable proteome (47).
Of all tegument proteins, the composition of the outer, amorphous tegument exhibited the largest degree of interstrain variability in general, whereas the tegument proteins in close interaction with the capsid showed very little variation, with the exception of pUS3 (6, 64). Remarkably, the tegument proteins that displayed the least variation in virion incorporation (pUL7, pUL11, pUL16, pUL21, VP1/2, pUL37, pUL51, VP13/14, and VP16) were all described to participate in virion morphogenesis (6, 913, 6567), whereas highly variable tegument proteins often do not appear to be involved in this process (pUS2, VP11/12, and VP22) (42, 6870).
The reduced incorporation of VP22, pUS3, and VP11/12 in Bartha virions was previously shown to be caused by mutations in the UL21 gene in the Bartha genome (42). Interestingly, recent work showed that pUS3 in PRV virions is required for rapid retrograde transport in neurons (71), potentially explaining how repair of the Bartha UL21 locus increases retrograde transport of Bartha virions (72). However, although our data also indicate a role for pUL21 in packaging of tegument proteins in PRV virions, we found that UL21 mutations do not entirely explain the reduced tegument protein incorporation in Bartha, since incorporation of IE180 and even pUS3 was only modestly increased by rescue of the Bartha UL21 locus. We therefore argue that at least one other mechanism is involved in this process. In addition, we also observed less pUS1 and pUL40 in Bartha virions. These proteins were presumed to be nonstructural (3, 73), making their exact location in the virion uncertain. However, they may reside in the tegument layer, as they lack a transmembrane domain and were also detected in PRV virions even after proteinase K treatment (40). Noteworthy, pUL40 (RR2) interacts with pUL39 (RR1), creating the ribonucleotide reductase (RNR) complex, which participates in nucleotide metabolism (74, 75). Nevertheless, incorporation of pUL40 in virions may occur independently of the functional RNR, since pUL39 is one of the few (or only) tegument proteins that appears to be incorporated to higher levels in Bartha virions (Fig. 3 and 4). It is somewhat curious that the loss of the observed tegument proteins in Bartha does not appear to be compensated by other viral proteins such as VP16, since this was described in PRV virions with VP22 deleted (50). Moreover, host actin has also been noted to act as a cellular ‘stuffer’ in PRV virions in the absence of VP22 or pUS3 (16, 50), yet no altered abundance of actin was observed in virion preparations of Bartha compared to those of other viruses (Table 4). Interestingly, it has been described before that PRV envelope proteins pUS9 and possibly gE/gI, which are absent in Bartha, trigger reorganization of cholesterol-rich host membrane lipid rafts and affect incorporation of viral and cellular proteins in these membrane microdomains (7678). This has been reported to contribute to efficient recruitment of microtubule motors and adaptors to virions, thereby affecting anterograde axonal transport of virus (7678). Although it is unclear whether lipid rafts represent sites of PRV assembly, it has been shown that at least some PRV glycoproteins associate with lipid rafts (77, 79, 80). Hence, although speculative, it is possible that the lack of pUS9 and/or gE/gI and their associated effect on lipid rafts may contribute to the reduced incorporation of particular proteins in Bartha virions.
We recently showed that the extracellular infectious virus titers of Bartha virions increase more rapidly early in infection of epithelial cells compared to those of Kaplan or Becker WT PRV (60). Despite this increased production of extracellular virus particles in Bartha-infected ST cells, intracellular production of structural viral proteins was similar in Bartha- versus WT PRV-infected cells, which led to the hypothesis of a more efficient assembly of Bartha virions (60). The current observation that fewer (tegument) proteins are incorporated in Bartha virions appears to be in line with this hypothesis. Indeed, it is tempting to speculate that, by producing more minimalistic virions that lack or show reduced incorporation of several (tegument) proteins, assembly of infectious virions may be more efficient for the Bartha strain compared to that of WT strains. This line of thoughts generates the question as to what the evolutionary benefit could be for the more complex composition of WT virions. Interestingly, several of the viral proteins that show reduced incorporation in Bartha virions have been described to play a role in immune evasion of alphaherpesviruses, including PRV (81). Examples include the suppression of type I interferon (IFN) signaling and NK cell activity by pUS3, VP22-mediated inhibition of cGAS-DNA phase separation and inhibition of inflammasome activation, VP11/12-mediated evasion of the STING DNA-sensing pathway, reduced tumor necrosis factor alpha (TNF-α) production by pUL41, inhibition of MHC-I expression by pUL56 and inhibition of CD80 expression by pUS1 (8291). Although speculative, it is possible that the extensive cell culture passage procedure, which is at the basis of the generation of the attenuated Bartha strain, has led to an in vitro evolution of the virus toward a simplified, more minimalistic virion that has omitted immune evasion tools that may be critical for successful infection of the host, but possibly unnecessary ballast in cell culture. Intriguingly, such a process may have contributed to the efficacy of Bartha as a vaccine strain. Indeed, a reduced ability of a virus to evade the host immune response is likely to contribute to a reduced ability of the virus to spread in the host while at the same time eliciting a particularly robust immune response (9294). This idea is in line with the observation that Bartha PRV triggers a strongly increased type I IFN response by pDC compared to Becker and Kaplan PRV (95) and the finding by Laval and colleagues that Bartha-infected mice display an increased type I IFN response compared to WT Becker-infected mice (96). Hence, although speculative and open for further exploration, it is possible that the altered virion composition of Bartha virions contributes to its potency as a vaccine (97).
Despite the fact that Kramer et al. (porcine PK-15 cells) and Yu et al. (hamster BHK-21 cells) used different cell lines compared to the present study (porcine ST cells) to produce the virion preparations for proteomic analysis (40, 41), several types of host proteins that were detected in these samples were similar. In fact, several of the host protein groups detected in association with PRV virion preparations have also been found in other herpesviruses (98), increasing the likelihood that these host protein groups are of biological relevance during herpesvirus infection. Examples of recurring host proteins include Rab GTPases, actin, annexins, mRNA binding proteins, chaperones, and signaling molecules such as kinases and phosphatases (98). A broad siRNA-mediated screen revealed that at least 15 of the 49 discovered host proteins in HSV-1 virions are involved in viral morphogenesis and release (99). For PRV, the Rab GTPases Rab6a, Rab8a, and Rab11a have been confirmed to participate in virus exocytosis (100). We could confirm the presence of these proteins in our virion preparations since Rab6a and Rab11a were identified by DDA (Table 4), whereas all three Rab GTPases were discovered by DIA (see Data Set S1). Moreover, Yu et al. reported the involvement of IRSp53, CDC42, Fascin, and Rac1 in the production of extracellular virus (41). Whereas only Rac1 was identified by DDA (Table 4), we were able to confirm the presence of all these host proteins within our virion preparations using DIA (see Data Set S1). Recently, it was reported that nascent HSV-1 and PRV virions acquire kinesin-1 upon assembly in epithelial cells, which is then utilized for retrograde axonal transport in neurons (101). In the present study, we detected the kinesin-1 heavy chain KIF5B by DIA in all four of the virus strains (see Data Set S1), in line with these intriguing results.
Even though our present analysis did not favor strong conclusions regarding interstrain differences in host protein incorporation, some interesting observations were made. Becker virion preparations appeared to contain less host proteins, and several specific proteins also appeared to be drastically less abundant in Bartha virion preparations, including MK03 (mitogen-activated kinase 3 or ERK1), CSK23 (casein kinase II subunit α), and PP1A (protein phosphatase 1 α) (Fig. 8). Interestingly and in line with this, ERK1 interacts with pUS2, a viral tegument protein that is absent in Bartha virions (35), and was shown to be packaged into virions in a pUS2-dependent manner (102). Casein kinase II on the other hand phosphorylates HSV-1 VP22 (103), a tegument protein that is also strongly reduced in Bartha virions (Fig. 3). In addition, IE180, another tegument protein that is virtually absent in Bartha virions (Fig. 3), has been reported to activate the phosphatase PP1A to dephosphorylate the translation initiation factor eIF2α (104). This raises the hypothesis that host proteins such as ERK1, CSK23, and PP1A may be incorporated in PRV virions through interactions with viral tegument proteins and may therefore be less abundant in Bartha virions because of a reduced virion incorporation or complete lack of their viral counterpart (i.e., pUS2, VP22, and IE180).
In conclusion, differential proteome analysis of three common WT PRV strains and the widely used attenuated Bartha vaccine strain revealed a remarkable conservation of most viral (and host) proteins in virions of WT PRV strains. However, the attenuated PRV vaccine strain Bartha showed a reduced incorporation of several tegument proteins, which correlates with a reduced size and increased icosahedral morphology of Bartha virions. This leads us to speculate that cell culture adaptations of the Bartha strain may have resulted in a simplified version of the PRV virion that displays reduced levels of proteins that are noncritical for replication in cell culture but may be of particular relevance in host infection and immune evasion. These data will therefore facilitate identification of viral and cellular proteins that may be of specific importance during in vivo alphaherpesvirus infection of their host species, which in turn may help to rationally design vaccines against alphaherpesviruses.


Cells and viruses.

Epithelial swine testicle (ST) cells were cultured in Earle’s minimal essential medium (MEM) supplemented with 10% fetal calf serum (FCS), 1 mM sodium pyruvate, and antibiotics (100 U/mL penicillin, 0.1 mg/mL streptomycin, and 0.05 mg/mL gentamicin) (Life Technologies) (= ST medium). All viruses used in study this have been described before. The WT PRV Kaplan strain (27) and the Bartha rescue strain Ba43/25aB4 (US region + UL21) (105) were kindly provided by T. Mettenleiter (Friedrich-Loeffler Institute, Germany). The WT Becker strain (28) was kindly provided by L. Enquist (Princeton University, USA). The WT NIA3 strain (29) was kindly donated by the ID-DLO (The Netherlands). The Bartha vaccine strain (30) was kindly provided by H. Nauwynck (Ghent University, Ghent, Belgium). All viral stocks were titrated on monolayers of ST cells.


The antibody directed against the PRV gE (18E8 and mIgG1) was described before (106). Antibodies directed against VP5 (3C10) and pUS3 (8F86, mIgG1) (107) were kindly provided by L. Enquist (Princeton University, USA). Antibodies raised against PRV VP16 (polyclonal rabbit) (10) were kindly donated by W. Fuchs (Friedrich-Loeffler Institute, Germany), and those raised against IE180 (polyclonal rabbit) (108) were kindly provided by E. Tabares (Autónoma University of Madrid, Madrid, Spain). Antibodies against histone H3 were purchased from Proteintech (polyclonal rabbit, 17168-1-AP) and horseradish peroxidase (HRP)-labeled alpha-tubulin antibodies were obtained from Abcam (Ab40742). HRP-labeled goat anti-mouse and goat anti-rabbit (Dako, catalog numbers P0447 and P0448, respectively) were used as secondary antibodies in Western blot analyses.

Isolation of virions.

The isolation of PRV virions (H-particles) was performed as previously described with some minor adjustments (40). Briefly, two 175-cm2 flasks with confluent ST cells were infected with Becker, Kaplan, NIA3, or Bartha PRV at an MOI of 10 in MEM at 37°C for 2 h; the cells were then washed twice and overlaid with ST medium without FCS. The supernatant was collected at 24 hpi, unless mentioned otherwise, and cell debris was removed by centrifugation for 10 min at 1,000 × g and 0.45-μm-pore size filtration. In contrast to the earlier described protocol (40), no DNase treatment was performed. The virions were pelleted for 1 h at 20,000 × g using a Type-35 rotor (Beckman Coulter), resuspended in phosphate-buffered saline (PBS), briefly sonicated, carefully layered onto a 10% Ficoll 400 solution, and subsequently centrifuged for 2 h at 116,000 × g in a SW41-Ti rotor (Beckman Coulter). Pelleted PRV virions were resuspended in HEPES buffer (20 mM HEPES [pH 7.4]) and pelleted for 1 h at 20,000 × g in the same rotor. Pelleted virions were resuspended in HEPES buffer, aliquoted, and stored at −80°C until further use.

SDS-PAGE and Western blotting.

ST cells were inoculated with the indicated PRV strains at an MOI 10 in MEM at 37°C for 2 h; the inoculum was then removed, and the cells were washed twice and overlaid with prewarmed ST medium. At the indicated time points, the cells were scraped into the medium and pelleted by centrifugation for 10 min at 700 × g (4°C). The cell pellet was washed with ice cold PBS and lysed for 1 h at 4°C in TNE lysis buffer (50 mM Tris, 150 mM NaCl, 1 mM EDTA, 10% NP-40 [Roche]) and protease inhibitor cocktail (Sigma-Aldrich). Nuclei were pelleted by spinning 10 min at 10,000 × g, and the supernatant was collected as the cytoplasmic lysate. If appropriate, the pelleted nuclei were washed twice in PBS and lysed with radioimmunoprecipitation assay buffer supplemented with protease inhibitor cocktail (Abcam ab156034) for 1 h at 4°C. Purified PRV virions were lysed and heated for 5 min at 95°C in SDS-PAGE loading buffer without β-mercaptoethanol or bromophenol blue. Protein concentrations of the cell and viral lysates were determined using a Pierce BCA kit according to the manufacturer’s instructions (Thermo Fisher), and the cell lysates were then mixed with SDS-PAGE loading buffer and heated for 5 min at 95°C. β-Mercaptoethanol and bromophenol blue were added to the viral lysates used for Coomassie staining and Western blotting. A total of 20 μg (cell lysates) or 5 μg (virion lysates) of protein was loaded and run on a polyacrylamide gel (10%) via SDS-PAGE and either blotted on a Hybond-P polyvinylidene difluoride membrane (GE Healthcare) or directly visualized using Coomassie brilliant blue staining (Thermo Fisher). For SDS-PAGE gels, samples were further processed for MS analysis as described below. The membranes for Western blotting were blocked in blocking buffer (PBS with 5% milk powder [Nestlé] and 0.1% Tween 20 [Sigma-Aldrich]) for 1 h at room temperature. Next, blots were incubated with primary antibodies at 4°C overnight, washed, and subsequently incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. Primary and secondary antibodies were diluted in blocking buffer. Finally, the blots were developed using chemiluminescence.

In-gel protein digestion.

Virions of each strain were purified as described above in two independent runs, and three replicates of each run were included, creating a total of six replicates that were divided over six different SDS-PAGE gels. For each gel, the entire lane containing the fractionated proteins was cut into small pieces of about 1 × 1 mm and transferred to protein low-bind tubes (Eppendorf). Gel pieces were washed three times for 10 min with 25 mM triethylammonium bicarbonate buffer (TEABC; Sigma-Aldrich) in 50% acetonitrile (ACN; Sigma-Aldrich) under mild agitation. The proteins were then reduced in 10 mM dithiothreitol (Sigma-Aldrich) in 25 mM TEABC for 1 h at 56°C. Next, the proteins were alkylated by incubation in 200 mM methyl methanethiosulfonate in isopropanol (both from Sigma-Aldrich) for 1 h at room temperature, and then the samples were washed in 25 mM TEABC in 50% ACN for 10 min under mild agitation. The samples were next dehydrated by a twice-repeated suspension in 100% ACN until the gel pieces turned opaque and then dried. Next, the gels were resuspended in a trypsin/Lys-C-solution containing 10 ng/μL trypsin/Lys-C mix (Promega; V507A) in 1 mM CaCl2–5% ACN in 50 mM TEABC, followed by incubation overnight at 37°C. The peptides were sequentially extracted from the gel with 50 and 100% ACN in 25 mM TEABC for 30 min, and the samples were vacuum dried and prepared for mass spectrometry.

Mass spectrometry.

For LC-MS analysis, the previously described SCIEX Triple-TOF 6600+ coupled with capillary flow LC setup was used with minor adaptions (109). In brief, the digested samples were resuspended in 20 μL of 0.1% formic acid (FA), of which 8 μL was loaded onto the LC system. The samples were trapped on a YMC capillary guard column (5 × 0.5 mm, 3-μm particle size) for 5 min with a mobile phase A at a 10-μL/min flow rate prior to LC separation using a gradient elution from 3 to 40% mobile phase B over 30 min. Mobile phase A consisted of 0.1% FA in water, and mobile phase B consisted of 0.1% FA in ACN. For DDA acquisition, MS1 spectra were acquired between 400 and 1,200 m/z for 250 ms, followed by selection and fragmentation of the 30 most intense precursor ions to acquire MS2 fragment spectra between 100 and 1,500 m/z for 50 ms. Selected precursor ions were dynamically excluded for 10 s. For SWATH acquisition (DIA), a 99-isolation window acquisition scheme was used to acquire MS2 spectra from 100 to 1,500 m/z for 37.5 ms. Before each 4-s cycle, an MS1 survey scan was acquired for 250 ms. The samples were randomized prior to LC-MS analysis, and a mixture of all samples was analyzed every six samples to monitor LC-MS performance over time. In addition, 40 fmol of PepCalMix (Sciex, catalog no. 5045751) was analyzed every five samples to calibrate the instrument in between analyses.

LC-MS data analysis.

The obtained DDA raw data were imported into the Progenesis QIP software. Next, the features—clusters of signal with a clear isotope distribution—were aligned along the retention time and m/z dimension, and the obtained peak list was exported as an MGF file after peak picking. This file was used to identify the features using the Mascot Daemon database search engine. The databases used were the pig reference proteome (UniProt ID UP000008227 at 23FEB2021) and the FASTA protein sequence proteomes of Becker (JF797219.1 at 10JUN2020), Kaplan (JF797218.1 at 10JUN2020), Bartha (JF797217.1 at 10JUN2020), and NIA3 (KU900059.1 at 10JUN2020). In addition, the common repository of adventitious proteins was also added to control for lab contaminants. One outlier sample of the Becker strain was omitted.
Several Progenesis QIP projects were created during data analysis, each serving a separate research question. First, to obtain only those peptides that were identical in all PRV strains, we performed a sequential search with all strains separately, while each time exporting and searching an MGF file from Progenesis QIP that only contained identified features. A second project was created to include the four proteins, pUS2, gI (pUS7), gE (pUS8), and pUS9, which are (partially) deleted in the Bartha genome. To this end, the Bartha search was omitted from the sequential searches described above.
For all searches, at least two unique peptide identifications were required to confidently identify the protein.

TEM analysis.

Samples were prepared for TEM as described previously (110). Virion area and circularity were calculated using ImageJ. Circularity was defined as follows:
Statistical analysis was performed using a two-tailed Student t test.


J.L.D. is supported by a Ph.D. grant from Research Foundation Flanders (FWO-Vlaanderen; This research was supported by grants to H.W.F. from FWO-Vlaanderen (grants G017615 and G.019617N []) and the Special Research Fund of Ghent University (GOA grant 01G01317 and grant BOFBAS2018000301 []).
We thank ProGenTomics for the mass spectrometry analysis and insightful discussions (Ghent University, Belgium). We also thank Lynn Enquist (Princeton University, USA) for donating the Becker strain and the pUS3 and VP5 antibodies, Thomas Mettenleiter (Friedrich-Loeffler-Institut, Germany) for donating the Kaplan strain, Hans Nauwynck (Ghent University, Belgium) for donating the Bartha strain, the ID-DLO for donating the NIA3 strain, Walter Fuchs (Friedrich-Loeffler-Institut, Germany) for donating the pUL48 antiserum and Enrique Tabares for donating the IE180 antiserum. Furthermore, we thank Liesbeth Couck and Wim Van Den Broeck (Ghent University) for the assistance in the TEM assays.

Supplemental Material

File (jvi.01158-22-s0001.xlsx)
ASM does not own the copyrights to Supplemental Material that may be linked to, or accessed through, an article. The authors have granted ASM a non-exclusive, world-wide license to publish the Supplemental Material files. Please contact the corresponding author directly for reuse.


Pellett PM, Roizman B. 2001. The family Herpesviridae: a brief introduction. In Fields virology, 5th ed. Wolters Kluwer Health, New York, NY.
Nauwynck H, Glorieux S, Favoreel H, Pensaert M. 2007. Cell biological and molecular characteristics of pseudorabies virus infections in cell cultures and in pigs with emphasis on the respiratory tract. Vet Res 38:229–241.
Pomeranz LE, Reynolds AE, Christoph J, Hengartner CJ. 2005. Molecular biology of pseudorabies virus: impact on neurovirology and veterinary medicine. Microbiol Mol Biol Rev 69:462–500.
Homa FL, Huffman JB, Toropova K, Lopez HR, Makhov AM, Conway JF. 2013. Structure of the pseudorabies virus capsid: comparison with herpes simplex virus type 1 and differential binding of essential minor proteins. J Mol Biol 425:3415–3428.
Heming D, Conway F, Homa L. 2017. Herpesvirus capsid assembly and DNA packaging, p 119–142. In Osterrieder K (ed), Cell biology of herpes viruses. Springer International Publishing, New York, NY.
Owen DJ, Crump CM, Graham SC. 2015. Tegument assembly and secondary envelopment of alphaherpesviruses. Viruses 7:5084–5114.
Coller KE, Lee JI, Ueda A, Smith GA. 2007. The capsid and tegument of the alphaherpesviruses are linked by an interaction between the UL25 and VP1/2 proteins. J Virol 81:11790–11797.
Scholtes LD, Yang K, Li LX, Baines JD. 2010. The capsid protein encoded by UL17 of herpes simplex virus 1 interacts with tegument protein VP13/14. J Virol 84:7642–7650.
Klupp BG, Granzow H, Klopfleisch R, Fuchs W, Kopp M, Lenk M, Mettenleiter TC. 2005. Functional analysis of the pseudorabies virus UL51 protein. J Virol 79:3831–3840.
Fuchs W, Granzow H, Klupp BG, Kopp M, Mettenleiter TC. 2002. The UL48 tegument protein of pseudorabies virus is critical for intracytoplasmic assembly of infectious virions. J Virol 76:6729–6742.
Kopp M, Klupp BG, Granzow H, Fuchs W, Mettenleiter TC. 2002. Identification and characterization of the pseudorabies virus tegument proteins UL46 and UL47: role for UL47 in virion morphogenesis in the cytoplasm. J Virol 76:8820–8833.
Klupp BG, Bo S, Granzow H, Kopp M, Mettenleiter TC. 2005. Complex formation between the UL16 and UL21 tegument proteins of pseudorabies virus. J Virol 79:1510–1522.
Kopp M, Granzow H, Fuchs W, Klupp BG, Mundt E, Karger A, Mettenleiter TC. 2003. The pseudorabies virus UL11 protein is a virion component involved in secondary envelopment in the cytoplasm. J Virol 77:5339–5351.
Fuchs W, Granzow H, Klopfleisch R, Klupp BG, Rosenkranz D, Mettenleiter TC. 2005. The UL7 gene of pseudorabies virus encodes a nonessential structural protein which is involved in virion formation and egress. J Virol 79:11291–11299.
Smiley J, Elgadi M, Saffran H. 2001. Herpes simplex virus Vhs protein. Methods Enzymol 342:440–451.
del Rio T, DeCoste CJ, Enquist LW. 2005. Actin is a component of the compensation mechanism in pseudorabies virus virions lacking the major tegument protein VP22. J Virol 79:8614–8619.
Robbins AK, Dorney DJ, Wathen MW, Whealy ME, Gold C, Watson RJ, Holland LE, Weed SD, Levine M, Glorioso JC. 1987. The pseudorabies virus gII gene is closely related to the gB glycoprotein gene of herpes simplex virus. J Virol 61:2691–2701.
Schreurs C, Mettenleiter TC, Zuckermann F, Sugg N, Ben-Porat T. 1988. Glycoprotein gIII of pseudorabies virus is multifunctional. J Virol 62:2251–2257.
Petrovskis EA, Timmins JG, Armentrout MA, Marchioli CC, Yancey RJ, Post LE. 1986. DNA sequence of the gene for pseudorabies virus gp50, a glycoprotein without N-linked glycosylation. J Virol 59:216–223.
Fuchs W, Klupp BG, Granzow H, Hengartner C, Brack A, Mundt A, Enquist LW, Mettenleiter TC. 2002. Physical interaction between envelope glycoproteins E and M of pseudorabies virus and the major tegument protein UL49. J Virol 76:8208–8217.
Klupp BG, Baumeister J, Dietz P, Granzow H, Mettenleiter TC. 1998. Pseudorabies virus glycoprotein gK is a virion structural component involved in virus release but is not required for entry. J Virol 72:1949–1958.
Fuchs W, Klupp BG, Granzow H, Mettenleiter TC. 1997. The UL20 gene product of pseudorabies virus functions in virus egress. J Virol 71:5639–5646.
Babic N, Klupp BG, Makoschey B, Karger IA, Flamand A, Mettenleiter TC. 1996. Glycoprotein gH of pseudorabies virus is essential for penetration and propagation in cell culture and in the nervous system of mice. J Gen Virol 77:2277–2285.
Zuckermann FA, Mettenleiter TC, Schreurs C, Sugg N, Ben-Porat T. 1988. Complex between glycoproteins gI and gp63 of pseudorabies virus: its effect on virus replication. J Virol 62:4622–4626.
Mettenleiter TC. 2002. Herpesvirus assembly and egress. J Virol 76:1537–1547.
Granzow H, Klupp BG, Fuchs W, Veits J, Osterrieder N, Mettenleiter TC. 2001. Egress of alphaherpesviruses: comparative ultrastructural study. J Virol 75:3675–3684.
Kaplan A, Vatter A. 1959. A comparison of herpes simplex and pseudorabies viruses. Virology 7:394–407.
Platt K, Maré C, Hinz P. 1979. Differentiation of vaccine strains and field isolates of pseudorabies (Aujeszky’s disease) virus: thermal sensitivity and rabbit virulence markers. Arch Virol 60:13–23.
Mcferran JB, Dow C, McCracken R. 1979. Experimental studies in weaned pigs with three vaccines against Aujeszky’s disease. Comp Immunol Microbiol Infect Dis 2:327–334.
Bartha A. 1961. Experiments to reduce the virulence of Aujeskzy’s virus. Magy Allatorvosok Lapja 16:42–45.
Freuling CM, Müller TF, Mettenleiter TC. 2017. Vaccines against pseudorabies virus (PrV). Vet Microbiol 206:3–9.
Qiu HJ, Tian ZJ, Tong GZ, Zhou YJ, Ni JQ, Luo YZ, Cai XH. 2005. Protective immunity induced by a recombinant pseudorabies virus expressing the GP5 of porcine reproductive and respiratory syndrome virus in piglets. Vet Immunol Immunopathol 106:309–319.
Card JP, Enquist LW. 2014. Transneuronal circuit analysis with pseudorabies viruses. Curr Protoc Neurosci 68:1–39.
Szpara ML, Tafuri YR, Parsons L, Shamim SR, Verstrepen KJ, Legendre M, Enquist LW. 2011. A wide extent of inter-strain diversity in virulent and vaccine strains of alphaherpesviruses. PLoS Pathog 7:e1002282-23.
Lomniczi B, Blankenship LEE, Ben-Porat T. 1984. Deletions in the genomes of pseudorabies virus vaccine strains and existence of four isomers of the genomes. J Virol 49:970–979.
Robbins AK, Ryan JP, Whealy ME, Enquist LW. 1989. The gene encoding the glll envelope protein of pseudorabies virus vaccine strain Bartha contains a mutation affecting protein localization. J Virol 63:250–258.
Schröter C, Klupp BG, Fuchs W, Gerhard M, Backovic M, Rey FA, Mettenleiter TC. 2014. The highly conserved proline at position 438 in pseudorabies virus gH is important for regulation of membrane fusion. J Virol 88:13064–13072.
Dijkstra JM, Brack A, Jöns A, Klupp BG, Mettenleiter TC. 1998. Different point mutations within the conserved N-glycosylation motif of pseudorabies virus glycoprotein M result in expression of a nonglycosylated form of the protein. J Gen Virol 79:851–854.
Klupp BG, Lomniczi B, Visser N, Fuchs W, Mettenleiter TC. 1995. Mutations affecting the UL21 gene contribute to avirulence of pseudorabies virus vaccine strain Bartha. Virology 212:466–473.
Kramer T, Greco TM, Enquist LW, Cristea IM. 2011. Proteomic characterization of pseudorabies virus extracellular virions. J Virol 85:6427–6441.
Yu FL, Miao H, Xia J, Jia F, Wang H, Xu F, Guo L. 2019. Proteomics analysis identifies IRSp53 and fascin as critical for PRV egress and direct cell-cell transmission. Proteomics 19:1900009–1900014.
Michael K, Klupp BG, Karger A, Mettenleiter TC. 2007. Efficient incorporation of tegument proteins pUL46, pUL49, and pUS3 into pseudorabies virus particles depends on the presence of pUL21. J Virol 81:1048–1051.
Walther TC, Mann M. 2010. Mass spectrometry-based proteomics in cell biology. J Cell Biol 190:491–500.
Aebersold R, Mann M. 2003. Mass spectrometry-based proteomics. Nature 422:198–207.
Nesvizhskii A. 2007. Protein identification by tandem mass spectrometry and sequence database searching. Methods Mol Biol 367:87–119.
Ludwig C, Gillet L, Rosenberger G, Amon S, Collins BC, Aebersold R. 2018. Data-independent acquisition-based SWATH-MS for quantitative proteomics: a tutorial. Mol Syst Biol 14:1–23.
Van Puyvelde B, Willems S, Gabriels R, Daled S, De Clerck L, Vande Casteele S, Staes A, Impens F, Deforce D, Martens L, Degroeve S, Dhaenens M. 2020. Removing the hidden data dependency of DIA with predicted spectral libraries. Proteomics 20(3-4):e1900306.
Gillet LC, Navarro P, Tate S, Ro H, Selevsek N, Reiter L, Bonner R, Aebersold R. 2012. Targeted data extraction of the MS/MS spectra generated by data-independent acquisition: a new concept for consistent and accurate proteome analysis. Technol Innov Resour 11:1–17.
Loret S, Guay G, Lippe R. 2008. Comprehensive characterization of extracellular herpes simplex virus type 1 virions. J Virol 82:8605–8618.
Michael K, Klupp BG, Mettenleiter TC, Karger A. 2006. Composition of pseudorabies virus particles lacking tegument protein US3, UL47, or UL49 or envelope glycoprotein E. J Virol 80:1332–1339.
Demichev V, Messner CB, Vernardis SI, Lilley KS, Ralser M. 2020. DIA-NN: neural networks and interference correction enable deep proteome coverage in high throughput. Nat Methods 17:41–44.
Bigalke JM, Heldwein EE. 2015. Structural basis of membrane budding by the nuclear egress complex of herpesviruses. EMBO J 34:2921–2936.
Russell T, Bleasdale B, Hollinshead M, Elliott G. 2018. Qualitative differences in capsidless L-particles released as a by-product of bovine herpesvirus 1 and herpes simplex virus 1 infections. J Virol 92:1–22.
Birzer A, Kraner ME, Heilingloh CS, Mühl-Zürbes P, Hofmann J, Steinkasserer A, Popella L. 2020. Mass spectrometric characterization of HSV-1 L-particles from human dendritic cells and BHK21 cells and analysis of their functional role. Front Microbiol 11:1997.
Trus BL, Cheng N, Newcomb WW, Homa FL, Brown JC, Steven AC. 2004. Structure and polymorphism of the UL6 portal protein of herpes simplex virus type 1. J Virol 78:12668–12671.
Lyman MG, Demmin GL, Banfield BW. 2003. The attenuated pseudorabies virus strain Bartha fails to package the tegument proteins Us3 and VP22. J Virol 77:1403–1414.
Dijkstra JM, Mettenleiter TC, Klupp BG. 1997. Intracellular processing of pseudorabies virus glycoprotein M (gM): gM of strain Bartha lacks N-glycosylation. Virology 237:113–122.
Sticker A, Goeminne L, Martens L, Clement L. 2020. Robust summarization and inference in proteome-wide label-free quantification. Mol Cell Proteomics 19:1209–1219.
Goeminne LJE, Sticker A, Martens L, Gevaert K, Clement L. 2020. MSqRob takes the missing hurdle: uniting intensity- and count-based proteomics. Anal Chem 92:6278–6287.
Delva JL, Van Waesberghe C, Van Den Broeck W, Lamote JAS, Vereecke N, Theuns S, Couck L, Favoreel HW. 2022. The attenuated pseudorabies virus vaccine strain Bartha hyperactivates plasmacytoid dendritic cells by generating large amounts of cell-free virus in infected epithelial cells. J Virol 96.
Sedlackova L, Rice SA. 2008. Herpes simplex virus type 1 immediate-early protein ICP27 is required for efficient incorporation of ICP0 and ICP4 into virions. J Virol 82:268–277.
Zhou ZH, Chen DH, Jakana J, Rixon FJ, Chiu W. 1999. Visualization of tegument-capsid interactions and DNA in intact herpes simplex virus type 1 virions. J Virol 73:3210–3218.
Klupp B, Hengartner C, Mettenleiter TC, Enquist L. 2004. Complete, annotated sequence of the pseudorabies virus genome. J Virol 78:424–440.
Laine RF, Albecka A, Van De Linde S, Rees EJ, Crump CM, Kaminski CF. 2015. Structural analysis of herpes simplex virus by optical super-resolution imaging. Nat Commun 6:1–10.
Klopfleisch R, Klupp BG, Fuchs W, Kopp M, Teifke JP, Mettenleiter TC. 2006. Influence of pseudorabies virus proteins on neuroinvasion and neurovirulence in mice. J Virol 80:5571–5576.
Klupp BG, Granzow H, Mundt E, Mettenleiter TC. 2001. Pseudorabies virus UL37 gene product is involved in secondary envelopment. J Virol 75:8927–8936.
Fuchs W, Klupp BG, Granzow H, Mettenleiter TC. 2004. Essential function of the pseudorabies virus UL36 gene product is independent of its interaction with the UL37 protein. J Virol 78:11879–11889.
del Rio T, Werner HC, Enquist LW. 2002. The pseudorabies virus VP22 homologue (UL49) is dispensable for virus growth in vitro and has no effect on virulence and neuronal spread in rodents. J Virol 76:774–782.
Baumeister J, Klupp BG, Mettenleiter TC. 1995. Pseudorabies virus and equine herpesvirus 1 share a nonessential gene which is absent in other herpesviruses and located adjacent to a highly conserved gene cluster. J Virol 69:5560–5567.
Clase AC, Lyman MG, Rio T, Randall JA, Calton CM, Enquist LW, Banfield BW. 2003. The pseudorabies virus Us2 protein, a virion tegument component, is prenylated in infected cells. J Virol 77:12285–12298.
Esteves AD, Koyuncu OO, Enquist LW. 2022. A pseudorabies virus serine/threonine kinase, US3, promotes retrograde transport in axons via Akt/mToRC1. J Virol 96.
Curanović D, Lyman MG, Bou-Abboud C, Card JP, Enquist LW. 2009. Repair of the UL21 locus in pseudorabies virus Bartha enhances the kinetics of retrograde, transneuronal infection in vitro and in vivo. J Virol 83:1173–1183.
Orlando JS, Balliet JW, Kushnir AS, Astor TL, Kosz-Vnenchak M, Rice SA, Knipe DM, Schaffer PA. 2006. ICP22 is required for wild-type composition and infectivity of herpes simplex virus type 1 virions. J Virol 80:9381–9390.
De Wind N, Peeters BP, Zuderveld A, Gielkens AL, Berns AJ, Kimman TG. 1994. Mutagenesis and characterization of a 41-kilobase-pair region of the pseudorabies virus genome: transcription map, search for virulence genes, and comparison with homologs of herpes simplex virus type 1. Virology 200:784–790.
Lyu C, Li WD, Peng JM, Cai XH. 2020. Identification of interaction domains in the pseudorabies virus ribonucleotide reductase large and small subunits. Vet Microbiol 246:108740.
Lyman MG, Curanovic D, Brideau AD, Enquist LW. 2008. Fusion of enhanced green fluorescent protein to the pseudorabies virus axonal sorting protein Us9 blocks anterograde spread of infection in mammalian neurons. J Virol 82:10308–10311.
Lyman MG, Curanovic D, Enquist LW. 2008. Targeting of pseudorabies virus structural proteins to axons requires association of the viral Us9 protein with lipid rafts. PLoS Pathog 4:e1000065–16.
Tanneti NS, Federspiel JD, Cristea IM, Enquist LW. 2020. The axonal sorting activity of pseudorabies virus Us9 protein depends on the state of neuronal maturation. PLoS Pathog 16:e1008861–24.
Desplanques AS, Nauwynck HJ, Tilleman K, Deforce D, Favoreel HW. 2007. Tyrosine phosphorylation and lipid raft association of pseudorabies virus glycoprotein E during antibody-mediated capping. Virology 362:60–66.
Favoreel HW, Mettenleiter TC, Nauwynck HJ. 2004. Copatching and lipid raft association of different viral glycoproteins expressed on the surfaces of pseudorabies virus-infected cells. J Virol 78:5279–5287.
Yang L, Wang M, Cheng A, Yang Q, Wu Y, Jia R, Liu M, Zhu D, Chen S, Zhang S, Zhao X, Huang J, Wang Y, Xu Z, Chen Z, Zhu L, Luo Q, Liu Y, Yu Y, Zhang L, Tian B, Pan L, Rehman MU, Chen X. 2019. Innate immune evasion of alphaherpesvirus tegument proteins. Front Immunol 10:1–16.
Qin C, Zhang R, Lang Y, Shao A, Xu A, Feng W, Han J, Wang M, He W, Yu C, Tang J. 2019. Bclaf1 critically regulates the type I interferon response and is degraded by alphaherpesvirus US3. PLoS Pathog 15:e1007559–25.
Xie J, Zhang X, Chen L, Bi Y, Idris A, Xu S, Li X, Zhang Y, Feng R. 2021. Pseudorabies virus US3 protein inhibits IFN-β production by interacting with IRF3 to block its activation. Front Microbiol 12:1–10.
Grauwet K, Vitale M, De Pelsmaeker S, Jacob T, Laval K, Moretta L, Parodi M, Parolini S, Cantoni C, Favoreel HW. 2016. Pseudorabies virus US3 protein kinase protects infected cells from NK cell-mediated lysis via increased binding of the inhibitory NK cell receptor CD300a. J Virol 90:1522–1533.
Lin HW, Hsu WL, Chang YY, Jan MS, Wong ML, Chang TJ. 2010. Role of the UL41 protein of pseudorabies virus in host shutoff, pathogenesis and induction of TNF-α expression. J Vet Med Sci 72:1179–1187.
Xu G, Liu C, Zhou S, Li Q, Feng Y, Sun P, Feng H, Gao Y, Zhu J, Luo X, Zhan Q, Liu S, Zhu S, Deng H, Li D, Gao P. 2021. Viral tegument proteins restrict cGAS-DNA phase separation to mediate immune evasion Article Viral tegument proteins restrict cGAS-DNA phase separation to mediate immune evasion. Mol Cell 81:1–15.
Matundan H, Ghiasi H. 2019. Herpes simplex virus 1 ICP22 suppresses CD80 expression by murine dendritic cells. J Virol 93:1–19.
Claessen C, Favoreel H, Ma G, Osterrieder N, De Schauwer C, Piepers S, Van De Walle GR. 2015. Equid herpesvirus 1 (EHV1) infection of equine mesenchymal stem cells induces a pUL56-dependent downregulation of select cell surface markers. Vet Microbiol 176:32–39.
Said A, Azab W, Damiani A, Osterrieder N. 2012. Equine herpesvirus type 4 UL56 and UL49.5 proteins downregulate cell surface major histocompatibility complex class I expression independently of each other. J Virol 86:8059–8071.
Deschamps T, Kalamvoki M. 2017. Evasion of the STING DNA-sensing pathway by VP11/12 of herpes simplex virus 1. J Virol 91:1–17.
Maruzuru Y, Ichinohe T, Sato R, Miyake K, Okano T, Suzuki T, Koshiba T, Koyanagi N, Tsuda S, Watanabe M, Arii J, Kato A, Kawaguchi Y. 2018. Herpes simplex virus 1 VP22 inhibits AIM2-dependent inflammasome activation to enable efficient viral replication. Cell Host Microbe 23:254–265.
Piguet V, Trono D. 2001. Living in oblivion: HIV immune evasion. Semin Immunol 13:51–57.
Fleming SB. 2016. Viral inhibition of the IFN-induced JAK/STAT signaling pathway: development of live attenuated vaccines by mutation of viral-encoded IFN-antagonists. Vaccines 4:23–26.
Montaner-Tarbes S, del Portillo HA, Montoya M, Fraile L. 2019. Key gaps in the knowledge of the porcine respiratory reproductive syndrome virus (PRRSV). Front Vet Sci 6:1–15.
Lamote JAS, Kestens M, Van Waesberghe C, Delva J, De Pelsmaeker S, Devriendt B, Favoreel HW. 2017. The pseudorabies virus glycoprotein gE/gI complex suppresses type I interferon production by plasmacytoid dendritic cells. J Virol 91:1–12.
Laval K, Van Cleemput J, Vernejoul JB, Enquist LW. 2019. Alphaherpesvirus infection of mice primes PNS neurons to an inflammatory state regulated by TLR2 and type i IFN signaling. PLoS Pathog 15:e1008087–21.
Delva JL, Nauwynck HJ, Mettenleiter TC, Favoreel HW. 2020. The attenuated pseudorabies virus vaccine strain Bartha K61: a brief review on the knowledge gathered during 60 years of research. Pathogens 9:897–813.
Lippé R. 2012. Deciphering novel host-herpesvirus interactions by virion proteomics. Front Microbiol 3:1–14.
Stegen C, Yakova Y, Henaff D, Nadjar J, Duron J, Lippé R. 2013. Analysis of virion-incorporated host proteins required for herpes simplex virus type 1 infection through an RNA interference screen. PLoS One 8:e53276–12.
Hogue IB, Bosse JB, Hu J, Thiberge SY, Enquist LW. 2014. Cellular mechanisms of alpha herpesvirus egress: live cell fluorescence microscopy of pseudorabies virus exocytosis. PLoS Pathog 10:e1004535.
Pegg CE, Zaichick SV, Bomba-Warczak E, Jovasevic V, Kim D, Kharkwal H, Wilson DW, Walsh D, Sollars PJ, Pickard GE, Savas JN, Smith GA. 2021. Herpesviruses assimilate kinesin to produce motorized viral particles. Nature 599:662–666.
Lyman MG, Randall JA, Calton CM, Banfield BW. 2006. Localization of ERK/MAP kinase is regulated by the alphaherpesvirus tegument protein Us2. J Virol 80:7159–7168.
Elliott G, O’Reilly D, O’Hare P. 1999. Identification of phosphorylation sites within the herpes simplex virus tegument protein VP22. J Virol 73:6203–6206.
Van Opdenbosch N, Van Den Broeke C, De Regge N, Tabarés E, Favoreel HW. 2012. The IE180 protein of pseudorabies virus suppresses phosphorylation of translation initiation factor eIF2α. J Virol 86:7235–7240.
Lomniczi B, Watanabe S, Ben-Porat T, Kaplan AS. 1987. Genome location and identification of functions defective in the Bartha vaccine strain of pseudorabies virus. J Virol 61:796–801.
Nauwynck HJ, Pensaert MB. 1995. Effect of specific antibodies on the cell-associated spread of pseudorabies virus in monolayers of different cell types. Arch Virol 140:1137–1146.
Olsen LM, Ch TH, Card JP, Enquist LW. 2006. Role of pseudorabies virus Us3 protein kinase during neuronal infection. J Virol 80:6387–6398.
Gomez-Sabastian S, Tabares E. 2004. Negative regulation of herpes simplex virus type 1 ICP4 promoter by IE180 protein of pseudorabies virus. J Gen Virol 85:2125–2130.
Van Puyvelde B, Daled S, Willems S, Gabriels R, de Peredo AG, Chaoui K, Mouton-Barbosa E, Bouyssié D, Boonen K, Hughes CJ, Gethings LA, Perez-Riverol Y, Bloomfield N, Tate S, Schiltz O, Martens L, Deforce D, Dhaenens M. 2022. A comprehensive LFQ benchmark dataset on modern day acquisition strategies in proteomics. Sci Data 9:1–12.
Jansens RJJ, Van Den Broeck W, De Pelsmaeker S, Lamote JAS, Van Waesberghe C, Couck L, Favoreel HW. 2017. Pseudorabies virus US3-induced tunneling nanotubes contain stabilized microtubules, interact with neighboring cells via cadherins, and allow intercellular molecular communication. J Virol 91:1–13.

Information & Contributors


Published In

cover image Journal of Virology
Journal of Virology
Volume 96Number 2421 December 2022
eLocator: e01158-22
Editor: Felicia Goodrum, University of Arizona
PubMed: 36453884


Received: 26 July 2022
Accepted: 8 November 2022
Published online: 1 December 2022


Request permissions for this article.


  1. suid herpesvirus 1
  2. Aujeszky’s disease virus
  3. pseudorabies virus
  4. PRV
  5. Becker
  6. Kaplan
  7. NIA3
  8. Bartha
  9. proteomics
  10. mass spectrometry
  11. data-dependent acquisition
  12. data-independent acquisition
  13. proteome
  14. tegument



Jonas L. Delva
Department of Translational Physiology, Infectiology and Public Health, Faculty of Veterinary Medicine, Ghent University, Ghent, Belgium
Simon Daled
ProGenTomics, Laboratory of Pharmaceutical Biotechnology, Faculty of Pharmaceutical Sciences, Ghent University, Ghent, Belgium
Cliff Van Waesberghe
Department of Translational Physiology, Infectiology and Public Health, Faculty of Veterinary Medicine, Ghent University, Ghent, Belgium
Ruben Almey
ProGenTomics, Laboratory of Pharmaceutical Biotechnology, Faculty of Pharmaceutical Sciences, Ghent University, Ghent, Belgium
Robert J. J. Jansens
Department of Translational Physiology, Infectiology and Public Health, Faculty of Veterinary Medicine, Ghent University, Ghent, Belgium
Dieter Deforce
ProGenTomics, Laboratory of Pharmaceutical Biotechnology, Faculty of Pharmaceutical Sciences, Ghent University, Ghent, Belgium
Maarten Dhaenens
ProGenTomics, Laboratory of Pharmaceutical Biotechnology, Faculty of Pharmaceutical Sciences, Ghent University, Ghent, Belgium
Department of Translational Physiology, Infectiology and Public Health, Faculty of Veterinary Medicine, Ghent University, Ghent, Belgium


Felicia Goodrum
University of Arizona


Jonas L. Delva and Simon Daled share first authorship of the manuscript. The order was determined by the corresponding author after negotiation.
Maarten Dhaenens and Herman W. Favoreel share senior authorship of the manuscript.
The authors declare no conflict of interest.

Metrics & Citations


Note: There is a 3- to 4-day delay in article usage, so article usage will not appear immediately after publication.

Citation counts come from the Crossref Cited by service.


If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download.

View Options

Figures and Media






Share the article link

Share with email

Email a colleague

Share on social media

American Society for Microbiology ("ASM") is committed to maintaining your confidence and trust with respect to the information we collect from you on websites owned and operated by ASM ("ASM Web Sites") and other sources. This Privacy Policy sets forth the information we collect about you, how we use this information and the choices you have about how we use such information.
FIND OUT MORE about the privacy policy