Equine Herpesvirus 1 Entry via Endocytosis Is ... - Journal of Virology

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Apr 24, 2008 - Entry via endocytosis also depends on binding of gD to its receptor, which then results in activation of a ... ology and Immunology, College of Veterinary Medicine, Cornell Uni- versity, Ithaca, NY 14853. Phone: (607) 253-4045.
JOURNAL OF VIROLOGY, Dec. 2008, p. 11859–11868 0022-538X/08/$08.00⫹0 doi:10.1128/JVI.00868-08 Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Vol. 82, No. 23

Equine Herpesvirus 1 Entry via Endocytosis Is Facilitated by ␣V Integrins and an RSD Motif in Glycoprotein D䌤 Gerlinde R. Van de Walle,1,2 Sarah T. Peters,1 Brian C. VanderVen,1 Dennis J. O’Callaghan,3 and Nikolaus Osterrieder1,4* Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, New York 148531; Department of Virology, Parasitology, and Immunology, Faculty of Veterinary Medicine, Ghent University, 9820 Merelbeke, Belgium2; Department of Microbiology and Immunology, Louisiana State University Health Sciences Center, Shreveport, Louisiana 711303; and Institut fu ¨r Virologie, Freie Universita ¨t Berlin, 10115 Berlin, Germany4 Received 24 April 2008/Accepted 12 September 2008

Equine herpesvirus 1 (EHV-1) is a member of the Alphaherpesvirinae, and its broad tissue tropism suggests that EHV-1 may use multiple receptors to initiate virus entry. EHV-1 entry was thought to occur exclusively through fusion at the plasma membrane, but recently entry via the endocytic/phagocytic pathway was reported for Chinese hamster ovary cells (CHO-K1 cells). Here we show that cellular integrins, and more specifically those recognizing RGD motifs such as ␣V␤5, are important during the early steps of EHV-1 entry via endocytosis in CHO-K1 cells. Moreover, mutational analysis revealed that an RSD motif in the EHV-1 envelope glycoprotein D (gD) is critical for entry via endocytosis. In addition, we show that EHV-1 enters peripheral blood mononuclear cells predominantly via the endocytic pathway, whereas in equine endothelial cells entry occurs mainly via fusion at the plasma membrane. Taken together, the data in this study provide evidence that EHV-1 entry via endocytosis is triggered by the interaction between cellular integrins and the RSD motif present in gD and, moreover, that EHV-1 uses different cellular entry pathways to infect important target cell populations of its natural host. It has long been a dogma that alphaherpesviruses enter and productively infect permissive cells solely after direct fusion at the plasma membrane. Studies with herpes simplex virus type 1 (HSV-1), the prototypical alphaherpesvirus, have shown that basically five viral glycoproteins cooperate during viral entry at neutral pH: glycoprotein C (gC) and gB are important for the initial attachment of the virus, gD is necessary for stable binding to cell surface receptors and triggers fusion, and finally, gB in combination with gD and the gH-gL complex is responsible for the actual fusion of the viral envelope and the plasma membrane (38, 39). Recently, however, HSV-1 entry through an endocytic route was described for certain cell types, such as HeLa and Chinese hamster ovary (CHO-K1) cells (30, 31). Entry via endocytosis also depends on binding of gD to its receptor, which then results in activation of a number of cellular signaling pathways (29). Many viruses utilize the cellular endocytic machinery to gain entry into their target cell, and the first step generally involves the binding of the virus to receptors, in many cases members of the integrin family (34). Integrins are part of a large family of cell adhesion receptors that are involved in interactions between the cell and the extracellular matrix or between neighboring cells. They are of crucial importance for cell survival through a host of signaling pathways that are activated by binding to ligands (2, 27). Equine herpesvirus 1 (EHV-1) is another member of the subfamily Alphaherpesvirinae and is a major pathogen of horses worldwide. After initial infection, EHV-1 replicates in respira-

* Corresponding author. Mailing address: Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853. Phone: (607) 253-4045. Fax: (607) 253-3384. E-mail: [email protected]. 䌤 Published ahead of print on 24 September 2008.

tory epithelia and causes respiratory disease. Virus is then evident in the lymphatic tissues associated with the upper respiratory tract, reaches regional lymph nodes, and infects mononuclear cells that ultimately reach the bloodstream, resulting in a cell-associated viremia (4). Carried by the infected peripheral blood mononuclear cells (PBMC), EHV-1 can reach the vasculature of the central nervous system or the pregnant uterus, where viral replication in endothelial cells (EC) leads to disorders of the central nervous system or abortion as a result of vasculitis and ischemic thrombosis (37, 50). Similar to other alphaherpesviruses, EHV-1 also enters cells through direct fusion at the plasma membrane, a process that is mediated by gB, gC, gD, and possibly the gH-gL complex (6, 28, 32, 40, 49) and by a cellular receptor which has not been identified conclusively to date (10). Recently, it was shown by electron microscopy that EHV-1 can enter cells not only via direct fusion at the plasma membrane but also via a nonclassical endocytic/ phagocytic entry pathway (11). Which cellular and viral proteins are involved in entry via this endocytic pathway is not known. In this study, CHO-K1 cells were used as a model to evaluate the importance of cellular integrins during endocytic entry of EHV-1. Moreover, a possible integrin binding motif in the major alphaherpesviral entry mediator gD, consisting of arginine, serine, and aspartic acid (RSD) at amino acid positions 152 to 154, was evaluated by mutagenic analysis for its involvement in viral entry via endocytosis. Finally, the importance of this endocytic pathway for EHV-1 entry was evaluated in equine PBMC and EC, which represent cell types relevant during EHV-1 infection of the natural host. MATERIALS AND METHODS Viruses. EHV-1 strain L11⌬gp2 (46) and mutant viruses were grown on rabbit kidney cells (RK13) at 37°C under a 5% CO2 atmosphere. Virus was reconsti-

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J. VIROL. TABLE 1. Primers used in this study a

Name

Direction

Primers used for two-step Redmediated recombination gD152N

Forward Reverse

gD152D

Forward Reverse

Primers used for sequencing gD gD1 gD2

Forward Reverse

Sequenceb

CAACCCGAAGCTATCATTTGCAATGTGTGATGAGAGATCACACATACTAT GGCAAGCTAGTAGGATGACGACGATAAGTAGGG TCAGCAGCCATAGTAATTAAACTAGCTTGCCATAGTATGTGTGATCTCTC ATCACACATTGCAACCAATTAACCAATTCTGATTAG CAACCCGAAGCTATCATTTGCAATGTGTGATGAGAGATCAGACATACTAT GGCAAGCT AGTAGGATGACGACGATAAGTAGGG TCAGCAGCCATAGTAATTAAACTAGCTTGCCATAGTATGTCTGATCTCTC ATCACACATTGCAACCAATTAACCAATTCTGATTAG CAATTTTATGCCTGCTGTGCTG ACTCAGAGCTGGCGTTTAGGTA

a

Directionality of the primer. Underlined sequences indicate the template binding regions of the primers used for PCR amplification with pEPKanS. Bold letters indicate the nucleotides that were mutated. b

tuted after transfection of 5 ␮g bacterial artificial chromosome (BAC) DNA into RK13 cells, using the CaPO4 precipitation method (35). Baculoviruses were grown in Sf9 insect cells at 28°C as detailed earlier (21). Proteins, inhibiting agents, and antibodies. Oregon green 488-labeled dextran (molecular weight, 10,000) was obtained from Molecular Probes. Hypotonic K⫹ buffer at 150 mosM (142 mM KCl, 3.6 mM CaCl2, 20 mM HEPES, 0.34 mM K2HPO4, 0.35 mM KH2PO4, and 0.81 mM MgSO4) was used as an agent to block endocytosis/phagocytosis (48). The tetrapeptide Arg-Gly-Asp-Ser (RGDS), an antagonist of integrin function, and its control peptide, Ser-Asp-Gly-Arg-Gly (SNGRG), were purchased from Sigma. The anti-human CD51/61 monoclonal antibody (MAb), an ␣V␤3 integrin antagonist, and MAb P1F6, an ␣V␤5 integrin antagonist, were obtained from Biolegend and Millipore, respectively. An unrelated mouse immunoglobulin G (IgG) isotype control was obtained from Santa Cruz Biotechnologies. The MAb 20C4 was kindly supplied by G. P. Allen, University of Kentucky, and was used to detect expression of EHV-1 gD. The anti-EHV-1 ETIF MAb L3ab and anti-EHV-1 gD 19-mer polyclonal Ab were described earlier (8, 23). Peroxidase-labeled anti-mouse IgG was obtained from Jackson Immunoresearch Laboratories, and goat anti-mouse or anti-rabbit IgG antibodies conjugated with Alexa fluor 488 or 568 were obtained from Molecular Probes. Cells. RK13 and equine dermal (NBL6) cells were grown in minimal essential medium supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 0.1 mg/ml streptomycin (1% penicillin-streptomycin). CHO-K1 cells were grown in Ham’s F12K medium (Invitrogen) supplemented with 10% FBS. PBMC were isolated from heparinized blood collected from healthy horses by density gradient centrifugation over Histopaque 1077 (Sigma), following the manufacturer’s instructions. After two washing steps, cells were resuspended in RPMI 1640 supplemented with 10% FBS, 0.3 mg/ml glutamine, 0.1 mg/ml kanamycin, 1% 100⫻ nonessential amino acids (Mediatech), and 1% penicillinstreptomycin. To isolate primary equine vascular EC from the carotid arteries of healthy control horses, collagenase (type II; Sigma) treatment was used as described previously (24). To separate the EC from contaminating cells, cultures were labeled with 10 ␮g/ml of the low-density lipoprotein 1,1⬘-dioctadecyl3,3,3⬘,3⬘-tetramethylindo-carbocyanide perchlorate (DiI-Ac-LDL; Biomedical Technologies Inc.) for 4 h at 37°C. After trypsinization, EC were washed, resuspended in medium, and sorted by fluorescence-activated cell sorting (FACS) using a FACS Aria high-speed flow cytometer (Becton Dickinson). The DiI-AcLDL fluor signal was detected with a 585/45 BP filter, and the brightest 22% of cells were gated and sorted at 65 lb/in2 into medium. Coupling of recombinant gD to beads and fluorescent labeling. Suspension cultures of Sf9 insect cells were infected with either an EHV-1 gG-expressing baculovirus (44) or an EHV-1 gD-expressing baculovirus (7) at a multiplicity of infection (MOI) of 0.5. Seventy-two hours after infection, cells were collected and lysed in lysis buffer (1% NP-40, 50 mM Tris-HCl, and 150 mM NaCl, pH 7.4) for 10 min on ice. Cell lysates were clarified by centrifugation, and the protein concentration was determined. Washed, carboxylated, 1-␮m silica beads (Kisker Biotech, Steinfurt, Germany) were covalently coupled to 2 mg protein as described previously (41). Coated beads were subsequently labeled with Alexa fluor 488 succinimidyl ester (Molecular Probes) according to the manufacturer’s instructions.

Mutagenesis of pL11. The EHV-1 strain RacL11 was cloned as a BAC (pL11), with mini-F vector sequences containing the origin of replication, a chloramphenicol resistance gene (cat), and the egfp gene in lieu of the nonessential gene 71 (encoding glycoprotein gp2) (35). The pL11 BAC was maintained in Escherichia coli GS1783 cells (a kind gift from Gregory A. Smith, Northwestern University, Chicago, IL). Virus reconstituted from pL11 was used in this study to make use of enhanced green fluorescent protein (EGFP) expression for rapid identification of infected cells. A point mutation in gD was engineered by converting nucleotide 608 of gD (orf72) from a guanine to an adenine, changing the aspartic acid into an asparagine (gD152N), by employing two-step Red-mediated recombination exactly as previously described (20, 43) (see Fig. 4A). Primers used for the mutant (gD152N) and revertant (gD152D) viruses can be found in Table 1. The respective genotypes were confirmed by nucleotide sequencing (Table 1). IFA and Western blotting. For indirect immunofluorescence assay (IFA), NBL6 cells were seeded in six-well plates and infected with EFGP-expressing viruses at an MOI of 0.001. At 1 hour postinfection (p.i.), medium was removed and infected cells were overlaid with medium A supplemented with 0.25% methylcellulose. At 48 h p.i., cells were fixed with 10% formalin in phosphatebuffered saline (PBS) for 15 min at room temperature (RT). After a washing step with PBS, nonspecific binding sites were blocked with PBS supplemented with 0.5% bovine serum albumin for 30 min at RT and subsequently incubated with 20C4 at a 1/500 dilution in PBS for 30 min at RT. After being washed with PBS, cells were incubated with Alexa fluor 568-conjugated goat anti-mouse IgG antibody for 30 min at RT. Fluorescence was evaluated and recorded using an inverted fluorescence microscope (Zeiss Axiovert 25 and Axiocam). For Western blot analysis, pellets of infected NBL6 cells were resuspended in radioimmunoprecipitation assay buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1 mM EDTA, 0.1% sodium dodecyl sulfate [SDS]) with a protease inhibitor cocktail (Roche). Sample buffer (0.15 M Tris-HCl, pH 6.8, 1.2% SDS, 0.3% glycerol, 0.15% ␤-mercaptoethanol, 0.018% [wt/vol] bromophenol blue) was added, and samples were subjected to 12% SDS-polyacrylamide gel electrophoresis exactly as described before (45). Expression of gD was evaluated with 20C4 (1/1,000 dilution), and L3ab (1/2,000 dilution) was used as a control antibody. Anti-mouse IgG coupled to peroxidase (Sigma) (1/7,500 dilution) was used as the secondary antibody. In vitro replication and attachment assays. To determine replication of the EGFP-expressing viruses, single-step replication kinetics and plaque areas were determined exactly as previously described (45). Briefly, plaque areas on RK13 cells were measured after infection at an MOI of 0.001 of cells seeded in a six-well plate and overlaid at 1 h p.i. with medium A containing 0.25% methylcellulose (Sigma). At 3 days p.i., 50 fluorescent plaques were photographed for each virus, and average plaque areas were determined using ImageJ software (http://rsb.info.nih.gov/ij/). Values were compared to L11⌬gp2 plaque diameters, which were set to 100%. Average percentages of plaque areas were determined from at least three independent experiments. Single-step growth kinetics were determined after infection of 1 ⫻ 105 RK13 cells at an MOI of 3. Virus was allowed to attach for 30 min at 4°C, followed by a penetration period of 1.5 h at 37°C. At the indicated times p.i., supernatants were collected and extracellular

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viral titers were determined by plating onto RK13 cells. At 3 days p.i., cells were fixed with 10% formalin in PBS for a plaque assay and stained with 0.3% crystal violet, and plaques were counted. Single-step growth curves were determined in three independent experiments. For attachment assays, CHO-K1 and equine EC were preincubated with or without the RGDS peptide or the control SNGRG peptide (200 ␮g/ml) for 30 min and cooled to 4°C for 30 min, after which EGFP-expressing viruses at an MOI of 1 were allowed to attach to the cells at 4°C. After 30 min, supernatants were collected and unbound virus was titrated by being plated onto RK13 cells, exactly as described above. Flow cytometry. To evaluate uptake by endocytosis/phagocytosis, six-well plates of CHO-K1 cells were incubated with Oregon green-labeled dextran (1 mg/ml) for 4 h at 37°C in the presence or absence of hypotonic K⫹ buffer. CHO-K1 cells were detached using trypsin (Gibco) and washed twice. To evaluate uptake of gD-coated beads, CHO-K1 cells were incubated with 1 ⫻ 107 beads, coated with gD or irrelevant proteins as described above, for 3 h at RT. Where indicated, CHO-K1 cells or beads were preincubated with blocking Abs (10 ␮g/ml), peptides (200 ␮g/ml), or their respective controls for 30 min at RT. After being washed with PBS, CHO-K1 cells were split into two aliquots and diluted 1:1 with PBS (unquenched) or 0.4% trypan blue from Cellgro (quenched) for 10 min at RT. Unbound trypan blue was removed by centrifugation, and the percent uptake was determined by the following formula: 100 ⫻ (FL1 after quenching/FL1 without quenching). To evaluate integrin expression, CHO-K1 cells were incubated with the anti-integrin ␣V␤5 MAb (1 ␮g/ml) and EC or PBMC were incubated with the anti-integrin ␣V␤3 MAb (2.5 ␮g/ml) for 30 min at RT. The isotype control mouse IgG (1 ␮g/ml) was also included. After being washed twice with PBS, cells were incubated with Alexa fluor 488-labeled goat anti-mouse IgG (1/500 dilution) for 30 min at RT. After a final wash, 10,000 cells were analyzed with a FACSCalibur flow cytometer (Becton Dickinson), and the intensity of fluorescence was analyzed using Cell Quest software (Becton Dickinson). For infection experiments, six-well plates of CHO-K1 cell, EC, or PBMC suspensions were infected with L11⌬gp2 at an MOI of 1 or 0.025 (EC) for 2 h at 37°C. Where indicated, inhibiting agents were added 30 min before infection (peptides [200 ␮g/ml] or antibodies [10 ␮g/ml]) or 10 min before infection (hypotonic K⫹ buffer). In other experiments, cells were infected with the mutant L11gD152N or revertant L11gD152D virus. After being washed extensively, CHO-K1 cells and EC were overlaid with medium supplemented with 0.25% methylcellulose, and PBMC were thoroughly washed and resuspended in medium. CHO-K1 cells were detached at several times (2 to 16 h) p.i. and washed twice, and PBMC were washed twice at 48 h p.i. After centrifugation, cells were resuspended in PBS supplemented with 10 ␮g propidium iodide (PI; Molecular Probes), and the intensity of fluorescence of 10,000 cells was analyzed using FACScan analysis and Cell Quest software (both from Becton Dickinson) to determine the percentage of infected cells. Statistical analysis. Student’s t test for paired data was used to test for statistically significant differences. Data given are means, and bars show standard deviations.

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FIG. 1. (A) Hypotonic K⫹ buffer reduces EHV-1 entry into CHO-K1 cells. CHO-K1 cells were preincubated with (gray bars) or without (white bars) 150 mosM hypotonic K⫹ buffer for 10 min at 37°C, followed by infection with L11⌬gp2 at an MOI of 1 for 2 h at 37°C. At various times p.i. (2 to 16 h), cells were detached and the percentage of infected cells was determined by flow cytometry. All data are expressed as means ⫾ standard deviations for at least three independent experiments. Asterisks indicate statistically significant differences (P ⫽ 0.01). (B) Hypotonic K⫹ buffer reduces dextran uptake in CHO-K1 cells. CHO-K1 cells were preincubated with (gray bars) or without (white bars) 150 mosM hypotonic K⫹ buffer for 10 min at 37°C, followed by incubation with Oregon green-labeled dextran (1 mg/ml) for 4 h at 37°C. (a) The mean fluorescence intensity of at least 10,000 cells was determined by flow cytometry. All data are expressed as means ⫾ standard deviations for at least three independent experiments, and P values for statistical significance are given. (b) Representative histograms of CHO-K1 cells (solid line) and CHO-K1 cells with Oregon green-labeled dextran in the absence (dotted line) or presence (dashed line) of hypotonic K⫹ buffer.

Integrin ␣V␤5 is involved in endocytic EHV-1 entry into CHO-K1 cells. Recently, it was described that EHV-1 can productively infect CHO-K1 cells via a nonclassical endocytic/ phagocytic entry pathway (11). As no experimental data are available thus far on how this entry takes place, we sought to examine which cellular and viral proteins are of importance during this process. First, CHO-K1 cells were infected with the EGFP-expressing EHV-1 strain L11⌬gp2 in the presence or absence of hypotonic K⫹, an endocytosis/phagocytosis-inhibiting agent. After virus attachment, unbound virus was removed and cells were further incubated for as long as 16 h. EGFP expression in infected cells was detectable starting at 4 h p.i. and reached a plateau at 6 h p.i., with 17.3% ⫾ 3.2% of cells being positive for the marker (Fig. 1A). In the presence of hypotonic K⫹, the percentage of infected cells was significantly reduced (Fig. 1A) (P ⫽ 0.01 for 4 to 16 h p.i.). To ensure that the observed effect was a consequence of alterations in the rate

and efficiency of endocytosis rather than alteration of fusion kinetics at the plasma membrane, experiments were performed with Oregon green 488-labeled dextran, which is a valuable tool to evaluate uptake by phagocytic and endocytic pathways. It was observed that fluorescently labeled dextran was readily taken up by CHO-K1 cells, but this uptake was significantly reduced in the presence of hypotonic K⫹ (Fig. 1B) (P ⫽ 0.01). The control experiments suggested that the observed reduction of infectivity in the presence of hypotonic K⫹ was indeed caused by a reduction in the rate of endocytosis. Since several members of the integrin receptor family facilitate entry of a number of viruses, we evaluated the effect of the tetrapeptide RGDS, an antagonist of integrin function, on CHO-K1 cell infection. RGD is a motif present in several adhesion molecules, for example vitronectin, and is recognized

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FIG. 2. Integrin inhibition results in decreased EHV-1 infectivity in CHO-K1 cells. (A) Cells were preincubated with medium, the integrin antagonist peptide RGDS, or control peptide SNGRG for 30 min at 37°C, followed by infection with L11⌬gp2 at an MOI of 1 for 2 h at 37°C. At 16 h p.i., cells were detached and washed, and the percentage of infected cells was determined by flow cytometry. All data are expressed as means ⫾ standard deviations for at least three independent experiments. P values for statistical significance are given. (B) Cells were preincubated with medium (control), RGDS, or SNGRG for 30 min at 4°C. Next, L11⌬gp2 at an MOI of 1 was allowed to attach to the cells for 30 min at 4°C, after which supernatants were collected and titrated on RK13 cells. Data are expressed as percentages of virus yield compared to the control and are means ⫾ standard deviations for at least three independent experiments. (C) CHO-K1 cells in suspension were incubated with anti-␣V␤5 MAbs for 30 min at RT, followed by incubation with Alexa fluor 488-labeled goat anti-mouse IgG for 30 min at RT (dashed line and arrow). As controls, cells were not incubated with primary antibodies (solid line) or were incubated with IgG isotype control MAbs (dotted line). Integrin expression was determined by flow cytometry. (D) Cells were preincubated with medium, ␣V␤5 function-blocking MAbs, or IgG control MAbs for 30 min at 37°C, followed by infection with L11⌬gp2 at an MOI of 1 for 2 h at 37°C. At 16 h p.i., cells were detached and the percentage of infected cells was determined by flow cytometry. All data are expressed as means ⫾ standard deviations for at least three independent experiments. P values for statistical significance are given.

by several integrins, such as ␣V␤3, ␣V␤5, and ␣5␤1 (36). Pretreatment of CHO-K1 cells with the RGDS peptide before infection resulted in a significantly decreased rate of infection (Fig. 2A) (P ⫽ 0.03), while the control peptide SNGRG did not alter EHV-1 infectivity compared to that of nontreated cells (Fig. 2A). To ensure that the observed effect was not caused merely by a decrease in binding of the virus to integrins,

J. VIROL.

attachment experiments were performed. As seen in Fig. 2B, no difference in virus attachment was observed in the presence of the integrin-blocking peptide RGDS (P ⫽ 0.6). Similarly, the control peptide SNGRG did not influence the amount of virus that could bind to the cells either (Fig. 2B) (P ⫽ 0.3). Based on these results, we concluded that integrins do not play a major role in attachment of the virus to cells but during endocytosis, at a postbinding event, potentially by inducing conformational changes of the receptor and initiating signaling cascades. In addition, the experiments indicated that EHV-1 entry into CHO-K1 cells appears to require the use of integrins via recognition of an RGD motif. The RGD-binding integrin ␣V␤5 is known to be expressed on CHO-K1 cells (1), and therefore function-blocking antibodies against ␣V␤5 were used to investigate its role during EHV-1 infectivity. First, IFA was performed to ensure proper expression of ␣V␤5 with the function-blocking antibodies (Fig. 2C). We observed that pretreating CHO-K1 cells with these antibodies resulted in a significant decrease of infected cells (Fig. 2D) (P ⫽ 0.01). Since preincubating CHO-K1 cells with IgG isotype control antibodies did not have any effect on EHV-1 infectivity (Fig. 2D), we concluded that the ␣V␤5 integrin plays an important role during EHV-1 entry in CHO-K1 cells, presumably by interaction with an RGD or RGD-like motif present in a viral envelope protein. The RSD motif in gD is a viral ligand for EHV-1 endocytic entry in CHO cells. A search for RGD motifs in EHV-1 envelope glycoproteins revealed the presence of such motifs in gB and gD, which are both essential for viral entry and cellto-cell spread (6, 28). However, because the RGD motif in gB appeared to be located in the predicted signal sequence, we focused on gD. gD of EHV-1 contains an RSD amino acid sequence, a motif described to be functionally equivalent to RGD, at position 152 (15). First, the possible involvement of gD during endocytic entry in CHO-K1 cells was investigated using beads coated with recombinant EHV-1 gD. Flow cytometric analysis was used to evaluate the uptake of beads coated with cell lysates from insect cells infected with gD-expressing baculovirus. Western blot analysis and IFA demonstrated the presence of gD (i) in cell lysates and (ii) on coated beads (data not shown). As controls, beads coated with cell lysates from mock-infected insect cells or insect cells infected with soluble gG (sgG)-expressing baculovirus were used. Beads were incubated with CHO-K1 cells, and significantly more gD-coated beads than mock- or sgG-coated beads were taken up by CHO-K1 cells (Fig. 3A) (P ⫽ 0.04). Moreover, uptake of gD-coated beads could be inhibited by preincubating cells with ␣V␤5 antibodies or RGD peptide. Similarly, preincubating gD-coated beads with an anti-gD antibody prevented uptake of beads (Fig. 3A) (P ⫽ 0.05). In contrast, the IgG isotype control or the scrambled SNGRG peptide did not influence the uptake of gD-coated beads (Fig. 3A). We therefore concluded that uptake in CHO-K1 cells is indeed mediated by a specific interaction between EHV-1 gD and the ␣V␤5 integrin. To ensure that only uptake of beads, not merely attachment, was studied, quenching with trypan blue was used as described in Materials and Methods, and fluorescence microscopy was performed for comparison (Fig. 3B). To test the hypothesis that the RSD motif present in gD is responsible for the observed effect, we mutated the motif in the

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FIG. 3. gD-coated beads are readily taken up by CHO-K1 cells. (A) Silica beads coated with fluorescently labeled EHV-1 gD were fed to CHO-K1 cells for 3 h at RT. As controls, mock- or sgG-coated beads were used. In some instances, gD-coated beads were preincubated with anti-gD antibodies (anti-19-mer; 10 ␮g/ml) or CHO-K1 cells were preincubated with ␣V␤5 function-blocking MAbs (10 ␮g/ ml) or RGDS peptide (200 ␮g/ml). The IgG isotype control (10 ␮g/ml) and SNGRG peptide (200 ␮g/ml) were included as controls. The percentage of bead uptake was determined using trypan blue as a quencher, exactly as described in Materials and Methods. All data are expressed as means ⫾ standard deviations for at least three independent experiments. (B) Representative light and immunofluorescence pictures of CHO-K1 cells incubated with gD-coated beads or control sgG-coated beads. Bar, 10 ␮m.

EHV-1 RacL11 BAC by changing the aspartic acid into an asparagine (gD152N) (Fig. 4A). In addition, the gD152N mutant was used to engineer a revertant virus (gD152D). Both the mutant and revertant viruses expressed gD at levels comparable to that of the original L11⌬gp2 virus, as determined by IFA (Fig. 4B) and Western blot analysis (Fig. 4C). In addition, three standard plaque size measurements and independent single-step growth kinetic experiments were performed with RK13 cells (45). Plaques formed by mutant and revertant viruses were comparable to those of parental L11⌬gp2 (Fig. 5A). In addition, all viruses grew to similar virus titers in RK13 cells and produced virtually identical extracellular viruses (Fig. 5B). These results are in agreement with a previous study where it was reported that EHV-1 entry into RK13 cells occurs by direct fusion at the plasma membrane (11). Flow cytometry was then used to determine the infection rates of CHO-K1 cells, and it was observed that the percentage of cells infected with the L11gD152N mutant was significantly lower than that seen with the L11gD152D revertant (Fig. 5C) (P ⫽ 0.003). This difference could be attributed to a difference in EHV-1 entry via the endocytic pathway, since using the L11gD152D revertant virus in the presence of hypotonic K⫹ resulted in a comparable decrease in the number of infected cells (Fig. 5C) (P ⫽ 0.032). In addition, no difference (P ⫽ 0.1) in the percentage of infected CHO-K1 cells could be observed in the presence or absence of hypotonic K⫹ when the

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FIG. 4. Characterization of mutant virus L11gD152N. (A) Generation of mutant L11gD152N virus and L11gD152D revertant virus. The schematic shows the overall genome of the recombinant pL11 BAC, consisting of a unique long (UL) and a unique short (US) segment bracketed by inverted internal repeat (IR) and terminal repeat (TR) sequences. A depiction of the unique short segment of pL11, with arrowheads indicating the transcriptional direction of the genes, is shown. The aphAI gene was inserted at amino acid position 152 of gene 72 (encoding gD), which is located downstream of the mini-F vector sequences, which also contain the egfp gene. The nucleotide sequences of the gD proteins of the original L11 virus (gDWT), the mutant virus (gD152N), and the revertant virus (gD152D) are shown. (B) Wild-type and mutant viruses express gD at similar levels. NBL6 cells were infected with wild-type L11⌬gp2, mutant L11gD152N virus, or the revertant L11gD152D virus at an MOI of 0.0001. (a) For immunofluorescence analyses, plaques were stained with anti-gD MAb 20C4 (1/500) followed by anti-mouse Alexa fluor 568 and were recorded with an inverted fluorescence microscope (Zeiss Axiovert 25). (b) For Western blot analysis, cell lysates were prepared from each sample and analyzed by SDS-polyacrylamide gel electrophoresis under reducing conditions. Anti-gD MAb 20C4 (1/1,000) or the anti-ETIF MAb L3ab (1/2,000), followed by anti-mouse IgG peroxidase (1/7,500), was used. Cell lysates from noninfected NBL6 cells were included as a control.

L11gD152N mutant virus was used (Fig. 5C). Also, the relative percentages of virus yield recovered after attachment assays with these mutant viruses were virtually identical (91.6% ⫾ 29.6% for the L11gD152N mutant versus 86.3% ⫾ 5.4% for the L11gD152D revertant), indicating that we were looking at the early steps of entry and not at differences in attachment kinetics between the two viruses. Likewise, no statistically significant difference was observed in the percentage of nonviable cells (i) upon infection with the different viruses and (ii) in the presence or absence of hypotonic K⫹, as determined by propidium iodide staining, indicating that normal cellular processes, in-

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FIG. 5. (A and B) In vitro growth kinetics of wild-type and mutant viruses. (A) Plaque sizes were determined after 3 days p.i. with mutant L11gD152N or the revertant L11gD152D and compared to the plaque size of wild-type L11⌬gp2, which was set to 100%. (B) For single-step growth kinetic experiments, RK13 cells were infected at an MOI of 3 with wild-type L11⌬gp2, mutant L11gD152N, or the revertant L11gD152D virus. Supernatants were collected at the indicated time points, and virus titers were determined by standard plaque assay. The results are expressed as means ⫾ standard deviations for three independent experiments. (C) Infection with the L11gD152N mutant results in decreased infectivity in CHO-K1 cells. CHO-K1 cells were infected with the mutant L11gD152N virus or the revertant L11gD152D virus in the presence (gray bars) or absence (white bars) of hypotonic K⫹ buffer at an MOI of 1. At 16 h p.i., CHO-K1 cells were detached and washed, and the percentage of infected cells was determined by flow cytometry (FACSCalibur; Becton Dickinson). Data are expressed as means ⫾ standard deviations for at least three independent experiments. P values for statistical significance are given.

cluding endocytosis, were similar under the experimental conditions used and did not influence the results (data not shown). EHV-1 entry via endocytosis is cell type dependent. Because EHV-1 is able to infect a broad range of different cell types, we included two cell types relevant during EHV-1 pathogenesis, namely, equine PBMC and EC, to substantiate our discovery of EHV-1 entry via endocytosis, using cellular integrins and the RSD motif present in gD. First, PBMC (predominantly B and T lymphocytes) are highly relevant to EHV-1 pathogenesis, as they initiate and sustain cell-associated viremia (4). We started with infection experiments using the L11gD152N mutant virus and the L11gD152D revertant virus and analyzed the percentages of infected cells by flow cytometry. Very similar to the results obtained with CHO-K1 cells, the percentage of infected PBMC was significantly reduced upon infection with the L11gD152N mutant virus (Fig. 6A) (P ⫽ 0.01). The effect could be attributed to EHV-1 entry via endocytosis since (i) treat-

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FIG. 6. Infection with L11gD152N mutant or preincubation with ␣V␤3 function-blocking antibodies results in decreased infectivity in equine PBMC. (A) PBMC were infected with the mutant L11gD152N virus or the revertant L11gD152D virus in the presence (gray bars) or absence (white bars) of hypotonic K⫹ buffer at an MOI of 1 for 2 h at 37°C. At 48 h p.i., PBMC were washed and the percentage of infected cells was determined by flow cytometry (FACSCalibur; Becton Dickinson). Data are expressed as means ⫾ standard deviations for at least three independent experiments. P values for statistical significance are given. (B) PBMC were incubated with anti-␣V␤3 MAbs for 30 min at RT, followed by incubation with Alexa fluor 488-labeled goat antimouse IgG for 30 min at RT (dashed line and arrows). As controls, cells were not incubated with primary antibodies (solid line) or were incubated with IgG isotype control MAbs (dotted line). Integrin expression was determined by flow cytometry. (C) Cells were preincubated with medium, ␣V␤3 function-blocking MAbs, or IgG control MAbs for 30 min at 37°C, followed by infection with L11⌬gp2 at an MOI of 1 for 2 h at 37°C. At 48 h p.i., cells were washed and the percentage of infected cells was determined by flow cytometry. All data are expressed as means ⫾ standard deviations for at least three independent experiments. P values for statistical significance are given.

ment of L11gD152D-infected cells with hypotonic K⫹ resulted in a comparable decrease of infected cells (Fig. 6A) (P ⫽ 0.02) and (ii) no difference in the percentage of L11gD152N-infected cells could be observed in the presence or absence of hypotonic K⫹ (Fig. 6A) (P ⫽ 0.7). In addition, we used anti-integrin antibodies to investigate the role of integrins during endocytic entry of EHV-1 into PBMC. In contrast to CHO-K1 cells, PBMC express ␣V␤3 instead of ␣V␤5 integrin. First, crossreactivity of anti-human ␣V␤3 antibodies with equine cells was investigated, and flow cytometric analysis showed a mixture of low and high ␣V␤3 expression on the equine monocyte and lymphocyte populations (Fig. 6B). PBMC were then preincu-

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FIG. 7. Integrin inhibition and infection with the L11gD152N mutant do not alter infectivity in equine EC. EC were preincubated with medium, the integrin antagonist peptide RGDS, or the control peptide SNGRG (200 ␮g/ml) (A) or with medium, ␣V␤3 function-blocking MAbs (10 ␮g/ml), the anti-gD anti-19-mer Ab (10 ␮g/ml), or IgG control MAbs (10 ␮g/ml) (B, panel b) for 30 min at 37°C, followed by infection with L11⌬gp2 at an MOI of 0.025 for 2 h at 37°C. P values for statistical significance are given. (B, panel a) Cells were incubated with anti-␣V␤3 MAbs for 30 min at RT, followed by incubation with Alexa fluor 488-labeled goat anti-mouse IgG for 30 min at RT (dashed line and arrow). As controls, cells were not incubated with primary antibodies (solid line) or were incubated with IgG isotype control MAbs (dotted line). Integrin expression was determined by flow cytometry. (C) In another set of experiments, cells were infected with the mutant L11gD152N virus or the revertant L11gD152D virus in the presence (gray bars) or absence (white bars) of hypotonic K⫹ buffer at an MOI of 0.025 for 2 h at 37°C. At 16 h p.i., cells were detached and washed, and the percentage of infected cells was determined by flow cytometry. All data are expressed as means ⫾ standard deviations for at least three independent experiments.

bated with the ␣V␤3 function-blocking antibody, which resulted in a significant decrease in EHV-1-infected PBMC compared to nontreated PBMC (Fig. 6C) (P ⫽ 0.01). In contrast, the IgG isotype control did not alter EHV-1 infectivity (Fig. 6C). When similar experiments were performed using primary equine EC instead, no difference in infectivity could be observed under all conditions tested, indicating that endocytosis appears to be a rather insignificant entry pathway for EHV-1 with this cell type. Equine EC are crucial during EHV-1 pathogenesis since infection and replication of EHV-1 in this cell type ultimately lead to vasculitis, thrombosis, and ischemia in several organs (37, 50). For these experiments, an MOI of 0.025 was used to obtain around 15% infected EC, a percentage similar to what was observed for CHO cells and PBMC. Neither incubation with RGDS peptide (200 ␮g/ml) nor antibody treatment (10 ␮g/ml) reduced infectivity of the L11⌬gp2 virus (Fig. 7A and B). For the latter experiments, the functionblocking antibody against ␣V␤3 was used, as flow cytometric analysis revealed expression of this integrin on primary equine EC (Fig. 7B), whereas no expression of ␣V␤5 could be detected (data not shown). In contrast, using the neutralizing anti-19-mer Ab against EHV-1 gD (10 ␮g/ml) as a positive control, a significant decrease in the percentage of infected cells was observed (Fig. 7B) (P ⫽ 0.01). In addition, the mutant L11gD152N and revertant L11gD152D viruses infected EC to the same extent, and neither virus was sensitive to treatment with hypotonic K⫹ (Fig. 7C). Also with this cell type, no differences in virus yield were observed with the attachment as-

says in the presence or absence of the RGDS peptides (data not shown), indicating that at least RGD-binding integrins expressed on EC are not important for attachment of EHV-1 to this cell type. Taken together, these results demonstrated that entry of EHV-1 via the endocytic pathway is clearly cell type dependent and is the main entry pathway with PBMC. DISCUSSION During the last years, much progress has been made in determining the roles of different entry pathways of herpesviruses. It has been reported that HSV-1, the prototypical alphaherpesvirus, enters certain cells through fusion with the plasma membrane and others via endocytosis. Moreover, it was shown that the cell type and the respective receptors are the main determinants of whether the fusion process occurs at the plasma membrane or through an endocytic route of entry (13). Recently, it was shown that EHV-1 can enter cells by these two different pathways as well (11). In the present study, we could demonstrate that ␣V integrins are important during the early steps of EHV-1 entry via endocytosis and, more specifically, through the interaction of the virus with an Arg-Gly-Asp (RGD) recognition site. The RGD motif is the minimal peptide region of many proteins known to interact with subsets of host cell surface integrins, such as ␣V␤3, ␣V␤5, and ␣3␤1 (2, 42). Such RGD motifs are also essential for integrin receptor binding of many enveloped and nonenveloped viruses, such as foot-and-mouth disease virus and coxsackievirus (5, 9). Like the case for other herpesviruses,

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human herpesvirus 8 has been shown to enter certain cell types via endocytosis upon binding to integrin ␣3␤1, also mediated by an RGD sequence present in gB (3, 47). In the case of HSV-1, viral interaction with integrins was evaluated by employing peptides containing known integrin recognition sites and blocking antibodies. No effect on HSV-1-induced plaque formation in epithelial Hep-2 cells could be observed by using an RGD peptide or MAbs to the human ␤1 or ␤4 integrin subunit (19). This is in contrast to our results where the RGD peptide and the function-blocking ␣V␤3 antibody had a significant effect on productive EHV-1 infection in CHO-K1 cells. In subsequent experiments, we could demonstrate by mutational analysis that an RSD motif present in EHV-1 gD is involved in the early steps of EHV-1 uptake via endocytosis. Previously, mutational analyses of integrin binding motifs in viral glycoproteins were also done with HSV-1. In one study, a recombinant soluble form of HSV-1 gH was created and showed binding to Vero cells and CHO-K1 cells expressing human ␣V␤3 (33). Binding was abolished upon mutation of an RGD motif in the glycoprotein, indicating that HSV-1 can bind to integrins via an RGD motif present in gH. In another study, however, a recombinant HSV-1 with a point mutation targeting the exact same motif showed no growth deficits in vitro and was still able to enter cells at rates equivalent to those of wild-type virus (12), which raises questions about the functional significance of the gH RGD-mediated interaction with ␣V␤3 integrins. Based on the data obtained in this study, the RSD motif in EHV-1 gD does appear to be functionally important, as infection with the L11gD152N virus was clearly less efficient than that with wild-type EHV-1. Recently, HSV-1 gD was shown to be important for entry into C10 melanoma cells via low-pH-independent endocytic entry, and the presence of gD was absolutely necessary for the ability of HSV-1 to successfully infect CHO-K1 cells by endocytosis (26, 31). The precise motif(s) in HSV-1 gD responsible for the virus-receptor interaction during endocytosis, however, has not been identified to date. In the last part of this study, we extended our observations about the mechanisms by which EHV-1 enters permissive cells beyond hamster CHO-K1 cells to cell types that are more relevant to EHV-1 replication in vivo. We were able to show that EHV-1 entry in equine EC does not occur primarily via endocytosis but via fusion at the plasma membrane (Fig. 8). Interestingly, when we used very low MOIs to infect EC, a significant difference in the percentage of infected cells became apparent (data not shown), indicating that endocytic entry of EHV-1 in this cell type might occur, albeit at a very low efficiency. The presence of enveloped EHV-1 particles within uncoated vesicles has been reported before for equine brain microvascular EC, suggesting the utilization of an endocytic entry pathway for equine cells of endothelial origin (16). Similar experiments with equine monocytes and lymphocytes showed that in contrast to the case with EC, endocytosis appears to be a biologically relevant and important pathway for EHV-1 entry and infection in these cells (Fig. 8). Very similar to our observations with EHV-1, it has been reported that Epstein-Barr virus infects epithelial cells via fusion at the plasma membrane but requires pH-independent endocytosis for entry into B cells (17, 25). In the case of HSV-1, viral entry was also analyzed in two human cell types that seem relevant

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FIG. 8. Putative model of the steps involved in the different EHV-1 entry pathways in CHO-K1 cells, equine EC, and equine PBMC. (A) Initial attachment of EHV-1 occurs via binding of gC to proteoglycans. (B1) Fusion at the plasma membrane involves interaction of gB, gD, and possibly the gH/gL complex with one or more putative cellular receptors. This leads to the efficient release of nucleocapsids (hexagons) into the cytoplasm. Fusion at the plasma membrane appears to be the primary entry pathway used by equine EC and also by RK13 cells. (B2) For other cell types, such as CHO-K1 cells and PBMC, however, viral entry seems to occur primarily via endocytosis, which is at least partially mediated by an RSD gD-integrin interaction. This pathway might lead to the degradation of some viruses in lysosomes and could explain why cell types where endocytosis is the predominant entry pathway become infected at a low efficiency.

for pathogenesis, and the virus was shown to enter human keratinocytes by a low-pH endocytic pathway, whereas human neurons were infected by an entry pathway that was pH independent (29). The differential requirements for lymphotropic herpesviruses such as Epstein-Barr virus, human herpesvirus 8, and EHV-1 to enter their target cells via an endocytic pathway are interesting from a pathogenetic standpoint and may indicate that those cells are more readily amenable to accept large DNA viruses by the endocytic route. Although we clearly identified RGD-binding integrins such as ␣V␤5 or ␣V␤3 and an RSD motif in EHV-1 gD as important requirements for EHV-1 endocytosis, more research will be necessary to study this complex, multistep process of EHV-1 entry in more detail. In the experiments presented here, we could never completely abolish EHV-1 infection, suggesting that other receptors are likely involved in EHV-1 endocytic entry. In line with this observation, it is interesting that EHV-1 gB and gH, two glycoproteins important for herpesviral entry, each contain integrin binding motifs. These motifs, LDI in gB and YGL in gH, are sequences present in fibronectin and were shown to mediate binding to integrins ␣4␤1 and ␣4␤7 (22). In addition, such motifs are present in the VP4 spike protein of rotaviruses and were shown to mediate cell entry and therefore infectivity of several rotaviruses (14). In future experiments, we plan to target the LDI motif and/or the YGL motif, present in gB and gH, respectively, alone or in combination with the point mutation in the RSD gD motif. Evaluation of the efficiency of infection by such EHV-1 recombinant viruses, especially infection of equine PBMC, would be of interest since ␣4 integrins

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are dominantly and highly expressed on B and T lymphocytes and monocytes (18). Taken together, we provide evidence here that (i) EHV-1 entry via endocytosis involves at least an RSD-integrin interaction and (ii) EHV-1 uses at least two different cellular entry pathways to infect important target cell populations of the natural host. Viral entry via multiple pathways involving several receptors increases the chance of a virus to survive in the host. The different strategies for infection of different host cells seem necessary for this herpesvirus to be capable of establishing a successful infection and maintaining itself in the individual horse and the population. ACKNOWLEDGMENTS We are grateful to James Lee Smith II for sorting the equine EC, Robin Yates for help with the dextran experiments, and Benedikt Kaufer for fruitful discussions. This work was supported by the Harry M. Zweig Memorial Fund for Equine Research at Cornell University to N.O. and by NIH grants AI-22001 and P20-RR-018724 to D.J.O. G.R.V. is a postdoctoral fellow of the Fonds voor Wetenschappelijk Onderzoek Vlaanderen. REFERENCES 1. Ahrens, I. G., N. Moran, K. Aylward, G. Meade, M. Moser, D. Assefa, D. J. Fitzgerald, C. Bode, and K. Peter. 2006. Evidence for a differential functional regulation of the two beta(3)-integrins alpha(V)beta(3) and alpha(IIb)beta(3). Exp. Cell Res. 312:925–937. 2. Akiyama, S. K., K. Olden, and K. M. Yamada. 1995. Fibronectin and integrins in invasion and metastasis. Cancer Metastasis Rev. 14:173–189. 3. Akula, S. M., P. P. Naranatt, N. S. Walia, F. Z. Wang, B. Fegley, and B. Chandran. 2003. Kaposi’s sarcoma-associated herpesvirus (human herpesvirus 8) infection of human fibroblast cells occurs through endocytosis. J. Virol. 77:7978–7990. 4. Allen, G. P., and J. T. Bryans. 1986. Molecular epizootiology, pathogenesis, and prophylaxis of equine herpesvirus-1 infections. Prog. Vet. Microbiol. Immunol. 2:78–144. 5. Chang, K. H., C. Day, J. Walker, T. Hyypia, and G. Stanway. 1992. The nucleotide sequences of wild-type coxsackievirus A9 strains imply that an RGD motif in VP1 is functionally significant. J. Gen. Virol. 73:621–626. 6. Csellner, H., C. Walker, J. E. Wellington, L. E. McLure, D. N. Love, and J. M. Whalley. 2000. EHV-1 glycoprotein D (EHV-1 gD) is required for virus entry and cell-cell fusion, and an EHV-1 gD deletion mutant induces a protective immune response in mice. Arch. Virol. 145:2371–2385. 7. Flowers, C. C., S. P. Flowers, Y. Sheng, E. B. Tarbet, S. R. Jennings, and D. J. O’Callaghan. 1995. Expression of membrane-bound and secreted forms of equine herpesvirus 1 glycoprotein D by recombinant baculovirus. Virus Res. 35:17–34. 8. Flowers, C. C., and D. J. O’Callaghan. 1992. Equine herpesvirus 1 glycoprotein D: mapping of the transcript and a neutralization epitope. J. Virol. 66:6451–6460. 9. Fox, G., N. R. Parry, P. V. Barnett, B. McGinn, D. J. Rowlands, and F. Brown. 1989. The cell attachment site on foot-and-mouth disease virus includes the amino acid sequence RGD (arginine-glycine-aspartic acid). J. Gen. Virol. 70:625–637. 10. Frampton, A. R., Jr., W. F. Goins, J. B. Cohen, J. von Einem, N. Osterrieder, D. J. O’Callaghan, and J. C. Glorioso. 2005. Equine herpesvirus 1 utilizes a novel herpesvirus entry receptor. J. Virol. 79:3169–3173. 11. Frampton, A. R., Jr., D. B. Stolz, H. Uchida, W. F. Goins, J. B. Cohen, and J. C. Glorioso. 2007. Equine herpesvirus 1 enters cells by two different pathways, and infection requires the activation of the cellular kinase ROCK1. J. Virol. 81:10879–10889. 12. Galdiero, M., A. Whiteley, B. Bruun, S. Bell, T. Minson, and H. Browne. 1997. Site-directed and linker insertion mutagenesis of herpes simplex virus type 1 glycoprotein H. J. Virol. 71:2163–2170. 13. Gianni, T., G. Campadelli-Fiume, and L. Menotti. 2004. Entry of herpes simplex virus mediated by chimeric forms of nectin1 retargeted to endosomes or to lipid rafts occurs through acidic endosomes. J. Virol. 78:12268– 12276. 14. Graham, K. L., F. E. Fleming, P. Halasz, M. J. Hewish, H. S. Nagesha, I. H. Holmes, Y. Takada, and B. S. Coulson. 2005. Rotaviruses interact with alpha4beta7 and alpha4beta1 integrins by binding the same integrin domains as natural ligands. J. Gen. Virol. 86:3397–3408. 15. Hamidpour, M., M. Behrendt, B. Griffiths, L. Partridge, and N. Lindsey. 2006. The isolation and characterisation of antiplatelet antibodies. Eur. J. Haematol. 76:331–338.

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