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Journal of Virology, August 2007, p. 8367-8370, Vol. 81, No. 15
0022-538X/07/$08.00+0     doi:10.1128/JVI.00819-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

The N Terminus of the Herpes Simplex Virus Type 1 Triplex Protein, VP19C, Cannot Be Detected on the Surface of the Capsid Shell by Using an Antibody (Hemagglutinin) Epitope Tag

Marieta Solé,^1, Edward M. Perkins,² Augusto Frisancho,^1, Eugene Huang,^1, and Prashant Desai^1*

Viral Oncology Program, The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins,¹ Department of Biology and Integrated Imaging Center, Johns Hopkins University, Baltimore, Maryland²

Received 17 April 2007/ Accepted 14 May 2007

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The herpes simplex virus (HSV) triplex is a complex of three protein subunits, VP19C and a dimer of VP23 that is essential for capsid assembly. We have derived HSV-1 recombinant viruses that contain monomeric red fluorescent protein (mRFP1), a Flu hemagglutinin (HA) epitope, and a six-histidine tag fused to the amino terminus of VP19C. These viruses were capable of growth on Vero cells, indicating that the amino terminus of VP19C could tolerate these fusions. By use of immunoelectron microscopy methods, capsids that express VP19C-mRFP but not VP19C-HA were labeled with gold particles when incubated with the corresponding antibody. Our conclusion from the data is that a large tag at the N terminus of VP19C was sufficiently exposed on the capsid surface for polyclonal antibody reactivity, while the small HA epitope was inaccessible to the antibody. These data indicate that an epitope tag at the amino terminus of VP19C is not exposed at the capsid surface for reactivity to its antibody.

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Capsid assembly for herpesviruses is a nuclear event resulting in the production of four closed structures, the spherical procapsid and the angular A, B, and C capsids (5, 7). B capsids contain internal scaffold proteins (p22a and p21), the viral protease (VP24), and the capsid shell proteins (VP5, VP19C, VP23, and VP26). For C capsids, genomic DNA replaces the scaffold proteins, and A capsids are empty capsids (reviewed in references 10 and 13). A heterotrimeric complex of one molecule of VP19C and two molecules of VP23 is important for the assembly of the capsid shell structure; if either is absent, capsid shells do not form (4, 9, 15, 16). This complex, designated the triplex, is a unique feature of herpesvirus capsid architecture.

Previously, we discovered that a VP19C construct that expressed an N-terminal histidine handle was capable of participating in assembly to give icosahedral capsids in insect cells using recombinant baculoviruses (8). Spencer et al. (11) first demonstrated that the N-terminal 90 amino acids of VP19C were not required for capsid assembly in the baculovirus system; more recently, similar results were seen in an extensive mutational analysis by Adamson et al. (1). The goal of the present study was to take advantage of these data and to determine whether herpes simplex virus type 1 (HSV-1) recombinant viruses that express an N-terminally tagged VP19C could be made. Subsequently, the accessibility of the N-terminal tag on the capsid surface could be determined by immunoelectron microscopy (immuno-EM) methods. Using this approach of ligand-specific detection, one can elucidate the topography of a protein or a domain in a three-dimensional structure, such as the HSV-1 capsid.

For this study, a SpeI restriction enzyme site was inserted just after the start of VP19C translation using PCR-based methods. This restriction site (ACTAGT) encodes threonine/serine codons after the start of VP19C translation. This plasmid was designated pKUL38Spe1; the parental plasmid pKUL38 has been described before (9). Oligonucleotides which, once annealed, create the Flu hemagglutinin (HA) epitope (YPYDVPDYA) and a six-histidine domain (SSHHHHHHGS) were made and cloned into the SpeI site of pKUL38Spe1, giving plasmids pKUL38-HA and pKUL38-HIS, respectively. The monomeric red fluorescent protein (mRFP1) open reading frame (2) was amplified using PfuTurbo polymerase (Stratagene). The PCR product was digested with SpeI and cloned into pKUL38Spe1 to create pKUL38-mRFP. All constructs were sequenced for authentic amplification and orientation. These constructs were recombined into the HSV-1 genome using homologous recombination. The recipient genome used for this was K19C, which contains a null mutation in the gene encoding this protein (9). Cotransfection of plasmid and viral DNA was performed with C32 cells, a VP19C-complementing cell line (9). The transfection progeny were plated on both C32 and Vero cell monolayers to detect recombinant viruses. Plaques were detected on Vero cells for viruses that expressed VP19C-HA, VP19C-HIS, and VP19C-mRFP. The above three viruses, designated K19C-mRFP, K19C-HA, and K19C-HIS, were plaque purified, and insertion of the tag sequence in the genome was confirmed by PCR assays. The construct that encodes the SpeI restriction site after the start of VP19C translation was also recombined into the K19C virus, and a virus designated K19C-Spe1 was isolated on Vero cells and plaque purified further.

The growth properties of the recombinant viruses were examined by infecting Vero and C32 cells and determining virus yields at different times postinfection (Fig. 1). K19C-Spe1, K19C-HA, and K19C-HIS gave rise to virus yields that were comparable to those of the wild-type virus, KOS, at 24 h postinfection (Fig. 1). The growth of K19C-mRFP was reduced 14-fold relative to that of the wild-type virus (Fig. 1). The growth of K19C-mRFP was recovered partially (sevenfold) by replication in the complementing cell line C32 (Fig. 1). The VP19C trans complementation in C32 cells was never at the level seen for wild-type virus (data not shown). The growth of K19C-Spe1 was comparable to that of wild-type virus, indicating that the SpeI site did not alter virus replication (Fig. 1).

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FIG. 1. Single-step growth curves of the VP19C recombinant viruses. Vero cells (1 x 106) were infected at a multiplicity of infection of 10 PFU/cell with the viruses indicated in the key. The cells were harvested at 2, 6, 12, and 24 h postinfection. The virus yield was enumerated by plaquing on Vero or C32 monolayers. The data plotted are the average results for two infections.

To confirm the expression of the tagged VP19C polypeptides, infected Vero cell lysates were analyzed at different times postinfection using antibodies to the different tags in Western blot assays (Fig. 2). In the case of antibodies to HA, six-histidine, and DsRed, a polypeptide with the correct mobility was detected in extracts derived from cells infected with the respective virus (Fig. 2). The DsRed antibody also recognizes mRFP protein. There was little reactivity of the antibodies to either the wild-type protein or mock-infected cell proteins. There was a corresponding mobility shift in the VP19C-mRFP polypeptide due to the fusion of the mRFP (25 kDa) to VP19C (50 kDa). VP19C-HA and VP19C-HIS polypeptides also exhibited slightly slower mobilities relative to that of the wild-type protein (Fig. 2). Since VP19C is expressed from a late gene, there is very little protein accumulation at 8 h postinfection. The highly reactive HA antibody was able to detect low amounts of this protein at early times (Fig. 2, HA panel).

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FIG. 2. Western blot analysis of the K19C-tagged viruses. Vero cells (1 x 106) were infected with the viruses indicated at the top of each panel at a multiplicity of infection of 10 PFU/cell, and the cells were harvested 8 and 24 h postinfection (MI, mock infected). Total cell lysates prepared were transferred to nylon membranes and then probed using antibodies. Blots probed with HA, HIS, and DsRed were processed using the enhanced chemiluminescence method, and the blot probed with UL38C was processed using 125I-Protein A (8). The UL38C rabbit antiserum (UL38C) was raised against a C terminus peptide (VILEGVVWRPGEWRA) spanning amino acids 449 to 463 of VP19C. The DsRed polyclonal rabbit antiserum, anti-HA mouse monoclonal antibody, and anti-HIS mouse monoclonal antibody were obtained from BD Biosciences, Sigma, and Molecular Probes, respectively.

[³⁵S]methionine-radiolabeled lysates were sedimented through sucrose gradients in order to isolate and analyze intranuclear capsids (9, 17). The polypeptide composition of the capsids was examined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (Fig. 3). The mature C capsid fractions for all the viruses were examined. The capsids isolated all contained the capsid shell proteins; the only difference between the different capsids was the lower mobility of the tagged VP19C polypeptides, which was more evident for the VP19C-mRFP polypeptide (Fig. 3, filled circle). The levels of C capsids derived from K19C-mRFP-infected cell lysates were much lower than the levels of C capsids derived from infected cell lysates of the other viruses (Fig. 3).

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FIG. 3. Analysis of capsids derived from infected cells. [35S]methionine-radiolabeled lysates derived from infected cells (viruses indicated at the top) were sedimented through sucrose gradients (9, 17). The polypeptide composition of C capsids was examined by SDS-polyacrylamide gel electrophoresis (15% acrylamide). The positions of the capsid proteins are indicated on the right of the figure; the only difference between the capsids was the lower mobility of the tagged VP19C, which was more evident for VP19C-mRFP (marked by the filled circle in the K19C-mRFP lanes). Molecular mass standards are shown in lane M, and the visible ones are 200, 96, 65, 46, 33, and 18 kDa.

In order to determine the surface topology of the different tags fused to VP19C, we carried out immuno-EM for purified capsids using antibodies to the various tags (Fig. 4). B capsids were purified after sedimentation of infected cell lysates through sucrose gradients. Capsids were adsorbed by placing a drop on freshly ionized carbon- and Formvar-coated nickel grids for 5 min, washed eight times by floating grids on a series of drops of deionized H₂O, and blocked for 1 h on a drop of 10% fetal calf serum in phosphate-buffered saline. Grids were then floated on a 10-µl drop of either rabbit anti-DsRed (mRFP), 10 µg/ml (Chemicon AB3216), mouse anti-HA (33 µg/ml; Covance 12CA5), or mouse anti-VP5 (diluted 1:20; LP12) and incubated at 4°C overnight. Grids were washed eight times in a wash buffer (1% fetal calf serum in phosphate-buffered saline) and floated on 10-µl donkey anti-rabbit 12-nm Au conjugate (diluted 1:30) or donkey anti-mouse 12-nm Au conjugate (diluted 1:40) (Jackson Research Laboratories) for 2 h at room temperature, washed eight times with wash buffer and five times with deionized H₂O, briefly floated on a drop of 2% uranyl acetate, partially dried by touching the side of the grid to filter paper, allowed to air dry, and observed with a Phillips EM410 or EM420 transmission electron microscope (FEI) operating at 100 kV. Images were recorded with a Megaview III digital camera (Olympus Soft Imaging Systems). Fields of greater than three intact capsids were selected by systematically scanning the grid and were recorded as described above. Gold particles visible on each intact capsid were counted, and the data were analyzed with Microsoft Excel's t test function. Numerous gold particles associated with KOS B capsids (Fig. 4E) were evident when VP5 antibodies (LP12) were used. None or very few gold particles were bound to KOS capsids when HA (Fig. 4C) or DsRed (Fig. 4A) antibodies were used. K19C-HA capsids exhibited little or no labeling when the capsids were reacted with HA antisera (Fig. 4D, arrowhead). This was not due to the loss of the epitope, because with similar sucrose gradient fractions, a 50-kDa polypeptide reacted with HA antisera in Western blots (data not shown). Many gold particles were observed bound to K19C-mRFP B capsids when DsRed antibody was used (Fig. 4B). A quantitative analysis was performed for these capsids as well as for KOS B capsids reacted with DsRed antibody. Intact capsids were enumerated, and the number of gold particles bound to the capsids was determined. The data revealed that KOS capsids exhibited little or no labeling (KOS, 0.27 gold particles/capsid) but that K19C-mRFP capsids exhibited significant labeling compared to KOS capsids (K19C-mRFP, 4.41 gold particles/capsid; t = 9.12; P value, 2.85 x 10^–17; n = 41 [one-tailed t test]). The mRFP tag was the only one that reacted with its cognate antibody, as judged by gold binding. We also generated a virus in which the HA tag was followed by a small peptide spacer sequence [Pro (Ser, Ala)₄ Pro] prior to the VP19C sequence. It was thought that this spacer sequence may expose the HA sequence at the capsid shell surface. B capsids isolated from this virus similarly did not react with antibodies to HA (data not shown).

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FIG. 4. Immuno-EM analysis of VP19C-tagged capsids. Immuno-EM was used to determine the accessibility at the surfaces of the capsid of HA and mRFP tags fused to the N terminus of VP19C. Purified B capsids isolated from sucrose gradients (17) were adsorbed to grids, reacted with antisera to DsRed (A and B), HA (C and D) or VP5 (LP12 [E]) and detected with 12-nm gold-labeled secondary antibody following negative staining with uranyl acetate. Gold particles were readily detected bound to KOS capsids when VP5 antibody (LP12) was used (E). Gold was not detected bound to KOS capsids when DsRed (A) and HA (C) antibodies were used. Occasionally, nonspecific gold particles were shown to bind to KOS capsids (arrowhead in panel A). Gold was detected bound to the K19C-mRFP capsids (arrowheads in panel B) but not to the K19C-HA capsids (D). Scale bar = 200 nm.

Because many of our studies have generally focused on studying bimolecular protein-protein interactions in isolated systems, our goal was to probe a multiprotein complex to ascertain the topology of protein domains. Hence, our aim was to determine the location of the N terminus of VP19C on the capsid shell. Use of this method of residue-specific ligand attachment to locate a protein domain in conjunction with high-resolution cryo-EM could potentially yield a three-dimensional map of the structural motif in the capsid shell. This method has been used successfully to locate the protein domains in the capsids of viruses (3, 6, 12, 14, 18). However, by using immuno-EM methods, it was not possible to detect the tag in the assembled capsid, indicating that the HA epitope was not exposed on the surface of the capsid. It is also possible, in the absence of a positive result from the HA tag, that the epitope is masked in the assembled shell even though the N terminus of VP19C is on the surface. However, the larger mRFP fusion protein somehow allowed for exposure of some or all epitopes of mRFP (polyclonal sera) on the capsid surface and gave reactivity to the antibody. Thus, one conclusion from the data is that the amino terminus of VP19C may not be exposed on the surface of the capsid shell, as judged by immuno-EM methods.

 
This work was supported by NIH Public Health Service grant AI33077.

We thank Stan Person for his critical comments on the manuscript and his strong support, insight, and enthusiasm for this work. We acknowledge the generous gift of the mRFP-1-expressing plasmid from Roger Tsien (UCSD). We thank Tony Minson (University of Cambridge) for the generous gift of antibody LP12.

 
* Corresponding author. Mailing address: Viral Oncology Program, The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, 353 CRB 1, 1650 Orleans Street, The Johns Hopkins University, Baltimore, MD 21231. Phone: (410) 614-1581. Fax: (410) 955-0840. E-mail: pdesai{at}jhmi.edu

Published ahead of print on 23 May 2007.

Present address: Division of Pulmonary, Allergy and Critical Care Medicine, Columbia University, New York, NY 10027.

Present address: Johns Hopkins Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205.

Present address: College of Dentistry, New York University, New York, NY 10010.

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Journal of Virology, August 2007, p. 8367-8370, Vol. 81, No. 15
0022-538X/07/$08.00+0     doi:10.1128/JVI.00819-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Solé, M. Articles by Desai, P. Search for Related Content PubMed PubMed Citation Articles by Solé, M. Articles by Desai, P.