Structural Rearrangement within an Enveloped Virus upon Binding to the Host Cell

We have made the surprising discovery that the interactionsof herpes simplex virus with its initial cell attachment receptorinduce a rapid and highly efficient structural change in thetegument, the region of the virion situated between the membraneand the capsid. It has been known for nearly a decade that virusescan trigger host signaling pathways when they bind to receptorson the cell surface; however, until now there has been no evidencethat a signal can be sent in reverse—from the "outsidein"—across a viral membrane. Evidence for this signalingevent was found during studies of UL16, a tegument protein thatis conserved among all the herpesviruses. Previous work hasdemonstrated that UL16 is bound to capsids isolated from thecytoplasm of infected cells, but this interaction is destabilizedduring subsequent egress steps, leading to release of the extracellularvirion. Pretreatment with N-ethylmaleimide, a small, membrane-permeatingcompound that covalently modifies free cysteines, restabilizesthe interaction, thereby permitting the capsid-UL16 complexto be isolated following disruption of virions with NP-40. Inthe experiments described here, we found that the natural signalfor release of UL16 from capsids is sent when virions merelybind to cells at 4°C. The internal change was also observedupon binding to immobilized heparin in a manner that requiresviral glycoprotein C. This represents the first example of signalingacross a viral envelope following receptor binding.

INTRODUCTION

Top

INTRODUCTION

It is well established that viruses can trigger cellular signalingpathways when they bind to their receptors on the plasma membrane(reviewed in reference 26). For example, binding can inducevirion movement across the cell surface, trigger polymerizationof cortical actin filaments needed for endocytic uptake, andmodify host gene expression to make the cellular environmentmore conducive for replication. It is also quite clear thatbinding triggers changes in the virion proteins that directlyinteract with the receptor, namely, the capsids of nonenvelopedviruses (44) and the glycoproteins found on the surfaces ofenveloped viruses (10). In contrast, there has been no evidenceto suggest that signals are transmitted in reverse—acrossthe membrane to the interior of any enveloped virus—uponreceptor binding. Here we report that the interaction of herpessimplex virus (HSV) with its initial cell attachment protein(heparan sulfate) induces a rapid and highly efficient structuralchange in the tegument, the large proteinaceous region situatedbetween the viral membrane and the DNA-containing capsid.

Our evidence for receptor-induced changes in the tegument wasfound during studies of UL16, a tegument protein that is conservedamong all herpesviruses (47). The function of this protein ispoorly understood, but based on its stable association withcytoplasmic capsids (27, 35) and its ability to interact witha membrane-bound tegument protein named UL11 (24), we proposedthat UL16 might provide a "bridging" function during virionbudding events at the trans-Golgi network (24). More recentlywe found that the UL16-capsid interaction changes during virionegress from the cell. Specifically, very little of the UL16present in extracellular virions was found to be capsid associated,even though the detergent-lysis conditions were the same asthose used to isolate capsids from the cytoplasm (27).

Although the previous study would seem to suggest that UL16is free from capsids in extracellular virions, there are twoobservations that made us suspect otherwise (27). First, studiesof other herpesviruses have revealed that their homologs ofUL16 are associated with capsids in detergent-disrupted, extracellularvirions (21, 47, 52). Second, we found that UL16 is almost entirelycapsid associated when extracellular virions are pretreatedwith either N-ethylmaleimide (NEM) or low pH, conditions thatblock the reactivity of "free" cysteine residues (27). Thus,it seems more likely (but still not proven) that treatmentsas different as pH and NEM would preserve an existing capsidinteraction rather than inducing UL16 into a particular conformationthat just happens to reenable capsid binding.

In the experiments described below, we show that following thebinding of HSV to host cells at 4°C, the interaction ofUL16 with the capsid was no longer detected when NEM was added.This change in the interior of the virus was also observed uponbinding to immobilized attachment receptors (i.e., heparin,a surrogate for heparin sulfate) and required the viral glycoproteinC (gC). Taken together, the data presented here represent thefirst evidence for the transmission of a signal across a virusenvelope as a result of glycoprotein-receptor interactions.

MATERIALS AND METHODS

Top

MATERIALS AND METHODS

Virus and cells. Vero and HeLa cells were maintained in Dulbecco's modified Eagle'smedium (Invitrogen) supplemented with 5% fetal bovine serum,5% bovine calf serum, penicillin, and streptomycin. Virus stockswere prepared by infecting confluent monolayers of Vero cellswith the KOS strain of HSV type 1 or the Becker strain of pseudorabiesvirus (PRV) at a multiplicity of infection of 0.01 for 1 h.Following infection, cells were incubated in Dulbecco's modifiedEagle's medium supplemented with 2% fetal bovine serum, 25 mMHEPES buffer, glutamine (0.3 µg/ml), penicillin, and streptomycinuntil a complete cytopathic effect was visualized ( $~$ 3 days).Cells were then scraped into the medium, frozen (–80°C)and thawed (37°C) three times, and then cup sonicated (4°C)at moderate power for three 1-min pulses. Cell debris was removedby centrifugation, and aliquots were then frozen at –80°Cuntil use.

Virus mutants. The gC-null mutant and revertant (gC-R) used in these studieswere propagated in Vero cells as described above (kindly providedby Harvey Friedman [13]). The gB-null mutants (KO82 and F-BACgB–) and complementing cells (VB38) used for their growthwere generously supplied by David Johnson (12). To generatevirions lacking gB, progeny of the gB-null viruses were obtainedfollowing infection of noncomplementing Vero cells. The ${Delta}$ UL16mutant used in the supporting studies (see the supplementalmaterial) was a gift from Joel Baines and has been describedpreviously (4).

NEM treatment of herpesviruses. Extracellular virions harvested at 18 to 24 h postinfectionwere treated with 10 mM NEM for 30 min at 37°C, either beforeor after removal of the envelope with NP-40 (final concentration,0.5%; I3021; Sigma). Previous work revealed that this concentrationand time of treatment efficiently modify free cysteines (5),and in our system they also maintain the UL16-capsid interactionin virions (27). Following NEM treatment, virions and capsidswere pelleted at 83,500 x g for 1 h and the samples were separatedin sodium dodecyl sulfate (SDS)-10% polyacrylamide gels andelectrotransferred to nitrocellulose membranes. The enhancedchemiluminescence method of immunoblot analysis was performedaccording to the manufacturer's instructions (Amersham). Rabbitanti-HSV UL16 and anti-VP5 sera were used at dilutions of 1:6,000and 1:7,500, respectively. PRV UL16-specific antibodies werekindly provided by Thomas Mettenleiter and used at a dilutionof 1:15,000.

Virus-cell binding assay. Confluent monolayers of cells on 100-mm plates were incubatedwith 1 ml of virus stock ( $~$ 5 x 10⁸ PFU) at 4°C with rockingfor 45 min to allow virus binding. NEM was added to the viruseither prior to or after incubation with the cells for 30 minat 4°C. Unbound virus and residual NEM were washed off thecells with 5 ml of phosphate-buffered saline (PBS). Viral membranesthat remained bound to cells were solubilized with 4 ml of NP-40lysis buffer (0.5% NP-40, 150 mM NaCl, 50 mM Tris-HCl [pH 8.0])for 15 min at 37°C. The lysates were removed from the dishby pipetting, and insoluble material and cellular debris wereremoved by centrifugation for 10 min at 3,829 x g. Capsids inthe supernatant were pelleted through a 700-µl 30%-sucrosecushion (wt/vol in TNE [20 mM Tris-HCl {pH 7.6}, 100 mM NaCl,1 mM EDTA]) in a SW55ti rotor at 83,500 x g for 1 h. Pelletswere dissolved in SDS sample buffer (2% SDS, 62.5 mM Tris-HCl,10% glycerol, 0.025% bromophenyl blue, 50 mM dithiothreitol[DTT], and 5% β-mercaptoethanol [BME]), boiled, separatedby SDS-polyacrylamide gel electrophoresis (PAGE) in 10% gels,and analyzed by immunoblotting using antibodies specific forVP5 and UL16. The amount of UL16 was determined by densitometryand normalized for VP5 levels.

Virus-heparin binding. Virus stocks ( $~$ 1 x 10⁸) were incubated with NEM for 30 min at37°C either before or after a 30-min incubation with 40µl of heparin agarose slurry (20207; Pierce). Followingincubations, the virus-coated beads were pelleted by centrifugationfor 30 s in a microcentrifuge, washed in 1 ml of PBS, and resuspendedin 1 ml of NP-40 lysis buffer for 15 min at 37°C. Beadsand insoluble material were pelleted for 4 min at 18,000 x g.Capsids remaining in the supernatant were pelleted through a150-µl 30%-sucrose cushion for 1 h in a TLA 100.3 rotorat 83,500 x g for 1 h. Pellets were dissolved in sample buffer,boiled, separated by SDS-PAGE in 10% gels, and analyzed by immunoblottingusing antibodies specific for VP5 and UL16. In some experiments,soluble heparin or heparan sulfate (Sigma) was used at concentrationsof 10 to 20 µg/ml or 10 to 1,000 µg/ml, respectively,prior to isolation of capsids as described above.

Analysis of capsids under nonreducing conditions. To analyze disulfide-bonded proteins, capsids harvested as describedabove were dissolved in sample buffer lacking reducing agentsand split into two equal portions, and reducing agents (BMEand DTT) were added to one. Both samples (reduced and nonreduced)were then boiled for 5 min at 95°C, separated by SDS-PAGEin 10% gels, and analyzed by immunoblotting.

RESULTS

Top

RESULTS

The capsids of herpesviruses are stable in nonionic detergents,but proteins in the viral membrane and many of the proteinsin the tegument are released by such treatments. We recentlyshowed that most of the UL16 molecules present in extracellularHSV do not pellet with the capsid following NP-40 treatment;however, the opposite is found when free cysteines are covalentlymodified with the small, membrane-permeating compound NEM (resultingin a shift in gel mobility) prior to envelope disruption (Fig.1, HSV) (27). In contrast, the UL16 homologs of beta- and gammaherpesviruseshave been found to be mostly capsid associated even in the absenceof NEM in similar types of experiments (21, 47, 52). To ascertainwhether sensitivity to NP-40 and NEM might be a property thatis specific to alphaherpesviruses, PRV was examined, and itsUL16 homolog indeed behaved similarly to that of HSV (Fig. 1).On average, $~$ 85% of the PRV molecules were released by NP-40treatment, but $~$ 80% pelleted with capsids when the virions werepretreated with NEM. Although the HSV and PRV homologs differdramatically in their total numbers of cysteines (20 versus11, respectively), 8 of these appear to be conserved based onalignments of UL16-related sequences from all herpesviruses(see the supplemental material), and thus, it is likely thatone or more of these are targets for NEM.

View larger version (53K):
[in this window]
[in a new window]

FIG. 1. Sensitivity to NEM and NP-40 is conserved among alphaherpesviruses. Extracellular HSV and PRV were harvested from infected Vero cells and treated with NEM or vehicle for 30 min at 37°C. Virions were then treated with NP-40 (+) or left untreated (–) to solubilize membranes. Intact virions or released capsids were pelleted through a 30%-sucrose cushion. Pellets and infected cells were dissolved in sample buffer and analyzed by immunoblotting with anti-VP5 and anti-UL16 serum.

UL16 is released from capsids upon cell binding. Although NP-40-containing buffers are widely used in studiesof protein interactions (e.g., in the discovery of UL16 as abinding partner of UL11 [24]), there are other examples of interactionsbeing disrupted by NP-40 treatment. One that had a profoundinfluence on our studies is the interaction between the SU andTM proteins of murine leukemia virus (MLV). In brief, theseproteins are found on the exterior of MLV, where they mediatebinding and fusion with the host cell during the initial stepsof infection. Although they are linked by a single disulfidebond, this bond is broken when the virus is exposed to NP-40but preserved if the virus is first treated with NEM (36). Thefree cysteine important for this activity is located in SU,just two amino acids away from the cysteine that is disulfidebonded to TM. NP-40 artificially triggers a disulfide-exchangereaction in which the SU-TM disulfide bond is broken and transferredto the CXXC motif within SU; however, the natural trigger forSU-TM separation is binding of MLV to its receptor (36, 45).

Upon closer inspection of the conserved cysteines of UL16, wenoticed a CXXC motif (see the supplemental material). AlthoughUL16 is not present on the surface of HSV (27), we were neverthelesscompelled to test the possibility that cell binding might somehowsend a signal inward to trigger the capsid release mechanism.Accordingly, cells were exposed to either untreated or NEM-treatedvirions at 4°C, a temperature that allows binding but preventsfusion from occurring. After virus attachment, NEM was addedto the monolayer that had received untreated virions, and 30min later, unbound virions were washed away. The remaining virionswere then stripped of their membranes with NP-40, and theircapsids were pelleted through a sucrose cushion. Thus, in bothcases, the virions were exposed to NEM prior to NP-40 treatment;only the timing of addition differed. Capsids and their associatedproteins were separated by SDS-PAGE and detected by immunoblotanalyses.

If cell binding is the natural trigger for the release of UL16from capsids, then little or no UL16 would be expected to bepresent in the material pelleted from the cell lysate unlessNEM had been added prior to binding, and this was found to bethe case (Fig. 2A). As expected, NP-40 efficiently removed theviral envelope regardless of NEM treatment, since viral gC andthe outer tegument protein UL11 did not pellet with capsids(data not shown). Data from five independent experiments revealedthat only 5% (±3%) of the UL16 molecules were capsidbound after cell binding compared to results for the pretreatedcontrol. Overexposure of the film confirmed that NEM had beenadded to the culture following virion binding, because someof the UL16 remaining on the capsids was shifted to the expectedsize (Fig. 2A, bottom panel). Higher-molecular-weight bandswere detected with the anti-UL16 serum (see the supplementalmaterial), but these appear to be irrelevant because they werealso observed in experiments using a virus that lacks the UL16-codingsequence and in experiments with PRV using HSV-specific UL16antibodies (see the supplemental material).

View larger version (37K):
[in this window]
[in a new window]

FIG. 2. Binding of viruses to cells triggers release of UL16 from capsids. (A) Virus was mock (–) or NEM treated either prior to (P) or after (A) binding to confluent monolayers of cells for 45 min at 4°C. Following virus binding or NEM treatments, viral membranes were solubilized with NP-40 lysis buffer for 15 min. Capsids were then pelleted through a 30%-sucrose cushion, dissolved in sample buffer, and analyzed by immunoblotting. The bottom panel is an overexposed (OE) version of the middle one. (B) In a variation of the experiment, NEM was added to bound (A_b) and free (A_f) virions from the same culture dish prior to the addition of NP-40 and the isolation of capsids. As controls, NEM was not added (–) or was added to virions prior (P) to cell binding.

Although secretory pathways should be inhibited at 4°C,we considered the possibility that a factor in the growth medium(rather than direct physical contact with a component on thecell surface) was responsible for the release of UL16. For this,cells were incubated with HSV, and capsids from virions thatdid not bind were examined (Fig. 2B). UL16 remained capsid associatedin these virions even though it was released from those thathad bound to the cells or did not receive NEM (Fig. 2B, compareA_f to A_b and minus-NEM samples). Again, UL16 was detected withcapsids if NEM was added to virions prior to cell binding (Fig.2B, P sample).

To determine whether cysteines of host proteins are requiredfor the release mechanism, cells were treated with NEM and thenwashed prior to the addition of virus. This protocol did notprevent virus binding or release of UL16 from capsids (see thesupplemental material). Moreover, release was observed in experimentswith HeLa cells (see the supplemental material), where the virusenters by endocytosis rather than by direct fusion at the plasmamembrane (32). Thus, the mechanism does not appear to be celltype specific or to require fusion.

Binding to heparin beads is sufficient for release. Although virus fusion does not occur at 4°C, the cell surfaceis a complex environment, and it seemed possible (though notlikely) that a host factor might insert through the viral membraneto alter the tegument in a manner that does not directly involvecell receptors. To address this, we sought a cell-free systemfor binding virions to their receptors. Because the efficiencyof UL16 release was enormous even at low temperatures, we hypothesizedthat the earliest virus attachment factors would be involved.These are provided by the heparan sulfate moieties of proteoglycans(42), which are abundantly scattered over the cell surface (37).It is well known that herpesviruses can efficiently interactwith heparin-coated beads in a manner that mimics virus attachment(49), and therefore, we treated HSV with NEM either before orafter binding to such beads. The amount of UL16 that was capsidassociated in the collected virions was measured following solubilizationwith NP-40. The results clearly showed that engagement of thevirus with immobilized heparin was sufficient to trigger theefficient release of UL16 from capsids (Fig. 3A). On average(three experiments), only 12% (±11%) of the UL16 moleculeswere capsid associated after binding compared to results forthe pretreated control. Similar results were obtained usingPRV (Fig. 3B), where equal amounts of capsids were analyzed(as measured by Ponceau S staining; data not shown).

View larger version (52K):
[in this window]
[in a new window]

FIG. 3. Binding to immobilized heparin is sufficient to initiate UL16 release. HSV (A) or PRV (B) was incubated with or without (–) NEM treatment either prior to (P) or after (A) binding to heparin beads. The samples were pelleted, washed with PBS, and dissolved in NP-40 lysis buffer. Insoluble material was removed by centrifugation, and capsids were pelleted through a 30%-sucrose cushion, dissolved in sample buffer, and analyzed by immunoblotting. (C) HSV was incubated with increasing amounts of soluble heparan sulfate (HS) or heparin for 30 min and then treated with NEM. Virions were lysed with NP-40, and released capsids were pelleted through sucrose and analyzed by immunoblotting. (D) As a control, a portion (20%) of the heparin-treated virus from panel C was incubated with heparin agarose for 30 min. Virions bound to beads were pelleted, dissolved in sample buffer, and analyzed by immunoblotting with anti-VP5 serum.

Soluble heparin blocks the adsorption of HSV to host cells (20),and therefore, we considered the possibility that it might alsotrigger release of UL16 from capsids. Virions were incubatedfor 30 min with increasing amounts of soluble heparin, and thenNEM was added. Soluble heparan sulfate was also tested. Neithercompound was able to trigger the release of UL16 (Fig. 3C) despitebeing able to inhibit the binding of virions to the heparin-coatedbeads (Fig. 3D). This shows that the release mechanism requiresheparin (or heparan sulfate) to be immobilized, possibly toenable receptor-glycoprotein clustering on the exterior of theviral envelope (see Discussion).

gC is required for heparin-triggered release. It has been known for some time that the viral glycoproteinsthat interact with heparan sulfate are gB and gC (42). gC isthought to be the predominant heparan sulfate binding protein;however, not all gC-null stains are defective in virus attachmentto cells (23, 25). Additionally, in its absence, HSV can stillassociate with heparan sulfate through gB (23). To test theinvolvement of these glycoproteins in the release mechanism,mutant viruses that lack gC or gB (Fig. 4A) were analyzed inthe heparin-coated bead binding assay (6, 12, 13). In the absenceof gB, virus particles efficiently bound to immobilized heparinand UL16 was readily released from the capsids (Fig. 4B). Identicalresults were obtained with a different gB-null virus (data notshown) (12). In contrast, virus particles lacking gC that boundto beads still had capsid-associated UL16 regardless of thetiming of NEM addition (Fig. 4B). The wild-type revertant ofthis virus (gC-R) regained the ability to trigger release ofUL16, although not as efficiently as the wild-type HSV KOS strain(78% versus 88%, respectively). This difference may be due tostrain variation, since the gC-null and gC-R constructs werederived from the NS strain (13). Intriguingly, when the gC-nullvirus was incubated with cells at 4°C, the release mechanismwas efficiently triggered (Fig. 4C), suggesting that other,downstream glycoprotein-receptor interactions are capable ofsending the signal (see Discussion).

View larger version (46K):
[in this window]
[in a new window]

FIG. 4. gC is required for heparin bead- but not cell-binding-triggered release. (A) Wild-type (WT) or mutant ( ${Delta}$ gB, gC-R, or ${Delta}$ gC) viruses were pelleted through 30%-sucrose cushions and analyzed by immunoblotting for the presence of gB and gC. VP5-specific antibodies were used to confirm the presence of virus particles. (B) Virus stocks tested in panel A were incubated with or without (–) NEM treatment either prior to (P) or after (A) binding to heparin beads. Capsids were pelleted through a 30%-sucrose cushion, dissolved in sample buffer, and analyzed by immunoblotting. (C) Virus was mock (–) or NEM treated either prior to (P) or after (A) binding to cells for 45 min at 4°C. Following virus binding or NEM treatments, viral membranes were solubilized with lysis buffer, and capsids were pelleted and analyzed as described above.

UL16 is not disulfide bonded to capsids. To further investigate the signaling mechanism, the disulfide-bondedstate of UL16 was examined before and after binding of virionsto heparin beads (Fig. 5). In these experiments, intact virionswere analyzed to visualize the entire population of UL16 insidethe envelope (both capsid bound and released). The results obtainedfrom reducing (control) gels were as expected, with all of theUL16 molecules running as slower migrating, modified monomersregardless of the timing of the NEM treatments, confirming thatNEM had equal access to UL16 within virions under all conditions.The results from nonreducing gels showed that UL16 is also monomericafter virions bind to beads, whether NEM was added for 30 minbefore or after this step. However, when NEM was omitted, mostof the UL16 molecules were not visible at any position in thegel. This is likely due to the spontaneous formation of irrelevantdisulfide bonds during virion disruption. Similar results werefound for PRV, except that its UL16 protein did not form intermoleculardisulfide bonds in the absence of NEM, presumably because itcontains fewer cysteines. However, these conditions did producea striking increase in mobility for UL16 relative to the reducedform (compare the leftmost lanes in the bottom two panels: minus-NEMsamples), which is indicative of an efficient creation of irrelevantintramolecular disulfide bonds during virion disruption in theabsence of NEM (48). A small amount of such a fast-migratingspecies was also seen for HSV when gels were overexposed orloaded with larger amounts of virions (data not shown). Collectively,these data suggest that receptor binding does not trigger UL16to form static, intermolecular disulfide bonds following releasefrom the capsid. However, because we can detect only the beginningand final states of UL16 in these assays, it is possible thatdisulfide-bonded intermediates form in the release pathway.

View larger version (64K):
[in this window]
[in a new window]

FIG. 5. UL16 is not disulfide bound to the capsid or other proteins. HSV or PRV was incubated with heparin beads for 30 min along with NEM treatment either prior to binding (P), after binding (A), in the sample buffer (SB), or not at all (–). Virus-bound beads were pelleted, washed with PBS, and dissolved in sample buffer lacking reducing agents. Samples were split, with half receiving BME and DTT (reducing) and the other half receiving neither (non-reducing) prior to immunoblot analysis.

DISCUSSION

Top

DISCUSSION

The experiments described here have produced the first evidencefor "outside-in" signaling across the membrane of an envelopedvirus. Specifically, we discovered that binding of HSV to itsinitial cell receptor triggers a rapid and efficient changewithin the tegument, leading to destabilization of the UL16-capsidinteraction (Fig. 6A). While this finding was quite unexpected,it is reassuring that "signaling from within" has been reportedfrom studies of retroviruses. For example, the fusion activityneeded for MLV entry is absent until the viral protease cleavesthe C terminus of the TM protein on the inside of the virus(1, 38). And in the case of human immunodeficiency virus, fusionactivity is absent until the Gag protein on the inside is cleavedto create the mature virus (31, 50). Thus, it seems reasonableto expect that alterations on the outside of an enveloped virusmight be of great importance to functions located inside.

View larger version (32K):
[in this window]
[in a new window]

FIG. 6. Probable players in the UL16-capsid release mechanism. (A) UL16 is capsid associated in the extracellular virion (red circles) and contains numerous free cysteines (emanating lines). The observed rearrangement requires clustering of one or more viral glycoproteins (blue), which must transmit a signal either directly to UL16 or to one of their binding partners (yellow). (B) gC and gB bind heparin sulfate (HS) and also interact with other glycoproteins (dotted lines). UL11 (yellow), a known binding partner of UL16, may facilitate release of UL16 through its interaction with the cytoplasmic tails of gE and gD.

How is the signal transmitted into HSV to release UL16 fromcapsids? There are many interesting clues (Fig. 6B). Startingon the outside surface of the virus, it is well known that theviral glycoproteins gB and gC interact with heparan sulfate(42) while gD can bind to 3-O-sulfated heparan sulfate (41).Our data reveal that gC is needed for the release of UL16 inthe heparin-bead assay but not in the cell-binding assay, whereother virus-receptor interactions are available. In this regard,it is interesting that gC has been reported to be in close proximityto gD (18), the viral protein that recognizes the cell receptorneeded for entry (of which there are three alternatives: HVEM,nectin 1, or 3-O-sulfated heparan sulfate) and works in cooperationwith gB, gH, and gL to mediate fusion (42). Moreover, bindingto entry receptors leads to conformational alterations in gD(15, 22, 43) and changes in its association with gB, gH, andgL (3, 7). This complexity has only increased with the recentdiscovery of an HSV coreceptor, PILR ${alpha}$ , which binds to gB andappears to be vital for entry into certain cell types in conjunctionwith gD-receptor interactions (40). In light of the multitudeof dynamic interactions that occur on the virus surface, itis easy to imagine that these might alter the inside of thevirion, and thus, it is possible that one or more of these fourglycoproteins is capable of triggering the release of UL16,even in the absence of heparan sulfate (Fig. 6B). This is clearlysupported by the efficient release of UL16 in the gC-null virusfollowing cell binding.

Regardless of the mechanism used for transducing the signalthrough the membrane, there must be a means for getting thatinformation to capsid-associated UL16. Many tegument proteinshave been found to interact with the tails of viral glycoproteinsand also with one another (8, 11, 14, 17, 34, 53). Of particularinterest in this regard is the UL11 protein (Fig. 6B), a directbinding partner of UL16 (24, 51) that is also capable of interactingwith the tails of gD and gE (11). Moreover, the UL11-UL16 interactionis blocked by NEM (51). Thus, binding of HSV to cell receptorsmight induce conformational changes in viral glycoproteins,which in turn affect UL11-UL16-capsid interactions. Due to theshort cytoplasmic tail of gC, it is unlikely that the signalis sent directly through this glycoprotein. A more attractivemodel is that clustering of gC alters interactions between otherglycoproteins on the surface, which then transmit the signalto UL16 through UL11. In support of this hypothesis, there isevidence for potential interactions of gC with gB, gD, and gLon the virion surface (18). On the other hand, the structureof the tegument is very poorly understood, and therefore, itis difficult to have confidence in making these predictions.For example, it is unknown whether UL16 is actually bound toUL11 within the virion, either before or after receptor binding.

What features of UL16 are important for the capsid release mechanism?At the moment, all that can be said is that conserved, freecysteines must be involved. This is based on the ability ofNEM to block release and shift the mobility of UL16 from bothHSV and PRV. The presence of CXXC among the conserved residuesis intriguing because this motif is found in the active sitesof many enzymes, such as those that mediate disulfide-exchangereactions (2) and those that modify proteins with palmitate(28). However, the thiol group of cysteine is the most reactiveof all side chains (9), and single cysteines in combinationswith other amino acids constitute many active sites, such asthose of the cysteine proteases (29). Moreover, free cysteinesare found in metal-binding domains (e.g., zinc fingers), whichplay numerous structural roles (16). Given the large numberof free cysteines that appear to be present in UL16, it is possiblethat they serve multiple roles, and at this point it is difficultto rule any mechanism out. For example, although we found noevidence to suggest that UL16 is disulfide bound to any otherprotein before or after receptor binding, it is possible thattransient disulfide bonds are formed as the release mechanismproceeds. Future investigations of the role of the conservedcysteine residues are warranted; however, in doing so, it isimportant to keep in mind that free cysteines in other tegumentproteins (rather than UL16) may have critical roles in the releasemechanism.

What might be the purpose of receptor-induced rearrangementsof the tegument? There are two possibilities that spring tomind. In the simplest of these, inward signaling triggers theearliest steps of uncoating in preparation for postfusion events(e.g., targeting the capsid to the nucleus of the infected cell).The other possibility is that rearrangements in the tegumentserve to regulate the fusion activity of viral glycoproteinsin a manner analogous to that found in retroviruses (see above).In this model, fusion activity would be inhibited when tegumentproteins are bound to the tails of viral glycoproteins, butinteractions with proteoglycans or other receptors would triggerrearrangements that allow the subsequent events of fusion toproceed. In support of this model, mutations in the cytoplasmictails of gB and gH have been reported to enhance or abolishfusion activity in cell-cell and virus-cell fusion assays (19,33, 39, 46). Therefore, it seems likely that the interactionof tegument proteins with the tails of glycoproteins could alsoinfluence fusion activity. The two proposed models are not mutuallyexclusive, and given the complexity of the tegument, other rolesfor the rearrangements may be envisioned as more is learnedabout its organization. Of particular interest in this regardis the ability of NEM to completely eliminate virion infectivity(27) even though it does not prevent virion binding to the hostcells (this study).

In summary, our recent studies of UL16 have shown that the structureof the tegument changes in ways that no one anticipated. Duringvirus assembly and egress, the interaction of UL16 with thecapsid is initially quite stable in NP-40, but by the time thevirion is released from the cell, that interaction has beenaltered such that free cysteines have to be modified to preventNP-40- or receptor-induced release (27). And while it has longbeen hypothesized that fusion of the virus with the host cellleads to phosphorylation of tegument proteins to destabilizetheir interactions during uncoating (30), our data show thatthe structure of the tegument is altered when the virus merelybinds to receptors. Clearly, there is a tremendous amount ofwork to be done in elucidating the dynamic interactions of tegumentproteins.

ACKNOWLEDGMENTS

We extend thanks to our PSU colleagues Jacob Marsh, MichaelMurphy, Nicolas Baird, and Kevin O'Regan for helpful discussions.Special thanks go to Todd Wisner (David Johnson's laboratory)and Elizabeth Wills (Joel Baines' laboratory) for providingtechnical help on growing mutant viruses used in these studies.We also thank Gary Cohen and Rebecca Craven for careful reviewof the manuscript and Pat Spear for useful suggestions.

This work was supported by an NIH grant to J.W.W. (AI071286).D.G.M. was supported by a training grant from the NIH (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 17033. Phone: (717) 531-3528. Fax: (717) 531-6522. E-mail: jww4{at}psu.edu

Published ahead of print on 20 August 2008.

Supplemental material for this article may be found at http://jvi.asm.org/.

REFERENCES

Top