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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Virology. Author manuscript; available in PMC 2010 May 10.
Published in final edited form as:
PMCID: PMC2674122
NIHMSID: NIHMS100424

A conserved carboxy-terminal domain in the major tegument structural protein VP22 facilitates virion packaging of a chimeric protein during productive herpes simplex virus 1 infection

Abstract

Recombinant virus HSV-1(RF177) was previously generated to examine tegument protein VP22 function by inserting the GFP gene into the gene encoding VP22. During a detailed analysis of this virus, we discovered that RF177 produces a novel fusion protein between the last 15 amino acids of VP22 and GFP, termed GCT-VP22. Thus, the VP22 carboxy-terminal specific antibody 22-3 and two anti-GFP antibodies reacted with an approximately 28 kDa protein from RF177-infected Vero cells. GCT-VP22 was detected at 1 and 3 hpi. Examination of purified virions indicated that GCT-VP22 was incorporated into RF177 virus particles. These observations imply that at least a portion of the information required for virion targeting is located in this domain of VP22. Indirect immunofluorescence analyses showed that GCT-VP22 also localized to areas of marginalized chromatin during RF177 infection. These results indicate that the last fifteen amino acids of VP22 participate in virion targeting during HSV-1 infection.

Keywords: Tegument protein VP22, GCT-VP22, virion packaging, marginalized chromatin

Introduction

The tegument of the herpes simplex virus particle is the proteinaceous layer located between the capsid and envelope and is made up of approximately 20 virus-encoded polypeptides (Enquist et al., 1998; Mettenleiter, 2002; Mettenleiter, 2004; Roizman, 1974; Vittone et al., 2005). While it is unclear how HSV-1 virions assemble, a number of interactions involving HSV-1 tegument protein self- and nonself-associations have been identified (Elliott, Mouzakitis, and O’Hare, 1995; Hafezi et al., 2005; Lee et al., 2008; Mouzakitis et al., 2005; Smibert et al., 1994; Taddeo et al., 2007; Vittone et al., 2005). Some viral tegument proteins reportedly interact with the cytoplasmic tail of certain viral glycoproteins (Chi et al., 2005; Farnsworth, Wisner, and Johnson, 2007; Fuchs et al., 2002; Gross, Harley, and Wilson, 2003) and this may impact virion assembly (Leuzinger et al., 2005; Mettenleiter, 2004; O’Regan et al., 2007; Sugimoto et al., 2008).

VP22, which contains 301 amino acids, is the most abundant tegument protein of HSV-1; nearly 2,000 copies of VP22 are estimated to be present within each virion (Heine et al., 1974). VP22 is a posttranslationally modified phosphoprotein (Blaho, Mitchell, and Roizman, 1993; Elliott, O’Reilly, and O’Hare, 1996; Elliott, O’Reilly, and O’Hare, 1999; Hutchinson et al., 2002; Knopf and Kaerner, 1980; Morrison et al., 1998; Morrison, Wang, and Meredith, 1998; Pomeranz and Blaho, 1999; Spear and Roizman, 1972) that exhibits multiple subcellular localizations during productive viral infection (Elliott and O’Hare, 1997; Elliott and O’Hare, 1999; Hafezi et al., 2005; Kotsakis et al., 2001; Pomeranz and Blaho, 1999; Sugimoto et al., 2008; Yedowitz et al., 2005). VP22 accumulates to high levels in nuclei at late infection times (Pomeranz and Blaho, 1999; Sugimoto et al., 2008) and it associates with areas of dispersed nucleoli and marginalized chromatin (Lopez et al., 2008). Transiently expressed VP22 has been detected in both the cytoplasm and nuclei of cells (Blouin and Blaho, 2001; Brignati et al., 2003; Elliott and O’Hare, 2000; Fang et al., 1998; Harms et al., 2000). While the predicted molecular weight of VP22 is 32,000 (Elliott and Meredith, 1992; McGeoch et al., 1988), which is below the size exclusion limit (molecular weight of 40,000 to 45,000) for passive diffusion through the nuclear pore (Davis, 1995), VP22 contains two computer predicted nuclear localization signal motifs (Harms et al., 2000; Kotsakis et al., 2001).

In an attempt to determine the biological function of VP22 during viral infection, a recombinant virus, HSV-1(RF177), was generated which contains a carboxy-terminal truncation of VP22 and synthesizes amino acids 1 to 212, termed Δ212 (Pomeranz and Blaho, 2000). Tegument proteins VP13/14, VP16, and vhs were synthesized and incorporated into RF177 virions at wild-type levels but RF177 demonstrated a small plaque size phenotype. These initial studies suggested a cell-cell spreading defect for viruses containing defects in VP22 (Pomeranz and Blaho, 2000) which has since been definitively confirmed (Duffy et al., 2006; Duffy, Mbong, and Baines, 2009). A further analysis revealed that RF177 produced a novel VP22-GFP fusion protein, which we term GCT-VP22. Our goal was to characterize the behavior of GCT-VP22 during productive RF177 infection. GCT-VP22 was incorporated into RF177 virus particles, suggesting that the information required for virion targeting was located in the last 15 amino acids of VP22. We identified a motif of six consecutive amino acids in this region that was conserved among the HSV-1, HSV-2, and VZV VP22 homologues and we hypothesize that this motif might function as a virion packaging signal. Finally, we discovered that GCT-VP22 targeted marginalized chromatin during RF177 infection. These findings suggest that we have identified a multifunctional, carboxy-terminal domain in VP22 that likely influences its subcellular localization and virion packaging during productive HSV-1 infection.

Results

HSV-1(RF177) synthesizes a carboxy-terminal VP22-GFP fusion protein termed, GCT-VP22

We previously reported that the recombinant virus HSV-1(RF177), termed RF177, synthesizes the amino-terminal 212 amino acids of VP22 (Δ212) which localizes to nuclei and incorporates into virions during RF177 infection of Vero cells (Pomeranz and Blaho, 2000). RF177 has been a useful research tool as it defined the roles of VP22 in viral cell-cell spreading (Pomeranz and Blaho, 2000) and efficient microtubule reorganization during HSV-1 infection (Yedowitz et al., 2005). To generate RF177, a cytomegalovirus promoter-driven green fluorescence protein (GFP) cassette was inserted after amino acid 212 of VP22 (Pomeranz and Blaho, 2000). This strategy left approximately 45 bp at the 3′ end of the UL49 gene (Fig. 1, line 3). A careful inspection of the nucleotide sequences of GFP, the remaining region of the multiple cloning site (linker), and the remaining portion of VP22 indicated that a likely in frame fusion occurred when generating recombinant virus RF177 (Fig. 1, line 5). To confirm the existence of this putative GFP-carboxy-terminal VP22 fusion (line 4), Vero cells were mock-infected or infected (MOI=5) with HSV-1(F) or RF177, whole cell extracts were prepared at 24 hpi, separated in a denaturing gel, transferred to nitrocellulose, and probed with the antibody 22-3, specific for the carboxy-terminal portion of VP22 (Geiss et al., 2001) and control RGST49 antibody, specific for the amino-terminal portion, as described in Material and Methods. We then reprobed the blots with anti-GFP polyclonal and monoclonal antibodies, accordingly. The results (Fig. 2) from theses experiments were as follows.

Fig. 1
Schematic representation of the HSV-1(F) genome and details of the mutated gene loci of HSV-1(RF177). Line 1 - the HSV-1 (F) genome with the unique long (UL), unique short (US), and terminal repeat segments (a, b, c, a′, b′, and c′) ...
Fig. 2
Immune reactivities of RF177-infected cell extracts. Vero cells were infected (MOI of 5) for 24 hpi, equal amounts of whole cell extract were separated in a denaturing gel, transferred to nitrocellulose, and probed with polyclonal (pAb) RGST49 (A), monoclonal ...

No viral proteins were detected in mock-infected cells (Fig. 2A–D, lane 3). As expected, both RGST49 and 22-3 antibodies reacted with full-length VP22 in HSV-1(F)-infected cells (Fig. 2A, lane 1 and Fig. 2C, lane 2). Multiple forms of VP22 were observed as described previously (Blaho, Mitchell, and Roizman, 1994; Pomeranz and Blaho, 1999). Low levels of Δ212 were detected in RF177-infected cells using RGST49 (Fig. 2A, lane 2) while no immune reaction was observed with the 22-3 antibody in the portion of the blot where Δ212 migrates (Fig. 2C, lane 3). However, the VP22 carboxy-terminal specific antibody 22-3 reacted with an approximately 28,000 molecular weight protein in the RF177-infected cells (Fig. 2C, lane 3). That this new protein reacted with the carboxy-terminal specific antibody implies that this portion of VP22 was being synthesized. When we reprobed the blots (Fig. 2A, C) with different anti-GFP antibodies, we found that both of these antibodies also reacted with approximately 28,000 molecular weight proteins in RF177-infected cell extracts (Fig. 2B, D, lane 2). Taken together, these findings indicate that an in-frame GFP fusion occurred with the carboxy-terminal 15 amino acids of VP22. We propose the term, GCT-VP22, for this GFP-carboxy-terminal VP22 fusion (Fig. 1, line 4). GCT-VP22 was not detected in the original description of RF177 because only amino-terminal specific antibodies were used to characterize the VP22 forms produced by this virus.

Full-length VP22 is a highly posttranslationally modified phosphoprotein (Blaho, Mitchell, and Roizman, 1994; Elliott, O’Reilly, and O’Hare, 1996; Geiss et al., 2004; Geiss et al., 2001; Heine et al., 1974; Pomeranz and Blaho, 1999) whose modifications correlate with its translocation to nuclei (Pomeranz and Blaho, 1999). While most of the reported VP22 posttranslational modification target sites remain in Δ212, two serines (at amino acids 292 and 294) are located in GCT-VP22 (Fig. 1, line 6). Serine phosphorylation has been proposed to play a role in virion detegumentation (Morrison, Wang, and Meredith, 1998). Earlier computational analyses identified two possible nuclear localization signal motifs in the primary structure of VP22 (Kotsakis et al., 2001). The pat4 motif is a four residue pattern composed of three basic amino acids and either an histidine or a proline residue. The pat7 motif starts with a proline followed within three residues by a basic segment containing three lysine or arginine residues out of four. VP22’s pat7 motif starts at amino acid 82 (82PRTRRPV88) and pat4 motif starts at amino acid 295 (295RPRR298) (Fig. 1, line 2 and 6). It should be noted that certain viral nuclear localization signals may also act as nucleolar localization signal motifs (Hiscox, 2002; Rowland and Yoo, 2003). The GCT-VP22 protein produced during RF177 infection contains only the pat4 motif. Therefore, the initial goal of this study was to determine the cellular distribution and kinetics of synthesis of GCT-VP22 to determine whether pat4 might participate in its subcellular localization.

GCT-VP22 detected during RF177 infection

Fortuitously, because GCT-VP22 was formed during the generation of RF177, this now enables us to define the role of the last 15 amino acids of VP22 in the subcellular localization of GFP during live viral infection. Our next goal was to characterize the kinetics of GCT-VP22 production. Vero cells were synchronously infected either with HSV-1(F) or RF177, whole cell extracts were prepared at 1, 3, 5, 7, and 24 hpi, separated in denaturing gels, transferred to nitrocellulose, and probed with anti-ICP4, -VP22, and -GFP antibodies, as described in Material and Methods. The results (Fig. 3) were as follows.

Fig. 3
Immune reactivities of infected cell proteins extracted during the course of synchronized HSV-1(F) and RF177 infections. Vero cells were synchronously infected with HSV-1(F) and RF177 or mock infected. At 1, 3, 5, 7, and 24 hpi, whole-cell extracts were ...

Low levels of ICP4 were detected at 3 hpi in cells infected with HSV-1(F) or RF177 (Fig. 3A, B, lane 5) and ICP4 increased at 5 and 7 hpi, reaching a maximum at 24 hpi (lane 6–8). Using anti-VP22 (RGST49) antibody, VP22 in HSV-1(F)-infected Vero cells was first detected at 5 hpi, increased slightly at 7 hpi, and accumulated at high levels at 24 hpi (Fig. 3A, lane 6–8). As expected, no ICP4 or VP22 was observed in the mock-infected cells (Fig. 3A, lane 2 and 3). In RF177-infected cells, similar amounts of GCT-VP22 were detected at 5 and 7 hpi using anti-GFP antibody (Fig. 3B, lane 6 and 7) and its reactivity reached a high level at 24 hpi (lane 8). Unexpectedly, faint, but discernable bands were observed reacting with the anti-GFP-antibody at 1 and 3 hpi in the region of the blot where GCT-VP22 migrated (lane 4 and 5). These findings raise the intriguing possibility that GCT-VP22 might be packaged into virions and transported into the cell at the onset of infection. The implication of such an effect would be that, perhaps, some sort of a VP22 packaging signal may be located within the carboxy-terminal 15 amino acids of VP22.

Close inspection of the results in Panel 3B indicated a slight difference in GCT-VP22 mobility between 1, 3 hpi and 5–24 hpi (compare lane 4, 5 with 6–8), inasmuch as VP22 at 1 and 3 hpi migrated slightly slower (Pomeranz and Blaho, 1999). We previously reported electrophoretic differences of VP22 depending upon its subcellular localization and presence in virions (Blaho, Mitchell, and Roizman, 1994; Pomeranz and Blaho, 1999; Pomeranz and Blaho, 2000). As these data are derived from whole infected cell extracts, VP22 localization cannot be assessed. However, this observation seems to support the notion that VP22 detected as a very early, slower migrating form (Pomeranz and Blaho, 1999) is from input virus and may reflect immediate posttranslational modification, as proposed (Morrison, Wang, and Meredith, 1998).

GCT-VP22 incorporation into RF177 virions

The results above indicate that GCT-VP22 could be detected at very early times during RF177 infection, even before ICP4 was observed. We therefore set out to determine whether GCT-VP22 might actually be incorporated into RF177 virions (Pomeranz and Blaho, 1999). Vero cells were infected with either HSV-1(F) or RF177, preparations of purified HSV-1 and RF177 virions were made, virion proteins were separated in a denaturing gel, transferred to nitrocellulose, and sequentially probed with anti-VP22 polyclonal and -GFP monoclonal antibodies as described in Materials and Methods. The results (Fig. 4) were as follows.

Fig. 4
Immune reactivities of HSV-1(F) and HSV-1(RF177) virion proteins. Virions were prepared from HSV-1(F)- and RF177-infected Vero cells as described in Materials and Methods. Virion proteins were separated in a denaturing gel, transferred to nitrocellulose, ...

Abundant VP22 and a low amount of Δ212 was detected in the virion preparations with the anti-VP22 polyclonal antibody following HSV-1(F) and RF177 infections, respectively (Fig. 4A). As expected, no anti-GFP reactivity was observed in virions of HSV-1(F) (Fig. 4B). However, two bands reacting with the anti-GFP antibody were observed in virion preparations derived from RF177. The mobility of the upper band was consistent with that of GCT-VP22 (Fig. 2) and the lower band likely represents a degradation product bound by this antibody (as seen in Fig. 2). The lack of reactivity of GCT-VP22 with the anti-VP22 polyclonal antibody was expected as it is specific for the amino-terminal domain (Fig. 2). We recognize that since we did not set out to engineer the GCT-VP22 construct, we also did not create a control recombinant in which GFP alone is produced under the same regulatory control. However, the Courtney group has repeated shown that GFP protein alone does not get packaged into virions during productive HSV infection (O’Regan et al., 2007). Our data corroborate the kinetic results above (Fig. 3) and demonstrate that GCT-VP22 was incorporated into RF177 virions. Together, these results imply that sufficient amounts of virion associated GCT-VP22 are delivered into RF177-infected cells to be detected by immunoblotting methods.

Full-length VP22 increases GCT-VP22 nuclear localization observed during live RF177 infection

We performed two separate sets of experiments to document the behavior of GCT-VP22 inside cells during the course of productive virus infection. In our first study, Vero and VP22-expressing V49 cells were synchronously infected with RF177 or mock-infected, Hoechst 33258 DNA dye was added to stain nuclei, and cells were visualized live by fluorescence microscopy at 18 hpi as described in Material and Methods. The results (Fig. 5) were as follows.

Fig. 5
Live fluorescence microscopy of RF177-infected Vero and VP22-expressing V49 cells. Vero and V49 cells were synchronously infected with RF177, stained with Hoechst, and visualized live by fluorescence microscopy as described in Materials and Methods. Images ...

Inspection of the Hoechst-stained, live, infected cells indicated that RF177 infection led to chromatin marginalization (compare Panel H with B and E), consistent with that observed with wild type HSV-1 (Lopez et al., 2008). This represents the first description of this phenomenon using RF177. GCT-VP22 synthesized during live RF177 infection of Vero cells was observed in both nuclei and the cytoplasm (Panel A). The cytoplasmic GCT-VP22 was readily discriminated in the merged image (Panel C). Since GCT-VP22 had a observed mol wt of approximately 28,000 (Fig. 2), it should be small enough to pass freely through the nuclear pore by diffusion (Pomeranz and Blaho, 2000) and therefore might be expected to localize throughout the cell. However, the extent of the nuclear localization of GCT-VP22 in V49 cells appeared greater than that with infected Vero cells (compare Panel D and A). No GFP signal was detected in mock-infected V49 cells (Panel G and I). While these results may be slight, it is tempting to speculate that the full-length VP22 present in the VP22-expressing V49 cells may actually participate the nuclear localization of GCT-VP22. During the original creation and characterizations of VP22 expressing cell lines (Blouin and Blaho, 2001; Kotsakis et al., 2001; Pomeranz and Blaho, 2000), transient GFP expression in the presence of VP22 alone did not show any differences in GFP localization from that in GFP transfected non-VP22 expressing Vero cells. Based on this, it seems unlikely that VP22 and GFP directly interact such that VP22 might affect GFP subcellular localization.

In the second study, Vero and V49 cells were synchronized HSV-1(F)- or RF177-infected and stained with Hoechst, but the cells were fixed/permeablized for indirect immunofluorescence using the VP22 carboxy-terminal specific monoclonal antibody (22–3) as described in Material and Methods. We also monitored GFP fluorescence during the RF177 infections and controls included using the VP22 amino-terminal specific polyclonal antibody (RGST49) to detect full-length VP22 in V49 cells. The results (Fig. 6) were as follows.

Fig. 6
Indirect immunofluorescence and GFP fluorescence of HSV-1(F)- and RF177- infected Vero and V49 cells. Cells were uninfected (V49) (A), mock-infected (V49mock) (B. C), synchronously infected with HSV-1(F) (B) or RF177 (C), or uninfected (V49) (C) and then ...

Since we were interested in following GCT-VP22 only, we first titrated down the 22-3 antibody to a level where its reactivity with full-length VP22 in V49 cells was negligible. The dilution of the 22-3 antibody used in this study showed little to no reactivity with VP22 in mock-infected V49 cells (Fig. 6B, Panel 6 and Fig. 6C, Panel 8). VP22 in uninfected V49 cells was readily detected with a low dilution of the VP22 amino-terminal specific polyclonal antibody RGST49, as expected (Pomeranz and Blaho, 2000), and localized to nuclei in the absence of other viral proteins (Fig. 6A, Panel 2).

In HSV-1(F)-infected Vero and V49 cells, 22-3 detected nuclear and, perhaps, microtubule-associated (Kotsakis et al., 2001) VP22 (Fig. 6B, Panel 2 and 4). Marginalized chromatin was readily observed as bright, dense Hoechst staining in the infected cells (Fig. 6B, Panel 1 and 3) and a portion of nuclear full-length VP22 associated with it (compare Panel 2 with 1), as expected (Lopez et al., 2008). Based on our control studies described above, these signals represent full-length VP22 which was newly synthesized during infection and indicate that monoclonal antibody 22-3 could specifically detect VP22 in infected Vero and V49 cells by indirect immunofluorescence. In control immunoblotting studies, infection of the V49 cells with RF177 did not detectably increase the expression of full-length VP22 (data not shown). No 22-3 immune reaction or GFP signal was detected in mock-infected V49 cells (Fig. 6C, Panel 8 and 9).

Bright 22-3 antibody staining which directly corresponded to GFP fluorescence was observed in nuclei as well as in perinuclear regions of RF177-infected Vero cells (Fig. 6C, compare Panel 2 with 3), consistent with our previous observations in live-infected Vero cells (Fig. 5). Although both 22-3 staining and GFP fluorescence was observed in the cytoplasm of RF77-infected V49 cells, these signals were enhanced in V49 nuclei, compared with the staining patterns seen in Vero cells (Fig. 6C, compare Panel 5 and 6 with 2 and 3). It is of particular interest and significance that a portion of the nuclear GCT-VP22 signal was associated with regions of marginalized chromatin (compare Panel 6 with 4). VP22 was recently reported to localize to areas of marginalized chromatin and dispersed nucleoli (Lopez et al., 2008). Importantly, this represents a novel finding and implies that the 15 amino acid portion of VP22 present in GCT-VP22 may play a role in VP22’s association with marginalized chromatin.

Taking the results of Figs. 5 and and66 together, it appears that either in live or fixed cells, the presence of full-length VP22 in the V49 cells seemed to increase the nuclear detection of GCT-VP22. This enhanced GCT-VP22 localization in nuclei may suggest a possible interaction between the full-length VP22 stably expressed in the V49 cells and the 15 carboxy-terminal amino acids of VP22 in GCT-VP22. Thus, VP22 may serve as a binding partner for GCT-VP22. A possible self-interaction of VP22 has been reported recently based on a yeast two-hybrid analysis (Lee et al., 2008; Vittone et al., 2005). Our results may corroborate these findings and extend them, inasmuch as they imply that the VP22 multimerization domain, or at least a portion of a bi-partite domain, might be situated within the carboxy-terminal 15 amino acids of VP22. Since GCT-VP22 contains the pat4 sequence, it is possible that this motif also plays a role in the targeting of this chimeric molecule during infection.

Discussion

We previously described the generation of recombinant virus HSV-1(RF177) (Pomeranz and Blaho, 2000; Yedowitz et al., 2005). In this study, we characterized the biological behavior of GCT-VP22, a novel VP22-carboxy-terminal GFP fusion protein which is detected in RF177-infected cells. Our key findings may be summarized as follows.

(i) GCT-VP22 is packaged into RF177 virions

In general, it is relatively difficult to detect incoming VP22 (i.e., virion derived) by indirect immunofluorescence or immunoblotting (Fig. 3A) methods (Elliott and O’Hare, 1999; Morrison, Wang, and Meredith, 1998; Pomeranz and Blaho, 1999; Pomeranz and Blaho, 2000). The reason for this is unknown but it may imply that incoming VP22 is either rapidly degraded or diffused. That GCT-VP22 was detected at 1 and 3 hpi during RF177 infection was, therefore, surprising. Since GCT-VP22 is expressed from the strong CMV IE promoter (Pomeranz and Blaho, 2000), it was synthesized at a high enough levels that it was incorporated into virions. It is also conceivable the incoming GCT-VP22 may be more stable than VP22.

(ii) The VP22 portion of GCT-VP22 contains a six amino acid region of identity which is conserved among the human α-herpesvirus VP22 homologues

The important issue is how GCT-VP22 gets incorporated in virions. The previous results suggest that the 15 amino acid domain of VP22 present in GCT-VP22 is sufficient to target the chimeric protein into RF177 virions. Since such a signal might be expected to be conserved among other herpesvirus VP22 homologues, we performed a computer analysis (Fig. 7) using the ClustalW alignment algorithm as described in Materials and Methods. While other VP22 homologues, such as that of herpes B virus (Cercopithecine herpesvirus 1; the α-herpesvirus of macaque monkeys) (Perelygina et al., 2003), possessed regions of partial identity with GCT-VP22, seven out of fifteen amino acids, A-R-S-A-S-R (290ARSASR295), were conserved between the HSV-1, HSV-2, and VZV VP22 homologues (Fig. 7). While this sequence is contiguous with that of the HSV-1 and HSV-2 pat4 motif, the VZV homologue does not appear to possess pat4 (line 5). We also performed a similar analysis using the other HSV-1 tegument proteins VP16, VP13/14, and vhs. We did not detect the ARSASR sequence and were unable to identify a similarly conserved motif in each of these proteins. HSV-1 and HSV-2 are more closely related to each other than both of them are to VZV (Davison, 2002); full-length VP22 homologues of HSV-1 and HSV-2 share about 69% conserved identity with each other while the VP22 homologue of VZV shares about 20% conserved identity with the homologue from HSV-1 (as determined by the MatrixAlign algorithm of MacVector, data not shown). Therefore, it was not surprising that the sequence of the VZV VP22 homologue was aligned by insertion of a gap (Fig. 7, line 3). Based on our findings, we propose that the motif, 290ARSASR295, may represent a human α-herpesvirus VP22 virion packaging signal (VP22 VPS).

Fig. 7
Alignment of the carboxy-termini of three human α-herpesvirus VP22 homologues. The VP22 termini of HSV-1, HSV-2, VZV, and GCT-VP22 (RF177) (line 1–4) were aligned using a computational algorithm as described in Materials and Methods. Asterisks ...

The recent reports that the last 89 amino acids of VP22 (out of 301) was required for VP22 incorporation into virions (Hafezi et al., 2005; Murphy et al., 2008; O’Regan et al., 2007) strongly supports the validity of our hypothesis. Since it is generally assumed that HSV virions acquire their final tegument and envelope in the cytoplasm, packaging of GCT-VP22 should also occur in the cytoplasm at the trans Golgi compartment or after interacting with glycoproteins at the Golgi (Mettenleiter, 2002; Mettenleiter, 2004; Sugimoto et al., 2008; Vittone et al., 2005). As interactions of VP22 with gD, gE, gI, and gM have been reported (Chi et al., 2005; Farnsworth, Wisner, and Johnson, 2007; Pomeranz and Blaho, 1999; Vittone et al., 2005), GCT-VP22 might associate with these as well. Thus, further characterizations of the behavior of GCT-VP22 in our RF177 system should provide important new information regarding HSV virion assembly.

Several important questions remain. Assuming the putative VP22 VPS functions as a motif for virion targeting, do other tegument proteins share this or a similar VPS? Our initial survey of vhs, VP13/14, and VP16 did not identify clear homologues to the VP22 VPS. Of course, this does not preclude the possibility that VPS’s may be distinct for each protein. Another important observation was that Δ212 is also packaged in RF177 virions (Fig. 4) (Pomeranz and Blaho, 2000). Thus, the putative VP22 VPS in GCT-VP22 may function as a primary packaging signal but at least one other signal likely exists in Δ212. One interpretation of this is that the VPS in GCT-VP22 is sufficient but not necessary for packaging. Since our results imply that VP22 and GCT-VP22 may interact, then the proposed two packaging signals on full-length VP22 may interact. It may be that the VPS functions to enhance packaging. Thus, without it, Δ212 is only packaged at low levels as we observe {Fig. 4, (Pomeranz, 2000)}. Such a model would also be consistent with the recent studies of Elliott which conclude that the carboxy-terminal third of VP22 is sufficient for virion packaging but an additional internal domain likely also plays a role (Hafezi et al., 2005). Studies currently underway to molecularly characterize the putative VP22 VPS should critically evaluate these predictions.

(iii) Full-length VP22 may increase the amount of nuclear GCT-VP22

This conclusion is based in the observation that GCT-VP22 demonstrated somewhat enhanced live nuclear localization following RF177 infection of VP22-expressing cells. This finding suggests that the association with VP22 inside infected cells may influence the localization of GCT-VP22. Self-associations of a number of HSV-1 tegument proteins, including VP22, have been described based on results from two-hybrid analyses (Lee et al., 2008; Vittone et al., 2005). An earlier biochemical characterization of VP22 by O’Hare’s group indicated that, in solution, VP22 likely dimerizes and is capable of forming higher order complexes, which may include additional viral proteins (Mouzakitis et al., 2005). These authors concluded that carboxy-terminal determinants were required for stabilizing such assembles. Our results support these studies and extend them. It appears that the last 15 amino acids of VP22 are capable, in some way, of associating with full-length VP22. One consequence of this interaction may be to support nuclear translocation of GCT-VP22 but not GFP. This raises the intriguing possibility that regulated VP22 nuclear translocation during infection requires either self-association or complex formation with other viral proteins. It should be noted that recent indirect-immune (Yedowitz et al., 2005) and live fluorescence (Donnelly and Elliott, 2001a; Donnelly and Elliott, 2001b; Hutchinson et al., 2002; Sugimoto et al., 2008) studies indicate that the kinetics and environment of VP22’s subcellular localizations differ from that of the other major tegument proteins vhs, VP16, and VP13/14.

Cellular localizations of full-length VP22 appear tightly regulated since the protein is capable of both cytoplasmic and nuclear accumulations and it may interact with other proteins or even transcripts (Aints et al., 2001; Brignati et al., 2003; Elliott, Mouzakitis, and O’Hare, 1995; Harms et al., 2000; Kotsakis et al., 2001; Martin et al., 2002; Pomeranz and Blaho, 1999; Sciortino et al., 2002; Vittone et al., 2005). That GCT-VP22 localizes to the nucleus is consistent with previous reports that the carboxy-portion of VP22 facilitates the nuclear association of GFP-VP22 fusion constructs (Brignati et al., 2003; Elliott and O’Hare, 1997; Fang et al., 1998). Although, GCT-VP22 is small enough to pass freely through the nuclear pore by diffusion without use of energy, it also contains a putative pat4 (Kotsakis et al., 2001) nuclear localization signal motif, 295RPRR298 (Fig. 1). A recent study by Zheng et al., implicated a related motif, 130PRPR133, in the nuclear localization of transiently expressed BHV-1 VP22 fused to GFP (Zheng et al., 2005). Thus, the pat4 motif may play a role in the localization of GCT-VP22.

Of course, the interplay between different localization signals/domains of VP22 may only be part of the complex cellular localization of VP22 and GCT-VP22. Control mechanisms such as phosphorylation or other modification may influence the localization of VP22 and CGT-VP22 as well. Phosphorylation or dephosphorylation in the vicinity of an NLS may play a role in the intracellular distribution of proteins {reviewed in (Jans and Hubner, 1996; Jans, Xiao, and Lam, 2000)}. VP22 is a virion phosphoprotein (Blaho, Mitchell, and Roizman, 1994; Elliott, O’Reilly, and O’Hare, 1996; Elliott, O’Reilly, and O’Hare, 1999; Gibson and Roizman, 1974; Pomeranz and Blaho, 1999; Pomeranz and Blaho, 2000) and phosphorylation of VP22 in the incoming virion has been proposed to be a signal for detegumentation (Morrison, Wang, and Meredith, 1998). However, VP22 may also remain associated with the capsid through the disassembly step at the plasma membrane and might play a role in capsid association with the nuclear pore complex (Chi et al., 2005; Ojala et al., 2000). While two putative phosphorylation sites of VP22, serine 292 and serine 294 (Elliott, O’Reilly, and O’Hare, 1996; Elliott, O’Reilly, and O’Hare, 1999), are contiguous with the pat4 motif (Fig. 1, line 7), these residues are not phosphorylation sites on HSV-2 VP22 (Geiss et al., 2004; Geiss et al., 2001). Therefore, phosphorylation of GCT-VP22 may also occur and this might have an effect on its nuclear import (Hubner, Xiao, and Jans, 1997). Additional detailed biochemical analyses are currently underway to address this issue.

(iv) GCT-VP22 associates with marginalized chromatin

Since we only looked at late times post infection, this effect may simply be due to nucleopathic viral effect. It has been well described that an ordered marginalization of chromatin and dispersal of nucleoli occurs during wild type HSV-1 infection (Calle et al., 2008; Hampar and Ellison, 1961; Lopez et al., 2008; Lymberopoulos and Pearson, 2007; Roizman and Knipe, 2001; Roizman, 1974; Simpson-Holley et al., 2005). Full-length VP22 was shown to associate with dispersed nucleolin (Lopez et al., 2008). HSV-1 infection reduces the level of rRNA synthesis and the nucleolus may be gradually pushed to the nuclear border by the formation of replication compartments (Besse and Puvion-Dutilleul, 1996). While the nucleolus is the site of ribosome biogenesis and it has been implicated in controlling regulatory processes, such as the cell cycle (Mayer and Grummt, 2005; Olson, 2004; Pederson, 1998; Politz et al., 2005), a number of HSV-1 proteins interact with the nucleolus (Leopardi and Roizman, 1996; Mears, Lam, and Rice, 1995; Morency et al., 2005). VP22, along with VP13/14 and US11, has been reported to bind RNAs in vitro (Roller and Roizman, 1990; Sciortino et al., 2002). US11 is another virion tegument protein which is known to localize to the nucleolus (MacLean, Rixon, and Marsden, 1987; Roller et al., 1996). These circumstantial observations imply that VP22 and GCT-VP22 may also be nucleolar-interacting proteins.

A key question is how VP22 and GCT-VP22 interact with marginalized chromatin and, perhaps, nucleoli. GCT-VP22 contains the pat4 nuclear localization sequence motif (Fig. 7) which we have suggested above plays a role in GCT-VP22’s enhanced nuclear accumulation in the presence of VP22. However, it has been reported that signals like pat4 overlap, in part, with nucleolar localization signals (Hiscox, 2002; Rowland and Yoo, 2003). In addition, signals for nucleolar localization are often linked to nuclear localization signals, forming “extended” motifs (Yamada et al., 1999). Thus, it is conceivable that the pat4 in GCT-VP22 is acting to target this GFP fusion protein to marginalized chromatin (and nucleoli). It remains to be determined whether this potential association with condensed chromatin is important in the process by which GCT-VP22 and VP22 become packaged into infectious virions.

Materials and methods

Cells and virus

African green monkey kidney (Vero) cells were obtained from the American Type Culture Collection and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% fetal bovine serum (5% FBS). V49 are VP22-expressing Vero cells (Pomeranz and Blaho, 2000) and were maintained in 5% FBS and containing G418 (1 mg/ml; Gibco-BRL). HSV-1(F) is the wild type virus strain used in this study. HSV-1(RF177), referred to as RF177, synthesizes a VP22 truncation protein (Δ212) and expresses GFP under control of a cytomegalovirus promoter (Pomeranz and Blaho, 2000). The RF177 stocks used in these studies were generated using Vero cells and are, therefore, virion-negative for full-length VP22. Virus stocks were prepared and titered as described previously (Pomeranz and Blaho, 2000).

Synchronized infections

Synchronized infections are defined as uniform staining in all cells in a microscopic filed at a given time post infection, as determined by indirect immunofluorescence with antibodies specific for unique HSV-1 polypeptides (Pomeranz and Blaho, 1999). Vero or V49 cells were incubated on ice at 4°C for at least 15 min prior to the addition of virus. The cells were then inoculated with virus (MOI of 15) at 4°C. After allowing the virus to adsorb for 1 h, the cells were rinsed once with 4°C phosphate-buffered saline (PBS) and warm (37°C) DMEM supplemented with 5% newborn calf serum (5% NBCS) was added, and the cells were incubated at 37°C for 18 h.

Infected whole cell extracts

Approximately 2 × 106 infected cells were harvested directly into the medium by gentle scraping, briefly pelleted by low speed centrifugation, and washed once in 140 mM NaCl, 3 mM KCl, 10 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.5 (PBS). Whole cell extracts were prepared by adding PBS containing 1% deoxycholate and 1% NP40 and the following protease inhibitors; 10 mM L-1-chlor-3-(4-tosylamido)-7-amino-2-heptanon-hydrochloride (TLCK), 10 mM L-1-chlor-3 (4-tosylamido)-4-phenyl-2-butanone (TPCK), and 100 mM phenylmethylsulfonyl fluoride (PMSF), followed by sonication using a Bronson sonifier. Protein concentrations of infected cell extracts were determined using a modified Bradford assay (Bio-Rad) according to the manufacturer’s specifications.

Virion preparations

Preparations of purified virions were performed exactly as described previously (Pomeranz and Blaho, 1999). Approximately 2 × 108 infected Vero cells (MOI = 5) were harvested directly in medium by gentle scraping at 24 hpi and pelleted by low speed centrifugation. Cells were swollen on ice prior to lysis using a Dounce homogenizer. Intracellular virions were purified from dextran T10 (Pharmacia) gradients and isolated virions were concentrated by high speed ultracentrifugation (180,000 × g). This procedure yielded approximately 106 PFU per ml of virion stock per ml. Virion preparations were tested for the absence of contaminating protein by immunoblotting for tubulin; no anti-tubulin immune reactivity was detected in any virion preparation.

Denaturing gel electrophoresis and immunoblotting

Equal amounts (50 μg) of infected-cell protein were separated in 15% SDS-polyacrylamide gels cross-linked with N, N′-diallyltartardiamide (DATD), electrically transferred to nitrocellulose, and probed with relevant primary antibodies. Horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (Amersham) secondary antibodies were diluted 1:1000 in PBS and incubated with the blots for 1 h. Specific viral bands were detected following development with enhanced chemiluminescence reagents (Amersham) or Lumi-light western blotting substrate (Roche Diagnostics). Alkaline phosphatase-conjugated goat anti-rabbit and anti-mouse IgG secondary antibodies (Southern Biotech) were used at 1:1000 in PBS containing 5% milk. Prestained molecular weight markers (Gibco-BRL) were included in all gels.

Immunological reagents

RGST49 is a rabbit polyclonal antibody specific for the amino terminal portion of the VP22 protein (Blaho, Mitchell, and Roizman, 1994; Pomeranz and Blaho, 1999) and was used at dilutions of 1:750 for immunofluorescence and 1:1000 for immunoblotting. Purified hybridoma 22-3 monoclonal antibody specific for the VP22 carboxy-terminus (Geiss et al., 2001) was used at dilutions of 1:1000 for immunoblotting and 1:100 for immunofluorescence. Polyclonal anti-GFP antibody (Clontech) was used at 1:4000 and monoclonal anti-GFP antibody (JL-8; BD Biosciences) was used at 1:1000 for immunoblotting. Monoclonal anti-ICP4 antibody H1114 (Goodwin) was used at 1:1000. Anti-nucleolin monoclonal antibody (C23; Santa Cruz Biotechnology) was used at 1:50 for indirect immunofluorescence; for this procedure, cells were blocked in 1.5% normal mouse IgG (Santa Cruz Biotechnology). Alexa568-conjugated anti-rabbit IgG and anti-mouse IgG (Molecular Probes) and fluorescein isothiocyanate-conjugated IgG (Sigma) were used at dilutions of 1:750 in 1% BSA for immunofluorescence.

Indirect immunofluorescence and live cell microscopy

Cells were prepared for indirect immunofluorescence by washing twice in PBS prior to fixation in 2.5% methanol-free formaldehyde (Polysciences, Inc.) for 20 min at room temperature. Next, the cells were washed twice again with PBS, permeablized with 100% acetone at −20°C for 4 min, rinsed twice again in PBS, and then blocked for at least 8.5 h at 4°C in 1% BSA containing 10 μg of pooled human Ig (mainly IgG) (Sigma) per ml. As noted above, with anti-nucleolin antibody, cells were incubated for 20 min in 1.5% mouse pre-immune serum. The cells were then rinsed twice in PBS and each primary antibody was added for 1 h. After extensive rinsing with PBS, the appropriate secondary antibody was added and incubated for an additional 1 h. Finally, the cells were preserved in ProLong Antifade reagent (Molecular Probes). Hoechst 33258 DNA dye (Sigma) was used for staining nuclei at a final concentration of 0.05 μg/ml. Cells were visualized with the appropriate filter on an Olympus IX70/IX-FLA inverted microscope with an attached Sony DKC-5000 digital camera linked to a PowerMac G3 and processed through Adobe Photoshop.

Sequence analysis and computer imaging

Amino acid alignments of VP22 homologues were performed by the ClustalW alignment algorithm using the MacVector sequence analysis application. A file containing the last 15 amino acids of VP22 was compared to all proteins in the SwissProt database. The following are relevant genome accession numbers: HSV-1, AAT11799; HSV-2, NP_044519; Varicellovirus (VZV), P09272. Immunoblots were digitized at 600 to 2,400 dots per inch using an AGFA Arcus II scanner linked to a Macintosh G3 PowerPC workstation. Raw digital images, saved as tagged image files (TIF) using Adobe Photoshop were organized into figures using Adobe Illustrator.

Acknowledgments

We thank Elise Morton for excellent cell culture maintenance and Jamie Yedowitz for expert assistance with indirect immunofluorescence. We thank Erin Peterson and Tom Moran for expert assistance in purifying the 22-3 monoclonal antibody from its hybridoma cells. We thank Lynda Morrison and Brian Geiss (St. Louis University) for helpful comments/proof-reading preliminary portions of this study. These studies were supported by grants from the U.S. Public Health Service (AI038873 and AI048582 to J.A.B.).

Footnotes

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