Dynamic Interactions of the UL16 Tegument Protein with the Capsid of Herpes Simplex Virus

The UL16 tegument protein of herpes simplex virus is conservedthroughout the herpesvirus family. It has been reported to becapsid associated and may be involved in budding by providingan interaction with the membrane-bound UL11 protein. UL16 hasbeen shown to be present in all the major locations that capsidsare found (i.e., the nucleus, cytoplasm, and virions), but whetherit is actually capsid associated in each of these has not beenreported. Therefore, capsids were purified from each compartment,and it was found that UL16 was present on cytoplasmic but notnuclear capsids. In extracellular virions, the majority of UL16(87%) was once again not capsid associated, which suggests thatthe interaction is transient during egress. Because herpes simplexvirus (HSV) buds into the acidic compartment of the trans-Golginetwork (TGN), the effect of pH on the interaction was examined.The amount of capsid-associated UL16 dramatically increasedwhen extracellular virions were exposed to mildly acidic medium(pH 5.0 to 5.5), and this association was fully reversible.After budding into the TGN, capsid and tegument proteins alsoencounter an oxidizing environment, which is conducive to disulfidebond formation. UL16 contains 20 cysteines, including five thatare conserved within a putative zinc finger. Any free cysteinesthat are involved in the capsid interaction or release mechanismof UL16 would be expected to be modified by N-ethylmaleimide,and, consistent with this, the amount of capsid-associated UL16dramatically increased when virions were incubated with thiscompound. Taken together, these data suggest a transient interactionbetween UL16 and capsids, possibly modified in the acidic compartmentof secretory vesicles and requiring a release mechanism thatinvolves cysteines.

INTRODUCTION

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INTRODUCTION

Herpesviruses are assembled with more than 40 different virallyencoded proteins that comprise three morphologically distinctstructures: the icosahedral nucleocapsid containing the viralDNA; the lipid envelope containing virus-encoded glycoproteins;and the tegument, the assortment of proteins between the nucleocapsidand the envelope. The specific mechanisms by which these variousviral proteins come together to drive the assembly and buddingprocesses are poorly understood.

The addition of tegument proteins to capsids during assemblyis widely viewed as an ordered process of adding "layers" ofprotein (38). In this model, some tegument is added in the nucleus,more in the cytoplasm, and the rest at the trans-Golgi network(TGN) where maturation budding occurs (39). The data presentedin this report challenge this view and suggest that the processis more dynamic than previously recognized. Specifically, theUL16 protein of herpes simplex virus (HSV) may be the firstexample of a tegument protein that associates with and releasesfrom capsids during egress through low-pH compartments of thecell.

The UL16 protein is conserved throughout the herpesvirus family(41, 45, 69). As measured by immunofluorescence, this proteinhas been found to be primarily nuclear at early times postinfection,colocalizing with the major capsid protein VP5 and the scaffoldprotein VP22a, suggesting that UL16 may interact with capsidsin the nucleus (41). The localization of UL16 changes to a mostlypunctate perinuclear region at later times postinfection (41,45). The actual function of this protein remains elusive, butit has been suggested that it participates in genome packagingand nucleocapsid maturation due to its weak association withDNA-containing type C capsids isolated from whole-cell lysates(45), its ability to bind single-stranded DNA in vitro (45),and its potential zinc finger domain (69).

More recently, UL16 has been identified as a binding partnerof another conserved tegument protein, UL11 (30). This is amyristylated and palmitylated protein that targets to Golgi-derivedvesicles in the absence of other viral proteins (29). Therefore,it is conceivable that the interaction between UL16 and UL11might participate in targeting nucleocapsids to the site ofmaturation budding, where the virus obtains its final envelope.Consistent with roles in maturation, Vero cells infected withUL16 or UL11 deletion mutants have defects in virus productioncompared to wild type (3, 4, 6, 22, 23, 33, 34, 54, 59, 60).

The goal of the experiments described here was to ascertainat which cellular sites UL16 interacts with nucleocapsids. Thedata reveal that UL16 is associated with capsids isolated fromthe cytoplasm but not with those from the nucleus of infectedcells. Moreover, very little UL16 remains capsid associatedfollowing envelope removal of extracellular virions. The amountof UL16 present on extracellular capsids is considerably increasedwhen virions are exposed to mildly acidic pH, and this associationis reversed when pH is returned to neutral. Since herpesvirusesbud into TGN-derived vesicles, which are thought to have anacidic pH (16, 19), this may represent a possible mechanismfor the association or release of UL16 from capsids. These findingsraise the likely possibility that other changes within the tegumentoccur during passage through low-pH compartments during virusegress.

MATERIALS AND METHODS

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MATERIALS AND METHODS

Virus and cells. Vero cells (ATCC CCL-81) were maintained in Dulbecco's modifiedEagle's medium (Invitrogen) supplemented with 5% fetal bovineserum, 5% bovine calf serum, penicillin, and streptomycin (Gibco15140-148). Confluent monolayers of Vero cells were infectedwith the KOS strain of HSV type 1 (HSV-1) (61). Following infection,cells were incubated in Dulbecco's modified Eagle's medium supplementedwith 2% fetal bovine serum, 25 mM HEPES buffer, glutamine (0.3µg/ml), penicillin, and streptomycin. The HSV-1 UL31 deletionmutant and complementing cell line (clone 7) that expressesthis protein were generously provided by Joel Baines (CornellUniversity) (28).

Antibodies. UL16-, UL11-, VP22-, and VP5-specific antibodies were producedin rabbits and have been described previously (29, 30, 37).Rabbit polyclonal antibodies were generated against purifiedglutathione transferase-UL21 and glutathione transferase-UL46antigens. Rabbit polyclonal VP16-specific antibodies were obtainedfrom Clontech (product number 3844-1). Monoclonal VP5 antibodies,8F5 and 5C, were generated against conformational epitopes onthe capsid surface (kindly provided by Jay Brown, Universityof Virginia) (64). Monoclonal antibodies were used to detectthe scaffold proteins VP21/VP22a (MCA406; Serotec, Washington,DC) (42). Rabbit polyclonal antibody raised against glycoproteingE was a kind gift from Harvey Friedman (University of Pennsylvania).

UL16 chimeras. Construction of plasmid pCMV.UL16-GFP (where CMV is cytomegalovirusand GFP is green fluorescent protein) used in this study hasbeen described previously (30). To express UL16 lacking theGFP tag, pCMV.UL16 was generated by removal of the GFP sequence.

Confocal microscopy. Vero cells were transfected with Lipofectamine 2000 accordingto the manufacturer's instructions (Invitrogen). At 20 to 22h posttransfection, cells were infected with HSV at a multiplicityof infection (MOI) of 5 PFU. At the various times postinfection,cells were fixed with 3% paraformaldehyde in phosphate-bufferedsaline (PBS) and then viewed by using a Zeiss laser scanningmicroscope with a helium-argon laser (488-nm peak excitation).

Capsid analyses. HSV capsids from the nucleus and cytoplasm were isolated asdescribed by Sherman and Bachenheimer with some modifications(58). Two 850-cm² roller bottles of confluent Vero cells wereinfected at an MOI of 10. At 16 to 20 h postinfection, cellswere scraped into 20 ml of PBS, collected by centrifugationat 1,000 x g for 10 min, resuspended in 6 ml of NP-40 lysisbuffer (0.5% NP-40, 150 mM NaCl, 1 M Tris-HCl, pH 8.0) containingprotease inhibitors (P8340; Sigma), and incubated for 15 minon ice. The cytoplasmic fraction was separated from the nucleiby centrifugation at 1,000 x g for 10 min. The nuclei were washedthree times and resuspended in 6 ml of NP-40 lysis buffer withprotease inhibitors. The separated cytoplasmic and nuclear fractionswere treated identically throughout the experiments. Capsidswere released from the purified nuclei by freezing (–80°C)and then thawing three times (37°C), followed by cup sonicationfor 3 min at moderate power. Insoluble material from the nuclearand cytoplasmic fractions was cleared by centrifugation at 8,000x g for 30 min. The capsids remaining in the soluble supernatantof the nuclear and cytoplasmic fractions were pelleted througha 1.7-ml 30% (wt/vol in TNE buffer [20 mM Tris-HCl, pH 7.6,150 mM NaCl, 1 mM EDTA]) sucrose cushion in a SW41 rotor at83,500 x g for 1 h. Pellets were then resuspended in 500 µlof TNE buffer, sonicated for 2 min at moderate power, and layeredonto a 20 to 50% (wt/vol, sucrose in TNE buffer) continuousgradient. Gradients were then centrifuged at 74,000 x g for1 h in an SW41 rotor. All centrifugation steps were carriedout at 4°C. Fractions of 750 µl were collected fromthe top of the gradient with a piston gradient fractionator(Brandel). Trichloroacetic acid (TCA) was added to a final concentrationof 13%, and the samples were incubated overnight at 4°C.The precipitated proteins were collected by centrifugation ina microcentrifuge at 18,000 x g for 30 min, washed with 100%ethanol, resuspended in sodium dodecyl sulfate-polyacrylamidegel electrophoresis (SDS-PAGE) sample buffer (3.5% SDS, 8.5%ß-mercaptoethanol, 130 mM dithiothreitol, 0.5 M urea,290 mM Tris-HCl, pH 8.8), and boiled for 15 min at 95°C.Samples were separated in SDS-polyacrylamide (10% or 7%) gelsand electrotransferred to nitrocellulose membranes. The enhancedchemiluminescence method of immunoblot analysis was performedaccording to the manufacturer's instructions (Amersham). Anti-UL16and anti-VP5 were used as the primary antibodies at dilutionsof 1:3,000 and 1:7,500 (in 5% nonfat milk in buffer containing20 mM Tris, pH 7.6, 135 mM NaCl, and 0.1% Tween-20), respectively.The positions of B and C capsids in the gradient were confirmedby identification of the light-scattering bands as previouslydescribed (17, 58) and by immunoblotting for the scaffold proteinsVP21 and VP22a (42).

Immunoprecipitation of capsids. Cells were infected at an MOI of 10. At 16 to 20 h postinfectioncells were washed with 5 ml of PBS, scraped into 1 ml of PBS,and collected by centrifugation at 1,000 x g for 10 min. Thecells were resuspended into 1 ml of NP-40 lysis buffer withprotease inhibitors and incubated for 15 min on ice. Whole-celllysates were generated by sonication for 3 min. For nuclearand cytoplasmic separation, nuclei were first isolated by centrifugationat 1,000 x g for 10 min and then washed three times with NP-40lysis buffer. Nuclei were lysed as described above. The lysateswere clarified by centrifugation at 8,000 x g for 30 min, andsupernatants were transferred to new tubes for immunoprecipitationanalysis. Capsids and their associated proteins were immunoprecipitatedwith monoclonal antibodies 8F5 and C5, which recognize epitopespresent on the mature capsid structure (9, 15, 36, 64). Immunoprecipitatedproteins were separated by electrophoresis in SDS-10% polyacrylamidegels. The gels were then electrotransferred to nitrocellulosemembranes and subjected to immunoblot analysis.

Analysis of UL16 in virions. Purification of extracellular virions was performed as previouslydescribed with some modifications (31). Briefly, confluent monolayersof Vero cells were infected at an MOI of 10 for 22 h, the mediumwas collected, and cell debris was removed by centrifugationfor 10 min at 1,000 x g. To isolate extracellular capsids, mediumfrom infected plates was treated with NP-40 (final concentration,0.5%) for 15 min at room temperature. Extracellular virionsor capsids (in 10 ml) were pelleted from the medium by centrifugationfor 1 h at 83,500 x g in an SW41 rotor through a 30% (wt/vol)sucrose cushion (1.7 ml). Virions or extracellular capsids wereseparated by SDS-PAGE in 7% polyacrylamide gels and analyzedby immunoblotting using VP5-, UL16-, and VP16-specific antibodies.

Trypsin treatment of virions. Extracellular virions harvested as described above were resuspendedin TNE buffer and separated into six equal fractions. Fractionswere treated as previously described with minor modifications(see Fig. 5) (68). Following treatment, virions and capsidswere repelleted through an additional sucrose cushion as describedabove. Pelleted virions and capsids were separated by SDS-PAGEin 10% gels and analyzed by immunoblotting using antibodiesspecific for VP5, VP16, and UL16.

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FIG. 5. Effects of low pH on the association of UL16 with capsids. Extracellular virions were harvested 20 to 24 h postinfection, pelleted through a 30% sucrose cushion, and resuspended in medium buffered to the indicated pH values. (A) After an incubation of 1 h at 37°C, the viral envelopes were removed with NP-40 treatment for 10 min at room temperature. The released capsids were pelleted, dissolved in sample buffer, and analyzed by immunoblotting with anti-VP5 and anti-UL16 antibodies. (B) Virions were first incubated at the indicated pH values for 30 min at 37°C and then incubated for an additional 30 min, either at the same pH or at pH 7.4 (following titration with NaOH). The viral envelopes were removed with NP-40, and the released capsids were collected and analyzed by immunoblotting. (C) Virions were treated as described for the previous panel except that the timing of the NP-40 treatment was either before or after neutralization with NaOH, as indicated by the asterisks. Relative levels of UL16 quantitated by densitometry and normalized for VP5 are indicated bellow each figure. ${alpha}$ , anti.

Low-pH treatment of extracellular virus. HSV pelleted through a sucrose cushion was resuspended in cellculture medium buffered with 5 mM HEPES (Sigma), 5 mM 2-(N-morpholino)ethanesulfonicacid (Sigma), and 5 mM succinate (Sigma) to achieve a pH rangingfrom 7.4 to 5.0. Following a 1-h incubation at 37°C, virionswere stripped of their membrane with NP-40 (0.5% final concentration)to release capsids. Virions or capsids were then pelleted bycentrifugation, and the levels of UL16 remaining capsid associatedwere determined by immunoblotting. In reversibility experiments,the acid pH was titrated back to neutral pH with 1 M NaOH eitherbefore or after NP-40 treatment.

NEM treatment of extracellular virions. Extracellular virions harvested at 18 to 24 h postinfectionwere treated with N-ethylmaleimide (NEM) for 30 min at 37°C,either before or after removal of the envelope with NP-40 (0.5%final concentration). A 10 µM final concentration of NEMwas used to attain rapid modification of free cysteines (5).Following NEM treatment, virions and capsids were purified bycentrifugation for 1 h at 83,500 x g, separated by SDS-PAGEin 7% or 10% gels, and analyzed by immunoblotting using antibodiesspecific for VP5, VP16, and UL16. Using densitometry, the percentagesof UL16 and VP16 remaining bound to capsids following removalof the envelope were determined by dividing the amounts of proteinpresent with capsids following envelope removal by the totalamounts present in the virion, all normalized to VP5 levels.

RESULTS

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RESULTS

The conserved UL16 protein is of interest because it may providea physical link between the capsid and the membrane-bound UL11protein during HSV budding. However, very little is known regardingthe capsid association properties of UL16. This protein hasbeen reported to be present at all locations where capsids arefound (the nucleus, cytoplasm, and virion), but whether it isactually capsid associated in all these locations has neverbeen shown for any herpesvirus. Therefore, the following experimentswere pursued.

Subcellular localization of UL16-GFP in infected cells. Previous immunofluorescence studies suggested that UL16 accumulatesin the nuclei of infected cells up to 18 h postinfection butat later times is localized to punctate, cytoplasmic structures(41). Moreover, the early nuclear staining colocalized withthat of capsid proteins VP5 and VP22a, raising the possibilitythat UL16 is added to capsids in the nucleus. In an attemptto confirm the localization properties of UL16 during the courseof an infection, a transfection-infection assay was employed.Vero cells transfected with a plasmid that expresses UL16-GFPwere subsequently infected with HSV and visualized by confocalmicroscopy at various times postinfection. The GFP chimera wasfound in both the cytoplasm and nucleus early during the infection,similar to results in noninfected (transfected only) cells (Fig.1A). More intense nuclear fluorescence was observed until 18h postinfection (Fig. 1A and data not shown) and preceded expressionof the virus-encoded UL16, first detected at $~$ 8 h (Fig. 1B).Therefore, any viral factors responsible for nuclear retentionmust have been expressed earlier, which is consistent with thesuggestion that UL16 might have a role in DNA replication orpackaging (45). Localization of UL16-GFP changed dramaticallyat later times postinfection to cytoplasmic regions surroundingthe nucleus of infected cells with very little nuclear fluorescence(Fig. 1A). Thus, an assay that does not require the use of antiserumconfirmed the nuclear trafficking properties of UL16 and raisedthe possibility that this protein would be present on nuclearcapsids.

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FIG. 1. Change in UL16-GFP localization during the course of infection. (A) Vero cells were transfected with pUL16-GFP. At 20 to 22 h posttransfection, cells were infected with HSV, fixed with paraformaldehyde at the indicated times postinfection, and viewed by confocal microscopy. T.O., transfected only. (B) Vero cells were infected with HSV, and at the indicated times postinfection, they were harvested and resuspended in sample buffer. Proteins were separated by SDS-PAGE in 10% gels, transferred to nitrocellulose, and detected by immunoblotting with a polyclonal antibody raised against UL16.

Association of UL16 with capsids. To test the prediction, capsids were released from the nucleiof detergent-disrupted, HSV-infected cells by freeze-thaw cyclingand sonication. Capsids from this extract were first pelletedthrough a sucrose cushion and then sedimented in a sucrose gradient.Fractions were collected from the top, and proteins in eachwere precipitated with TCA prior to SDS-PAGE and immunoblottingwith antibodies specific for UL16 and the VP5 capsid protein.Despite the large amount of UL16 present in the nucleus at 18h postinfection, this protein was not found to be associatedwith the purified, intranuclear capsids (Fig. 2B). Indistinguishableresults were obtained when intranuclear capsids were harvestedat 16 h postinfection (data not shown). In contrast, UL16 wasreadily detected as a species cosedimenting with the heterogeneouspopulation of capsids isolated from the cytoplasm using identicalbuffer and detergent conditions (Fig. 2C). Capsids isolatedfrom the cytoplasm are thought to contain more tegument thanthose isolated from the nucleus, and this likely accounts forthe sedimentation differences shown in panels B and C of Fig.2. The additional tegument would increase the size of the particles,and therefore decrease the density, causing them to sedimentat a slower rate in the gradient. When UL16 was expressed aloneby transient transfection and subjected to sucrose gradientcentrifugation, it was found in the top fractions (Fig. 2A),suggesting that it cannot form large aggregates on its own.

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FIG. 2. Cosedimentation of UL16 with cytoplasmic but not nuclear capsids. Vero cells were transfected with pUL16 (A) or infected with HSV at an MOI of 10 (B to D) for 16 to 22 h (representative 18-h time point shown). Detergent lysates of the transfected cells were analyzed directly in a 20 to 50% (wt/vol) sucrose gradient whereas nuclear and cytoplasmic capsids from the infected cells were first pelleted through a 30% sucrose cushion prior to sedimentation. Fractions were collected, and proteins were concentrated by TCA precipitation. The precipitates were dissolved in sample buffer and separated either in a 7% (for detection of VP5) or 10% (for UL16) polyacrylamide-SDS gel prior to immunoblot analyses with anti-VP5 or anti-UL16 rabbit polyclonal antibodies. The locations of B and C capsids in the gradients are indicated. (D) To compare the levels of UL16 present on equal amounts of nuclear (Nuc) and cytoplasmic (Cyt) C capsids, the relevant species were collected from the gradients, repelleted through a 30% sucrose cushion, and resuspended in sample buffer. Undiluted (Undil) or 1:10 dilutions of the samples were analyzed by immunoblotting with anti-VP5 and anti-UL16 sera. ${alpha}$ , anti.

Most of the capsids in the nucleus are of the B rather thanthe mature, DNA-containing, export-competent C form (58), andif UL16 was associated only with the latter, then it might bedifficult to detect. Therefore, C capsids from nuclei and cytoplasmwere purified from a sucrose gradient and diluted so that equalamounts of VP5 from each compartment could be compared. Again,UL16 was readily detectable on the capsids from the cytoplasmbut not those from the nucleus, even though it was present inthe nuclear compartment (Fig. 2D). When quantitated by densitometry,the amount of UL16 normalized for VP5 levels was 40-fold higheron cytoplasmic capsids. Moreover, UL16 was not detected on intranuclearcapsids produced by a mutant defective for UL31 expression (datanot shown), which accumulates C capsids in the nucleus (8).Thus, UL16 copurifies with C capsids isolated from the cytoplasmof infected cells but not those from the nucleus.

Although the sedimentation pattern of UL16 suggests that itis physically associated with capsids isolated from the cytoplasm,it is possible that the protein is part of a large cytoplasmiccomplex that just happens to purify with capsids. To addressthis, capsids from HSV-infected cell lysates were immunoprecipitatedwith two different monoclonal antibodies that recognize VP5epitopes present only on the mature capsid surface (9, 15, 36,64). As a negative control, an irrelevant mouse monoclonal (anti-humanimmunodeficiency virus) antibody was used. Subsequent immunoblotassays showed that UL16 is indeed associated with capsids immunoprecipitatedfrom whole-cell lysates (Fig. 3A). As expected, envelope glycoproteingE and "outer" tegument proteins UL11 and VP22 (all of whichare released from virions in the presence of NP-40) did notcoimmunoprecipitate with capsids in these experiments (datanot shown). Moreover, the monoclonal antibodies did not immunoprecipitateUL16 from transfected cells (data not shown).

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FIG. 3. Coimmunoprecipitation of UL16 with cytoplasmic but not nuclear capsids. Vero cells were infected with HSV, and viral proteins were immunoprecipitated (IP) 18 h later from whole-cell lysates (A) or nuclear (Nuc) and cytoplasmic (Cyt) fractions (B) by using monoclonal antibodies 8F5 or 5C, which are specific for VP5 epitopes present only on mature capsids. A monoclonal antibody specific for human immunodeficiency virus (HIV) p24 was used as a negative control. The immunoprecipitated capsid and associated proteins were separated in an SDS-10% polyacrylamide gel and analyzed by immunoblotting with polyclonal rabbit serum specific for VP5 or UL16. WB, Western blotting; ${alpha}$ , anti.

To further test the hypothesis that UL16 is associated onlywith cytoplasmic capsids, the immunoprecipitation analysis wasrepeated on capsids from nuclear and cytoplasmic fractions.UL16 was easily detected on capsids isolated from the cytoplasmbut was not found associated with capsids from the nucleus,even though it was present in this compartment (Fig. 3B). Whilethe experiments described here clearly demonstrate that UL16is present on at least some of the capsids present in the cytoplasm,the results do not rule out the possibility that UL16 is weaklyassociated with nuclear capsids or is added just as the capsidsexit the nucleus, events that would make detection very difficult.

Analysis of UL16 in virions. Previous studies suggested that UL16 is associated with HSV-1(41) but not HSV-2 particles (45). To rigorously address whetherUL16 is inside the virion and capsid associated, a trypsin sensitivityassay was used (Fig. 4A). UL16 is sensitive to trypsin (datanot shown) but was found to be present in a form that was protectedfrom trypsin cleavage unless the envelope was removed by NP-40treatment, confirming that it was inside the virion. However,very little UL16 pelleted with extracellular capsids, even whentrypsin was not present. Quantitation by densitometry of fourindependent experiments showed that only 13% ± 6% ofUL16 remained associated with capsids after NP-40 treatment.

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FIG. 4. UL16-capsid interactions in virions. (A) Extracellular virions were harvested from HSV-infected Vero cells and pelleted through a 30% sucrose cushion. The samples were resuspended in TNE buffer, separated into six equal fractions, and treated as indicated for 15 min at room temperature. Following treatment, virions and capsids were pelleted through an additional sucrose cushion, resuspended in sample buffer, and separated by SDS-PAGE in 7% gels. Proteins were analyzed by immunoblotting using antibodies specific for VP5, VP16, and UL16. (B) Capsids were isolated from NP-40-treated cytoplasmic (cyt) lysates or extracellular medium by centrifugation through a 30% sucrose cushion. Capsid pellets were resuspended in sample buffer, and two different amounts were analyzed by immunoblotting using antibodies specific for VP5 and UL16. ${alpha}$ , anti; Tryp, trypsin; Inh, inhibitors; Undil, undiluted.

If the UL16 interaction is transient during egress, then capsidsisolated from the cytoplasm might contain more UL16 than thosefrom extracellular virions. Comparisons of equal amounts ofcapsids purified from the cytoplasm and virions (normalizedfor VP5) revealed similar levels of UL16 (within a twofold difference)(Fig. 4B); however, the interpretation of this result is difficultbecause the cytoplasm contains capsids in all stages of assembly:as they exit the nucleus, traverse the cytoplasm, bud at internalmembranes, and travel in vesicles to the cell surface for release.The low levels of capsid-associated UL16 present in extracellularvirions ( $~$ 13%) may be an indication that the majority of thisprotein ( $~$ 87%) is packaged via another mechanism. Alternatively,these results may be indicative of a transient capsid associationthat is subsequently reversed to release the majority of UL16into the "outer" virion space at some stage following budding(see Discussion).

Low-pH treatment of extracellular virions. HSV is thought to bud into the TGN, which has a slightly acidicpH of 6.0 (16, 19). During transit to the cell surface, thepH of the secretory vesicles continues to drop to $~$ 5.5 (11).Since low pH is known to trigger conformational changes in manyproteins (e.g., the hemagglutinin of influenza virus) (13, 53),it is possible that HSV might utilize this environment to triggervarious maturation events, including release of UL16 from capsids.With this hypothesis in mind, extracellular virions were collectedand incubated in medium buffered at various pH values. NP-40was used to solubilize the virus envelope, and the capsids werethen pelleted through a sucrose cushion. The amount of UL16copelleting with capsids increased as the pH of the medium wasmade more acidic (Fig. 5A).

Because low pH might alter the structure of UL16—causingit to precipitate and pellet nonspecifically with capsids—thereversibility of the reaction was examined. Precipitated proteinsoften have difficulty resolubilizing, but this was not the casewith UL16, which was completely released following neutralization(Fig. 5B). Moreover, the viral membrane was not needed for reversibility(Fig. 5C). Pelleting the pH 5.0-treated capsids through a sucrosecushion gave similar results whether the cushion had a pH of5.0 or 7.4. Presumably, this is because the cushion is small,and the speed of sedimentation through that cushion is fast.These data provide evidence that dynamic changes may occur insidethe virions as they travel through the secretory vesicles enroute to the extracellular medium.

NEM treatment of virions. Upon completion of budding, the capsid and other internal componentsof the virion first encounter an oxidizing environment, whichpromotes disulfide bond formation (14, 65). Because UL16 contains20 cysteines, 5 of which are conserved in a putative zinc finger(69), it seemed possible that one or more of these might beimportant for capsid association or release, perhaps as partof a disulfide-exchange reaction (18, 20, 27, 35, 40). Freecysteines are covalently modified when exposed to NEM, a group-specificchemical that has been used extensively in the study of proteinstructure and function (5, 7, 26, 56, 70). To examine the possibleinvolvement of cysteines in the UL16-capsid interaction, extracellularvirions were treated with NEM at 37°C. A dramatic increasein the amount of UL16 that copellets with capsids was observed(Fig. 6A). This was not observed with other tegument proteinstested: VP16, UL11, UL21, and UL46, which contain 6, 5, 5, and11 cysteines, respectively (Fig. 6A and data not shown). Thecorresponding increase in apparent molecular mass of UL16 isindicative of multiple NEM modifications, which are not sensitiveto reducing agents in sample buffer or to boiling prior to electrophoresis.NEM has a molecular mass of 125 Da, and the magnitude of theshift in mass of $~$ 1,500 Da suggests that approximately half ofthe 20 cysteines are modified. In several repeats of the experiment,the amount of capsid-associated UL16 within the virion (normalizedto VP5) ranged between 68 to 85% following NEM treatment, whereasthe amount was only 3 to 15% without NEM (a representative resultis shown in Fig. 6B). At the concentration of NEM used, thetiter of extracellular virions was reduced 4 orders of magnitudeafter 10 min of treatment at 37°C (data not shown), whichunfortunately provides little insight because the drug can potentiallyact on many viral proteins.

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FIG. 6. Effects of NEM on the UL16-capsid interaction. (A) Extracellular virions were treated with or without NEM at 37°C. Viral membranes were removed with NP-40 treatment, and the capsids were pelleted through a 30% sucrose cushion prior to immunoblot analyses with antibodies specific for VP5, VP16, and UL16. (B) Extracellular virions were treated with or without NEM for 30 min and then treated with or without NP-40. The released capsids or intact virions (C or V, respectively) were pelleted through a 30% sucrose cushion prior to immunoblotting with the indicated antiserum. (C) Virions present in the medium were concentrated by centrifugation through a sucrose cushion and resuspended in a small volume of TNE buffer. The sample was divided into two equal portions, and these were treated with or without NEM for 30 min. Both samples then received NP-40 for 15 min at room temperature prior to sedimentation through parallel 20 to 50% sucrose gradients. Fractions were collected from the top and analyzed by immunoblotting with antibodies specific for VP5 and UL16. (D) Gradient-purified capsids treated with NEM or carrier prior to lysis for 15 min at 37°C from infected cytoplasm and medium were analyzed by immunoblotting for levels of VP5 and UL16. Gradients were run in duplicate. ${alpha}$ , anti.

To determine whether NEM-modified UL16 is capsid associatedrather than present in an aggregate or large complex of anothersort, the samples were analyzed in a sucrose gradient followingsolubilization of the viral membrane with NP-40. In the absenceof NEM treatment, all detectable UL16 remained at the top ofthe gradient while the capsids moved toward the bottom (Fig.6C). In contrast, treatment with NEM resulted in all detectableUL16 cosedimenting with capsids. Attempts to demonstrate theUL16-capsid association in virions using monoclonal antibodiesspecific for VP5 (as described in the legend of Fig. 3) werenot possible, presumably because the presence of tegument proteinsthat blocked epitope recognition, with the result that lessthan 10% of all extracellular capsids were immunoprecipitatedfrom NP-40-treated virions (data not shown).

Because infectious virions can be obtained from the cytoplasmfollowing disruption of infected cells at neutral pH, it seemedlikely that the levels of UL16 associated with cytoplasmic capsidswould increase if the cells were pretreated with NEM. This wasfound to be the case, as the quantity of UL16 present on cytoplasmiccapsids (normalized to VP5) was fourfold higher if cells weretreated with NEM prior to lysis (Fig. 6D). This is likely dueto the action of NEM on virions present within secretory vesiclesbecause NEM did not cause UL16 to associate with intranuclearcapsids (data not shown).

DISCUSSION

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DISCUSSION

The experiments described here provide a detailed analysis ofthe interaction of UL16 with capsids. The major finding is thatthe interaction is dynamic, with a binding and release mechanismthat is pH regulated and likely involves cysteines. To our knowledge,this report provides the first evidence for a transient interactionof a tegument protein with a herpesvirus capsid during egress.However, the possibility that virus maturation events mighttake place as the virion travels through low-pH compartmentswas first suggested by the discovery that neutralization ofcellular organelles blocks production of infectious HSV, eventhough budding still occurs (19). It is likely that other maturationevents will be found to occur as the virion travels the egresspathway. The particular insights obtained regarding UL16 arediscussed below.

Nuclear localization of UL16. UL16-GFP was found to localize to the nucleus, even in the absenceof the other HSV proteins. Because this 69-kDa chimera is predictedto be too large for passive entry through the nuclear pore complex(12), it presumably contains a nuclear localization signal orinteracts with a host protein that enables its transport. Inthe context of an infection, there is an increase in the amountof UL16 (41) or UL16-GFP (this study) present in the nucleus;however, the interactions responsible for nuclear retentionremain unknown.

What is the function of UL16 in the nucleus? This tegument proteincontains a putative zinc finger (60) and has been shown to bindsingle-stranded DNA in vitro (45), suggesting that it may associatewith the DNA packaging machinery along with UL6, UL15, UL17,UL28, UL32, and UL33 (1, 2, 10, 24, 25, 46, 47, 52, 57, 63,67, 71). If so, then UL16 would be predicted to be associatedwith procapsids, which would not have been detected in the experimentsdescribed here because they are unstable, present in low numbers,and difficult to isolate (43, 50, 55). An association with procapsidswould also be consistent with the observed colocalization ofUL16 with capsid and scaffold proteins (41). To gain furtherinsight, studies to identify the binding partners of UL16 inthe nucleus are warranted.

Whatever its function in the nucleus, it is clear that UL16is not stably present on the C capsids isolated from this compartment.Moreover, it was not detected on capsids made by a UL31-nullvirus (data not shown), which accumulate in the nucleus becauseof a defect in nuclear egress (8). It remains possible thatUL16 associates with nuclear capsids, but this interaction cannotbe detected under the lysis conditions used in this study becausethe association is weaker than that with cytoplasmic capsids.Also, UL16 might be added to capsids just as they bud into—orexit from—the perinuclear space, but the capsids presentin that compartment at any given time are few in number andwould not be easily detected in the experiments described here.In any case, it seems likely that the function of UL16 in thenucleus is not required for the packaging of this tegument proteininto the mature virion. Therefore, it may be possible to findUL16 mutants that are packaged even though they fail to enterthe nucleus.

Mechanism of UL16 packaging. There are at least two potential mechanisms for the packagingof UL16 into virions. In the first, UL16 binds to C capsidsprior to their arrival at the TGN for maturation budding, amodel that is consistent with (but not proven by) the data presentedhere. From this point of view, it is particularly interestingthat antibodies specific for mature capsids were unable to efficientlyrecognize their epitopes when extracellular virions were solubilizedwith NP-40 (data not shown) under conditions that readily enabledrecognition of capsids from the cytoplasm (and nucleus). Ifantibody access is blocked following tegumentation, then thecapsids that are immunoprecipitated from the cytoplasm withUL16 attached probably represent intermediates en route to theTGN. In this model, subsequent interaction of UL16 with membrane-boundUL11 would provide a bridging function to promote budding (30).

In the other mechanism of packaging, UL16 travels to the TGNindependently of capsids, presumably by means of its interactionwith UL11 (30). When capsids arrive at the membrane, their bindingto the UL16-UL11 complex would then promote budding. If thismodel is correct, then the population of cytoplasmic capsidsthat has UL16 bound would be the capsids that have already enteredthe postbudding pathway. Because virions in this pathway havereceived their full complement of tegument proteins, they wouldnot be expected to react with the antibodies, which argues againstthis model. However, most of the UL16 in extracellular virions( $~$ 85%) was found to be free of capsids upon treatment with NP-40,and this raises the possibility that some is packaged withoutthe need for that interaction. Alternatively, it is possiblethat all UL16 molecules are transiently associated with capsidsduring transport to the TGN, with most being released by thetime the virion leaves the cell. The dramatic and reversiblechanges in UL16 binding that occur in response to pH clearlysupport the transient association model.

To gain further insight on the packaging mechanism, it willbe important to identify the protein that links UL16 to thecapsid. One candidate is UL21, a capsid-associated tegumentprotein that has recently been identified as a binding partnerof UL16 in pseudorabies virus (21, 66). The HSV homolog of UL21has been reported to interact (perhaps indirectly) with microtubules(62); hence, it is reasonable to speculate that UL16 and UL21might be involved in transporting capsids to the TGN. Subsequentrelease of the complex would prevent capsids from reenteringthe egress pathway when the next cell is infected.

Dynamic interaction of UL16 with capsids. Two entirely different methods were found for increasing theamount of UL16 that is associated with extracellular capsids(low pH and NEM), and the observations made with these provideevidence to suggest that the interaction is transient duringcapsid egress. Nevertheless, there is a fundamental but difficultquestion that remains unanswered: Are the UL16 molecules inan untreated virion actually bound to the capsid and merelyreleased upon exposure to NP-40? If so, then it still must bethe case that the structure of UL16 in extracellular virionsis different from that on cytoplasmic capsids because the sameNP-40 buffer conditions were used to isolate both forms.

It might be possible to ascertain the capsid association stateof UL16 in virions through the use of chemical cross-linkers;however, such experiments are complicated by the many cysteinesin this protein and their potential for creating abnormal disulfidebond linkages following virion disruption. NEM is often usedto block any free cysteines that are present, and one interpretationof the data presented here is that modification of UL16 blocksa cysteine exchange reaction that is needed for release (andtriggered by NP-40). However, it is possible that NEM changesthe conformation of free UL16 so that it becomes capsid associatedin the extracellular virion. If so, then the conformation ofUL16 in extracellular virions must still be different from thatin the cytoplasm, where capsid association was readily detectedeven in the absence of NEM. Moreover, NEM did not stimulateinteraction with capsids in the nucleus.

The effects of pH on the binding of UL16 to capsids does notshed light on the native state of UL16 within the virion. Thatis, low pH might prevent an NP-40-induced conformational changethat releases UL16, or it might cause UL16 to revert to a conformationthat once again becomes competent for capsid binding. However,it seems more likely that treatments as different as pH andNEM would preserve an interaction rather than induce the particularconformation needed for capsid binding. Moreover, the UL16 homologof human CMV (UL94) has been reported to be associated withextracellular capsids following NP-40 treatment (69), and thusthat virus appears to lack the release mechanism of HSV. Inany case, the reversibility of the pH effect does argue stronglythat UL16 is not merely precipitated upon treatment. Moreover,UL16 was readily found to be associated with capsids from thecytoplasm at neutral pH, and this once again argues that thestructure of this protein is very different by the time thevirion is released from the cell.

Based on all available data, we hypothesize that UL16 is stablyadded to capsids prior to their arrival at the site of buddingin the cytoplasm. Subsequently, something destabilizes thiscapsid interaction, but the mechanism is unknown. Treatmentswith NEM or low pH may provide clues as to what is involved(i.e., cysteine residues and transit through acidic compartmentsof the cell). Whatever the mechanism, it is clear that mostof the UL16 in extracellular virions is not stably associatedwith capsids. Although the data presented here demonstrate apH-dependent, reversible stabilization of UL16 with extracellularcapsids, it is important to understand that in vitro treatmentwith low pH does not restore UL16 to the stable state foundon cytoplasmic capsids. That is, when the pH is returned topH 7.4, the stable interaction of UL16 with extracellular capsidsis lost (unlike UL16 on cytoplasmic capsids, which are isolatedat this pH). The inability to "reset" UL16 to its cytoplasmicstate may be an indication that additional maturation eventsoccur among tegument proteins during capsid egress.

There are many examples of viral maturation events that occurwithin the Golgi complex and secretory vesicles (32, 44, 49,51). Most recently, structural changes in the West Nile viruscapsid have been determined by cryo-electron microscopy as thevirus particles travel through the low-pH environment of theTGN (72). Neutralization of organelle pH with ammonium chloridecauses these immature particles to accumulate within the cell(48). The idea that herpesviruses could be utilizing the acidicpH of the secretory vesicles in a similar fashion to bring aboutchanges in the tegument necessary for infectivity seems highlylikely (19).

ACKNOWLEDGMENTS

We extend special thanks to our colleagues Richard Courtney,Michael Murphy, Kevin O'Regan, and Nicholas Baird for helpfuldiscussions and to Rebecca Craven for careful review of themanuscript. In addition, we thank Michelle Bucks and Jacob Marshfor technical help on the capsid isolation protocol and theNEM infectivity experiments, respectively.

This work was supported by NIH grants to J.W.W. (CA47482, AI071286)and in part by a training grant from the National Cancer Institute(CA60395).

FOOTNOTES

* Corresponding author. Mailing address: Department of Microbiology and Immunology, The Pennsylvania State University College of Medicine, 500 University Drive, P.O. Box 850, Hershey, PA 17036. Phone: (717) 531-3528. Fax: (717) 531-6522. E-mail: jwills{at}psu.edu

Published ahead of print on 12 September 2007.

REFERENCES

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