Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA
Correspondence
Duncan W. Wilson
wilson{at}aecom.yu.edu
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ABSTRACT |
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Present address: Pfizer Global Research and Development, Groton Laboratories, Eastern Point Road, Groton, CT 06430, USA.
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INTRODUCTION |
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It is generally agreed that HSV capsids acquire their final tegument and envelope in the cytoplasm. However, the molecular mechanism of HSV-1 envelopment in the cytoplasm is poorly characterized. During infection, viral membrane glycoproteins are expressed and traffic via cytoplasmic organelles to the cell surface. Several lines of evidence suggest that the cytosolic domains of these glycoproteins may provide a platform for tegument assembly and recruitment of capsids followed by envelopment (Mettenleiter, 2002). In a recent study, a double mutant of pseudorabies virus (PrV) lacking viral glycoproteins gM and gE (or gM and the cytoplasmic tail of gE) showed a failure in envelopment, instead accumulating cytoplasmic nucleocapsids immersed in a dense layer of tegument (Brack et al., 1999
). Following single deletion of either glycoprotein, the production of infectious virions was not affected, suggesting that the cytoplasmic tails of gE and gM act in a redundant fashion during assembly, most likely interacting with the tegument proteins and, indirectly, with the surface of nucleocapsids. In HSV-1, simultaneous deletion of the same two glycoproteins did not dramatically affect production of infectious virions (Browne et al., 2004
), although simultaneous deletion of gD and gE resulted in a striking defect in particle assembly (Farnsworth et al., 2003
). This apparent redundancy makes analysis of specific glycoproteintegument interactions difficult to perform in vivo. To overcome this obstacle, we developed a glutathione S-transferase (GST) fusion protein-binding assay to investigate the interaction between envelope glycoprotein tails and tegument partners in vitro. This approach has previously been used to demonstrate the interaction between the cytoplasmic tail of glycoprotein H (gH) and the tegument component VP16 (Gross et al., 2003
). In the current study, we focused on analysing the tail of the glycoprotein D (gD) because it is a highly abundant component of the envelope (Spear & Roizman, 1972
) with a short cytoplasmic tail of 30 amino acid residues that can easily be manipulated in mutagenesis studies. Moreover, it has been shown to play a key, albeit redundant, role in assembly (Farnsworth et al., 2003
). Additionally, Pomeranz & Blaho (1999)
reported that gD co-localizes with at least one tegument protein, VP22, during infection.
We prepared GST fusion proteins that contained the cytoplasmic tail of gD or mutants thereof at their carboxy terminus. Following binding of the GST fusion proteins to glutathioneSepharose beads and incubation with HSV-infected cell extracts, these fusion proteins were tested for their ability to interact with tegument proteins. Here we report that the cytoplasmic tail of gD bound to the tegument protein VP22 in a manner that was independent of other viral polypeptides. Additionally, our results indicated that VP22 may be one of the tegument components that mediate the interaction between the cytoplasmic tail of gD and the HSV capsid.
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METHODS |
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Expression of GST fusion proteins.
Appropriate expression plasmids were transformed into Escherichia coli strain BL21 (Stratagene). Overnight stationary-phase cultures were used to inoculate fresh medium the following day to an OD600 of 0·2 and cultures were grown on a shaker to an OD600 of 0·9. IPTG was added to the cells to a concentration of 1 mM to induce expression of the fusion protein. After 3 h, cells were collected by centrifugation at 8000 r.p.m. for 10 min at 4 °C in a JA20 rotor, the medium was discarded and the pellet was frozen at 20 °C overnight. The following day, the pellet was thawed and resuspended in lysis buffer (0·05 % Tween 20, 50 mM EDTA, 1 mM PMSF, 5 µg aprotinin ml1, 5 µg leupeptin ml1 and 1 mg lysozyme ml1 in PBS) and sonicated using a probe sonicator six times for 10 s each, with 30 s intervals on ice. Debris and unbroken cells were pelleted at 10 000 r.p.m. for 10 min at 4 °C in a JA20 rotor. Supernatants were subjected to SDS-PAGE and stained with Coomassie blue to visualize the fusion proteins.
Preparation of HSV-1-infected cell extracts.
Confluent monolayers of COS cells were infected with HSV-1 strain SC16 at an m.o.i. of 10 in DMEM with 10 % fetal bovine serum (FBS) and 1 % penicillin/streptomycin (PS). After 1 h, fresh medium was added and the incubation proceeded for an additional 18 h at 37 °C. Cells were collected and a post-nuclear supernatant (PNS) was prepared as previously described (Harley et al., 2001). Briefly, cells were washed twice with cold homogenization buffer (0·25 M sucrose, 2 mM MgCl2, 10 mM Tris/HCl pH 7·6), scraped, pelleted at 1000 g for 5 min at 4 °C and resuspended in 1 ml homogenization buffer containing 5 µg leupeptin ml1, 5 µg aprotinin ml1 and 1 mM PMSF. Cells were broken by passing through a 25 gauge 5/8 inch needle eight times and then spun at 2000 g for 10 min at 4 °C to remove unbroken cells and nuclei. The PNS was adjusted to 150 mM NaCl, 0·5 % NP-40, 50 mM Tris/HCl pH 7·6, incubated for 1 h on ice to solubilize tegument proteins and centrifuged at 53 000 r.p.m. in a Beckman TLA 100.3 rotor for 1 h. The supernatant (S100) was aliquotted and frozen at 70 °C.
Production of radiolabelled VP22.
A VP22/mycHis-encoding plasmid (Invitrogen) and Redivue [35S]methionine were added to the TNT Rabbit Reticulocyte Lysate in vitro transcription/translation system (Promega) following the manufacturer's protocol. The resulting in vitro-translated VP22 was mixed with PBS containing 5 µg leupeptin ml1 and 5 µg antipain ml1 and incubated with GST fusion protein-coated glutathioneSepharose beads as described below. Material bound to the beads was subjected to SDS-PAGE and the gel treated with 30 % methanol, 10 % acetic acid for 30 min, En3Hance (NEN Life Sciences Products) for 1 h and 1 % glycerol for 30 min. The gel was then dried using a Bio-Rad gel drier and exposed to film for 12 days.
Preparation of [3H]thymidine-labelled capsids.
Prior to infection, confluent monolayers of COS cells were overlaid with DMEM containing 1 % dialysed FBS and 1 % PS for 2 h and then infected with HSV-1 strain SC16 at an m.o.i. of 10. After 1 h, fresh medium with [3H]thymidine (NEN) was added to a final concentration of 25 µCi ml1 and incubated for 18 h. Cells were harvested as above, and a PNS was prepared and adjusted to a final concentration of 2·5 mM magnesium acetate, 50 mM potassium acetate, 150 mM NaCl, 0·5 % Triton X-100 and 5 mM DTT and incubated on ice for 1 h. The treatment removed the membranes and solubilized some of the tegument associated with the cytoplasmic virions. Following incubation, debris was removed by a clearing spin at 1500 g. The treated PNS was used to test for binding of capsids to fusion proteins bound to glutathioneSepharose beads. The protein concentration of the PNS was determined using a BCA assay (Pierce) with BSA as a protein standard.
GST in vitro-binding assay.
GlutathioneSepharose beads were dispensed into a microcentrifuge tube and washed three times with cold PBS. BSA (100 µl of a 10 mg ml1 solution), an equivalent amount of GST fusion protein-containing bacterial extract and PBS to a total volume of 1 ml were added to the washed beads and incubated at 4 °C for 1 h on a rotator. The beads were then washed twice with wash buffer 1 (50 mM Tris/HCl pH 7·6, 150 mM NaCl, 0·5 % Triton X-100) and then with wash buffer 2 (50 mM Tris/HCl pH 7·6, 1·3 M NaCl, 0·5 % Triton X-100) and again with wash buffer 1. Infected-cell cytosol (S100), in vitro-translated VP22 or cell extracts containing [3H]thymidine-labelled capsids was then added to the GST fusion protein-bound beads, incubated at 37 °C for 1 h and then washed six times with wash buffer 1. For detection of bound tegument proteins, the beads were resuspended in 500 µl 50 mM Tris/HCl pH 7·6. SDS-PAGE sample buffer was added and the mixture was heated to 95 °C for 5 min, subjected to SDS-PAGE and Western blotted using appropriate antibodies.
Measurement of packaged viral DNA.
A TCA precipitation assay to measure DNA packaging was carried out as previously described (Church et al., 1998). Briefly, after performing the GST in vitro-binding assay with [3H]thymidine-labelled capsid-containing extracts, beads were incubated with 2 mM MgCl2 and 280 U DNase I (type II; Sigma) ml1 for 90 min at 37 °C. EDTA and SDS were added to final concentrations of 10 mM and 0·3 %, respectively, and incubation continued for an additional 15 min. The treated material was then spotted on to individual GF/C Whatman filter papers and allowed to dry. Each filter was subjected to one 4 °C wash and two consecutive 65 °C washes in TP buffer (5 % TCA, 20 mM sodium pyrophosphate) before rinsing in 70 % ethanol and drying. The level of TCA-precipitable radioactivity was determined by liquid scintillation counting.
Electron microscopy analysis of bead-associated capsids.
GST or GST fusion proteins were bound to glutathioneSepharose beads and incubated with HSV capsid-containing extracts and the beads were washed, pelleted and fixed in 2·5 % glutaraldehyde in SC (100 mM sodium cacodylate, pH 7·43) at room temperature for 45 min. The pellet was then rinsed in SC, post-fixed in 1 % osmium tetroxide in SC followed by 1 % uranyl acetate, dehydrated through a graded series of ethanols and embedded in LX112 resin (LADD Research Industries). Ultrathin sections were cut on a Reichert Ultracut E, stained with uranyl acetate followed by lead citrate and viewed on a JEOL 1200EX transmission electron microscope at 80 kV.
Co-immunoprecipitation of endogenous gD and VP22.
COS cells were infected with HSV-1 strain PAAr5 (Jones et al., 1995) at an m.o.i. of 10. At 15 h post-infection, cells were collected and resuspended in lysis buffer [50 mM Tris/HCl pH 7·6, 150 mM NaCl, 0·5 % NP-40, 0·5 mM EDTA, 0·5 mM PMSF, 2 mM DTT, 200 µg protease inhibitor cocktail tablets (Roche Diagnostics) ml1] and incubated on ice for 30 min. The resulting lysate was cleared by centrifugation for 20 min at 15 000 g and pre-cleared by incubation with Protein Gagarose beads (Sigma) for 20 min at 37 °C. After pelleting the beads, the supernatants were collected and mixed with fresh Protein G beads with either anti-gD (Virusys) or anti-HA (Roche Diagnostics) monoclonal antibody (mAb). After a 15 min incubation at 37 °C, beads were washed four times with a wash buffer and once in PBS. Bound material was then subjected to SDS-PAGE and Western blotted using anti-VP22 antibody.
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RESULTS |
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Binding of VP22 to the gD tail is independent of other viral proteins
The interaction between VP22 and gD may be direct or mediated by other viral proteins. To examine this question, we used a rabbit reticulocyte lysate in vitro transcription/translation system to generate 35S-labelled VP22 and added it to our in vitro GST-binding assay. Autoradiography showed that in vitro-translated VP22 (running at a molecular mass of 45 kDa) bound to the gD tail (Fig. 2). Since the rabbit reticulocyte lysate contained only the in vitro-translated VP22 and no other viral proteins, we could conclude that VP22 binding did not require any other viral polypeptides. Although we could not discount the possibility that a cellular polypeptide in the reticulocyte lysate mediated the gDVP22 interaction, no host protein with the abundance of VP22 has ever been described in the HSV particle.
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Alanine-scanning mutagenesis of the gD cytoplasmic tail
To simplify the identification of amino acids within the gD cytoplasmic tail that play an important role in VP22 binding, we generated a truncation mutant of the tail that retained only the most amino-terminal 17 residues (named T17) (Fig. 3a). When the GSTT17 fusion protein was tested for its ability to bind VP22 present in cytosolic extracts prepared from HSV-infected cells, it was found to bind VP22 in a similar manner to the full-length gD tail (Fig. 3b
). Next, to identify residues that might be critical for binding VP22, we carried out alanine-scanning mutagenesis. Clusters of amino acids were mutated simultaneously to alanine and named according to the amino acid residues that were mutated (Fig. 3a
). These mutants, along with gDc and GST, were expressed and incubated with infected-cell extracts and the relative amounts of VP22 binding were detected by Western blotting. Fig. 3(c)
shows that mutations in the arginine and lysine residues at positions 5/6 or 9/10 diminished the binding of VP22, indicating that these amino acid residues are important. Although the HRR mutant showed a slightly decreased binding of VP22, this level of binding was variable, and most commonly the HRR mutant bound VP22 at a level similar to the wild-type gD tail. Next, we examined the role of the key lysine and arginine residues in the context of the full-length gD tail (Fig. 3d
). Similar to the truncated mutant, mutation of R5/K6 showed a dramatic decrease in binding of VP22. Individual mutation of these residues had a less dramatic effect, indicating that these amino acid residues both contribute to VP22 binding but may play a somewhat redundant role.
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Visualization of HSV-1 capsids bound to gDc-coated glutathioneSepharose beads
Although rapid and convenient, monitoring the c.p.m. of labelled packaged viral DNA is an indirect method of testing for the presence of capsids. As a direct test of gD tailcapsid interaction, GST- or gDc-coated glutathioneSepharose beads were incubated with the capsid preparation, fixed and then processed for ultrastructural analysis. Fig. 6 shows representative images of capsids bound to gDc-coated beads. For gDc-coated beads, it was relatively easy to identify icosahedral capsids. Capsids were never found within beads, but always at the perimeter, suggesting they were not non-specifically trapped within the glutathioneSepharose matrix.
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DISCUSSION |
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We generated a fusion protein between GST and the cytoplasmic tail of gD in order to test its interaction with HSV-1 tegument proteins. Our results showed that the gD tail specifically bound the tegument protein VP22 but did not interact with the abundant cellular protein -tubulin nor with the capsid scaffold proteins ICP35 and Pra. Furthermore, co-immunoprecipitation experiments confirmed that the interaction between gD and VP22 could be detected in infected cells. Additionally, alanine-scanning mutagenesis of the gD tail identified two amino acid residues, arginine and lysine (from the amino-terminal end of the tail) at positions 5 and 6, respectively, that were critical for VP22 binding.
Since VP22 and VP16 form a complex (Elliott et al., 1995) and we observed VP16 binding to the gD tail, we were interested to test whether VP22 bound the gD tail directly or via VP16 or other viral proteins. Therefore, we synthesized 35S-labelled VP22 using a rabbit reticulocyte lysate in vitro transcription/translation system and tested its ability to bind the gD tail. We found that in vitro-translated VP22 did bind specifically to the gD tail, showing that this binding was independent of other viral polypeptides.
Under our conditions we also observed capsid binding to the gD tail. This binding was saturable, unlike the low level of binding seen with GST alone. We speculated that capsid binding to the gD tail was mediated by tegument components present in the crude capsid preparation. Our results showed that the gD mutant tail gDR5K6 that failed to bind VP22 also failed to bind capsids. This suggested that VP22 was mediating capsidtail interactions. However, it is also possible that other tegument proteins mediate binding of capsids to the gD tail, utilizing the same residues as VP22. Although VP16 is present on these capsids (Fig. 5a) and can bind to the gH tail (Gross et al., 2003
), it appeared unable to tether capsids to the gH tail (Fig. 5b
). Additionally, we observed that VP16 was capable of binding to the gD mutant tail gDR5K6, which does not bind capsids (data not shown). This also suggested that VP16 was not involved in capsidtail interactions under these conditions.
The association between VP22 and gD may be one of many interactions between glycoprotein tails and tegument proteins that facilitate the budding of capsids into organelles and the incorporation of tegument/glycoproteins into nascent virions. In HSV-1, deletion of the cytoplasmic tail of gD had relatively little effect on normal virus production (Feenstra et al., 1990). However, simultaneous deletion of gD and gE showed elimination of cytoplasmic envelopment and accumulation of large numbers of unenveloped nucleocapsids within a dense layer of tegument in the cytoplasm (Farnsworth et al., 2003
), indicating that gD and gE may share overlapping roles in cytoplasmic envelopment. Our data suggest that at least one of these roles may be in VP22 recruitment and predict that the cytoplasmic tail of gE may also bind VP22. Interestingly, Farnsworth et al. (2003)
reported that the cytoplasmic tail of gE, when fused to GST, was indeed able to bind VP22. Thus, gD and gE may act in a redundant manner to incorporate VP22 into the newly forming virions. Alternatively, it could be argued that VP22 serves to gather gD and gE to the sites of capsidenvelope interaction.
Our findings regarding HSV-1 are somewhat similar to data reported for the alphaherpesvirus PrV. gE and gM have been shown to interact with the UL49 protein, a homologue of HSV-1 VP22 (Fuchs et al., 2002), and, following simultaneous deletion of these two glycoproteins, the UL49 product does not become incorporated into the PrV particle (Fuchs et al., 2002
). However, in the absence of the UL49 protein, gE and gM are incorporated into virions, implying that they may interact redundantly with other tegument proteins for this purpose.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 15 July 2004;
accepted 25 October 2004.