JOURNAL OF , OCt. 1990, p. 4866-4872 Vol. 64, No. 10 0022-538X/90/104866-07$02.00/0 Copyright C) 1990, American Society for Microbiology Bovine Cells Expressing Bovine Herpesvirus 1 (BHV-1) Glycoprotein IV Resist Infection by BHV-1, , and Pseudorabies Virus CHRISTOPHER C. L. CHASE,t* KELLY CARTER-ALLEN,t CORTLAND LOHFF, AND GEOFFREY J. LETCHWORTH III Department of Veterinary Science, University of Wisconsin-Madison, Madison, Wisconsin 53706 Received 21 November 1989/Accepted 6 July 1990

We expressed the bovine herpesvirus 1 (BHV-1) glycoprotein IV (gIV) in bovine cells. The expressed was identical in molecular mass and antigenic reactivity to the native gIV protein but was localized in the cytoplasm. Expressing cells were partially resistant to BHV-1, , and pseudorabies virus, as shown by a 10- to 1,000-fold-lower number of plaques forming on these cells than on control cells. The level of resistance depended on the level of gIV expression and the type and amount of challenge virus. These data are consistent with previous reports by others that cellular expression of the BHV-1 gIV homologs, herpes simplex virus glycoprotein D, and pseudorabies virus glycoprotein gp5O provide partial resistance against infection with these . We have extended these findings by showing that once BHV-1 enters gIV- expressing cells, it replicates and spreads normally, as shown by the normal size of BHV-1 plaques and the delayed but vigorous synthesis of viral . Our data are consistent with the binding of BHV-1 gIV to a cellular receptor required for initial penetration by all three herpesviruses and interference with the function of that receptor molecule.

Bovine herpesvirus 1 (BHV-1) causes two major disease workers were only able to immunoprecipitate gD and not gE syndromes in cattle: infectious bovine rhinotracheitis and or gI, leading them to conclude that gD was responsible for infectious pustular vulvovaginitis (26). BHV-1 codes for at viral inhibition. However, the presence or absence of the least three glycoproteins that are present in the envelope of other gene products was not reported (1). The expression of the virus. These glycoproteins are likely to play a role in the only the gD-1 gene in murine or human cells resulted in cell-virus interactions necessary for a productive BHV-1 resistance to HSV-1 but not to HSV-2 (12). Both of these infection of bovine cells (26). Three of these glycoproteins, studies demonstrated that this resistance could be overcome gI, glll, gIV, have been biochemically characterized (18, with high multiplicities of infection (MOIs) with HSV-1. 25), antigenically mapped by monoclonal antibody analysis Petrovskis et al. demonstrated that simian or human cells (17, 24), and molecularly cloned (26; T. Zamb, Abstr. 68th expressing PRV gp5O, the homolog of HSV-1 gD, resist Annu. Meet. Conf. Res. Workers Anim. Dis., abstr. no. 330, infection by PRV or HSV-1 (22). This resistance also could p. 57, 1987). Genetic mapping and sequence analysis (T. be overcome with a higher MOI. In this report, we validate Zamb, Abstr. 68th Annu. Meet. Conf. Res. Workers Anim. and extend the idea of a common function for the gD-1 Dis. 1987) of the BHV-1 have shown that gIV is the homologs by demonstrating that bovine cells expressing gIV homolog of glycoprotein D (gD) of herpes simplex virus 1 partially resist infection by BHV-1, HSV-1, and PRV. Fur- (HSV-1) and pseudorabies virus (PRV) glycoprotein gpSO. thermore, our data suggest that this resistance is expressed We expressed gIV in bovine fibroblasts in order to further only during the initial infection event, since it fails to inhibit characterize the role of this glycoprotein in early infection synthesis, host protein shutoff, or viral spread events. from cell to cell. Second, the kinetics and level of resistance Resistance to viral infection by cells expressing virion of gIV-expressing cells are examined. This will provide envelope glycoproteins has been demonstrated previously valuable information for assessing the expression of this for two alphaherpesviruses. Results of experiments examin- protein in vivo as a potential herpesvirus resistance mecha- ing the resistance of gD-1-expressing cells to different nism. herpesviruses are contradictory (1, 12). Arsenakis et al. (1) demonstrated that the expression of the BamHI J fragment MATERIALS AND METHODS of HSV-1 inhibited infection with HSV-1 and HSV-2 in hamster cells. This fragment contained four complete open Cells and virus. Bovine fibroblasts were harvested from reading frames (gD, US5, gG, and gI) and two truncated bovine skin (7) and cultured in minimum essential medium open reading frames for gE and a protein kinase (20). Those (MEM; GIBCO Laboratories, Grand Island, N.Y.) supple- mented with 5% heat-inactivated fetal bovine serum (Hy- clone Laboratories, Logan, Utah). All cells were grown at 37°C in a humidified atmosphere of 5% (vol/vol) CO2. * Corresponding author. t Present address: U.S. Department of Agriculture, Agricultural The Cooper (Colorado-1) strain of BHV-1, obtained from Research Service, Arthropod-borne Animal Diseases Research Lab- the American Type Culture Collection (Rockville, Md.) at oratory, P.O. Box 3965, University Station, Laramie, WY 82071- passage 10, was plaque purified once in bovine fibroblasts, 3965. and a master stock was prepared. Work stocks were grown t Present address: Department of Medical Microbiology, Univer- in Madin-Darby bovine kidney cells and frozen in aliquots. sity of Wisconsin-Madison, Madison, WI 53706. The MacIntyre strain of herpes simplex virus 1 (HSV-1) was 4866 VOL. 64, 1990 CELLS EXPRESSING BHV-1 gIV RESIST BHV-1, HSV-1, AND PRV 4867

M gIV M H using the Klenow reaction, and ligated to BamHI linkers. This BamHI-linked fragment was ligated into the unique 1.4 kb pML-2 p341 fm vT BamHI site of the 11.3-kb pCGBPV9AB5 (19). Plasmid BamHI nerAdto5.3 kb Bglat BgII1 pCGBPVgAB5 contains the entire genome of bovine papil- lomavirus 1 (BPV-1), a neomycin resistance gene providing Ugation B SV40 kanamycin resistance in bacteria and G418 resistance in B eucaryotic cells, and a bacterial replication origin. Ligation SV40 of the BHV-1 gIV fragment in the opposite transcriptional orientation as the BPV early open reading frame resulted in pML-2 pgIV the plasmid pCGBPV-D#l (Fig. 1). Plasmid DNA for use in 6.7 kb transfection experiments was propagated in TB-1 cells } grown at 37°C on a shaker rack in L broth (16) containing 50 ,ug of kanamycin per ml. Plasmid DNA was prepared by H \ the B Hindlil + BamH alkaline lysis method (2), and supercoiled DNA was isolated Digestion by centrifugation on a CsCl-ethidium bromide gradient (16). pCGBPV9A neo Transfection and selection of cells expressing gIV. Twenty- four hours prior to transfection, bovine fibroblasts were BPV 11.3kb H ,H g_ B MMIT SV40 plated at a density of 2.5 x 105 per 25-cm2 flask. Bovine 4.1 kb fibroblasts were transfected with 20 Rxg of pCGBPV-D#1 per BamHl Digestion BamHl Unker Addtion 25-cm2 flask. Cells were transfected by the calcium phos- phate method, selected by survival in MEM containing G418, cloned as previously described (5), and subcloned by Ugation a limiting dilution. Cloned cells were screened for gIV H B expression in an enzyme-linked immunosorbent assay MMT (ELISA). For the ELISA, normal and transfected bovine fibroblasts were grown in 96-well plates. Eighteen hours prior to the assay, normal bovine fibroblasts were inoculated BPVI pCGBPV-D#1 gIV 15.4 kb for 30 min with BHV-1 at a MOI of 10 and one-half of the SV40 wells containing transfected cells were treated with 1 p.M of B CdCl2 (Sigma Chemical Co., St. Louis, Mo.). This concen- tration of CdCl2 was determined to be the maximum con- H flBO centration that had no obvious effect on the viability of FIG. 1. Procedure used for the construction of the expression control cells. Virally infected and mock-infected bovine plasmid pCGBPV-D#l. Arrows indicate the direction of transcrip- fibroblasts as well as CdCl2-treated and untreated trans- tion. Restriction enzyme symbols: B, BamHI; H, Hindlll; M, MaeI. fected cells were washed twice with phosphate-buffered MMT, Mouse metallothionein; SV40, simian virus 40. saline (PBS) and fixed for 30 min in methanol at 4°C. The methanol was removed, and the wells were filled with obtained from the State Laboratory of Hygiene, Madison, ELISA washing buffer (ELISA PBS; 15 mM Na2HPO4, 5 Wis., and workstocks were grown in HEp-2 cells. The mM NaH2PO4, 140 mM NaCl [pH 7.3], 0.05% Tween 20, Sullivan strain of PRV was provided by B. C. Easterday, 0.5% fish gelatin) and incubated at room temperature for 2 h. School of Veterinary Medicine, University of Wisconsin- The cells were washed three times with ELISA washing Madison. Vesicular stomatitis virus-New Jersey, Ogden buffer and incubated with 100 RI1 per well of a 1: 2,000 strain, was provided by Suzanne Vernon, University of dilution of anti-gIV mouse immunoglobulin G 2A monoclo- Wisconsin-Madison. nal antibody, 1106 (17), for 1 h at room temperature. The Construction of the recombinant plasmid pCGBPV-D#l. cells were then washed as described above and incubated Restriction enzymes, the Klenow fragment of Escherichia with 100 ,ul of 1: 2,000 dilution of biotinylated horse anti- coli DNA polymerase, oligonucleotide linkers, E. coli TB-1, mouse antibody (Vector Laboratories, Burlingame, Calif.) and DNA ligase were purchased from Bethesda Research for 2 h at room temperature. The cells were again washed Laboratories, Inc., Gaithersburg, Md., and used as recom- and incubated with 100 p.1 of 1: 20,000 dilution of peroxidase- mended by the supplier. All plasmids were propagated in E. conjugated avidin (Cooper Biomedical, Inc., West Chester, coli TB-1. The cDNA clone of BHV-1 gIV (T. Zamb, Abstr. Pa.) for 2 h at room temperature. The cells were washed five 68th Annu. Meet. Conf. Res. Workers Anim. Dis. 1987) was times with ELISA washing buffer, and 100 p.l of substrate obtained from Molecular Genetics, Inc., Minnetonka, (0.4 mg o-phenylenediamine dihydrochloride [Sigma] per ml Minn., in a carrier plasmid. The carrier plasmid was digested and 0.01% hydrogen peroxide in citrate phosphate buffer, with the restriction enzyme MaeI and the 1.4-kilobase (kb) pH 5.0) was added. The reaction was stopped with 2.5 M fragment containing gIV (Fig. 1) separated from the other H2SO4 as the mock-infected cells began to develop a color fragments by electrophoresis through a 1.2% low melting change. Absorbances were measured in an ELISA reader. temperature agarose gel (SeaPlaque; FMC Corp., Marine Immunoprecipitation of the gIV protein. Bovine fibroblasts Colloids Div., Rockland, Me.). Single-stranded ends were were grown to confluency in 25-cm2 flasks. Eighteen hours filled in by using the Klenow reaction (16) and ligated to prior to labeling, monolayers of transfected bovine fibro- BamHI linkers. The BamHI-linked fragment was ligated into blasts were treated with 1 p.M CdCl2. Nontransfected bovine the BglII site of the 5.3-kb p341 (6) between the mouse fibroblasts were inoculated with BHV-1, as described above. metallothionein promoter and the simian virus 40 polyade- Virally infected and mock-infected bovine fibroblasts as well nylation processing site (Fig. 1). This ligation resulted in a as CdCl2-treated and untreated transfected cells were la- 6.7-kb plasmid, pMD1-1.4, that was digested with HindlIl beled, lysed, clarified, and immunoprecipitated with gIV and BamHI. The 4.1-kb fragment was isolated, repaired by monoclonal antibody 1106 (17), as previously described (5). 4868 CHASE ET AL. J. VIROL.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was carried out by the procedure of Laemmli (14), by using a 5% acrylamide stacking gel and a 7.5% acrylamide resolv- ing slab gel. Molecular mass standards (Sigma) ranging in size from 205 to 29 kilodaltons were used as markers. Gels were dried and exposed to XAR-5 film (Eastman Kodak Co., Rochester, N.Y.) at -70°C. Indirect immunofluorescence. Normal and transfected bo- vine fibroblasts were grown on fetal bovine serum-coated glass cover slips or slides. Eighteen hours prior to the assay, normal bovine fibroblasts were inoculated with BHV-1 at an MOI of 10 and half of the cover slips with transfected cells were treated with 1 ,uM of CdCl2 (Sigma). At the end of 18 h, the cells were washed with PBS, either fixed with methanol at -20°C for 30 min and allowed to dry or placed on ice for the entire assay. The cells on the cover slips were treated with gIV-specific antibodies and then with fluorescein iso- thiocyanate-conjugated goat anti-mouse immunoglobulin G in an indirect immunofluorescence assay, as previously described (5). The fresh cells were then fixed with 1% paraformaldehyde for 30 min at room temperature. The monolayers were then washed with PBS, mounted in 90% (vol/vol) glycerol-100 mM Tris, pH 8.0, and examined with a standard or confocal fluorescence microscope. FIG. 2. Immunofluorescence analysis of gIV-expressing cells. To determine the percentage of gIV-expressing cells resis- (A) BHV-1-infected bovine fibroblasts; (B) gIV-expressing cell line tant to BHV-1, normal and transfected cells were grown as D1-1; (C) gIV-expressing cell line D1-4.1; (D) gIV-expressing cell described above. The cells were inoculated with BHV-1 at line D1-4.21. Cell immunofluorescence was demonstrated by using an MOI of 8 and fixed at 12 h with methanol as described pooled anti-gIV monoclonal antibodies in an indirect assay. above. The cells were treated and examined as described above, except that a gI-specific antibody, 5106, was used instead of a gIV-specific antibody. The cells were counted, stained prior to fixation revealed no membrane fluorescence and the percentage of infected cells was determined. (data not shown). Confocal fluorescence microscopy with Sequential [35S]methionine labeling of BHV-1-infected gIV- methanol-treated cells showed a large perinuclear cytoplas- expressing cells. Normal bovine fibroblasts and gIV-express- mic concentration of gIV while confocal analysis of para- ing cells were grown to confluency in 25-cm2 flasks and formaldehyde-treated cells demonstrated membrane fluores- infected with BHV-1 at an MOI of 2. Cells incubated for 1, cence on less than 5% of the cells (data not shown). 6, 14, and 22 h after BHV-1 infection were washed twice The Dl-1 and D1-4 parental cell lines were labeled with with PBS, incubated in methionine-free MEM for 1 h, and [35S]methionine and radiolabeled gIV was immunoprecipi- labeled with 40 ,uCi of [35S]methionine per ml for 5 to 7 h. tated with antibody 1106, analyzed by sodium dodecyl The cell lysate was harvested, clarified, resolved on a sulfate-polyacrylamide gel electrophoresis, and visualized sodium dodecyl sulfate-polyacrylamide gel electrophoresis by autoradiography. A single protein with a molecular mass gel, and visualized by autoradiography as described above. of 77-kilodaltons was present in the recombinant clones (Fig. 3). This recombinant protein was the same molecular mass RESULTS as the native gIV protein immunoprecipitated in BHV-1- infected cells (Fig. 3). No protein bands were present at this Expression of recombinant gIV. Bovine fibroblasts stably molecular mass for either mock-infected cells (Fig. 3) or cells transfected with pCGBPV-D#1 (Fig. 1) were selected with transfected with the shuttle vector pCGBPVgAB5 (Fig. 3). G418. Thirteen resistant foci were cloned and screened by an The addition of the heavy metal, Cd2", resulted in increased ELISA by using gIV monoclonal antibody 1106 (17). All 13 production of recombinant gIV (Fig. 3). foci were positive and 2, D1-1 and D1-4, were selected for To characterize the antigenic properties of the recombi- further characterization. The D1-4 parental line was sub- nant gIV, 125I-labeled monoclonal antibodies directed cloned, and the subclones were screened for the level of gIV against all five native gIV epitopes identified by Marshall et expression. Indirect fluorescent-antibody staining of the al. (17) were individually incubated with Cd2+-stimulated methanol-treated gIV-expressing cell lines with a gIV-spe- gIV-expressing cells. Antibodies directed against each cific monoclonal antibody showed a perinuclear distribution, epitope were bound above control cell levels by the gIV- suggesting the concentration of the protein in the Golgi expressing cells (data not shown), indicating the presence of apparatus (Fig. 2B to D). The Dl-1 cell line had the highest the same immunoreactive domains on the recombinant pro- level ofgIV fluorescence of the transfected cells (Figure 2B), tein as those seen on the native protein. while the D1-4.21 (Fig. 2D) and D1-4.10 (data not shown) cell Virus production by gIV-expressing cells. The gIV-express- lines had an intermediate level of gIV fluorescence. The ing cell line, D1-1, produced 1 log less BHV-1 than nontrans- D1-4.1 (Fig. 2C) and the D1-4.2 (data not shown) cell lines fected control fibroblasts (Fig. 4). Figure 4 is representative had the lowest amount of gIV fluorescence. All the trans- of assays done with both D1-1 and D1-4 cell lines at MOIs fected cell lines expressed gIV at a lower level than BHV- from 0.1 to 10. Increased expression of gIV following 1-infected cells (Fig. 2A). Mock-infected cells had no fluo- induction with Cd2+ for 18 h prior to infection did not affect rescence (data not shown). Stain localization and intensity the amount of virus produced by gIV-expressing cells (data was uniform among cells. Analysis of gIV-expressing cells not shown). VOL. 64, 1990 CELLS EXPRESSING BHV-1 gIV RESIST BHV-1, HSV-1, AND PRV 4869

0 10

0 la w to LLL OI) I + C++ 1-1 FE z 0 > Iv U) O = T- E. o m o 0 0 m a 0

205 kD- :z-

116 kD- 0 1 2 24 36 48 97 kD- Hours after infection FIG. 4. BHV-1 growth curve in gIV-expressing cells. Normal bovine fibroblasts ( ) and the D1-1 cell line ( - -) were infected z.. Om - g I V with BHV-1 at an MOI of 0.1. The inoculum was removed after 30 min. At 12-h intervals over 48 h, the supernatants D1-1 (0) and BF 66 kD- (O) were removed from the cells and replaced with MEM and the cells were frozen at -70°C. The cell lysates D1-1 (0) and BF (-) were recovered following quick thawing of the frozen monolayers. Viral titers were determined on bovine fibroblasts. TCID, Tissue culture infective dose.

Plaque reduction was directly related to the level of gIV expression. The high-expressing D1-1 cell line had a greater- 45 kD- _ than-1,000-fold reduction in plaque formation. Plaque num- FIG. 3. Characterization ofgIV from cells transfected with pCG- bers were reduced more than 100-fold in the D1-4.10 and BPV-D#1. Nontransfected cells were mock infected (CONTROL) D1-4.21 cell lines, while the lower-expressing D1-4.1 and or infected with BHV-1 for 18 h prior to labeling. Transfected cell D1-4.2 cell lines had a 10-fold reduction in plaque formation. lines D1-1 and D1-4 were left untreated or treated with CdCl2 for 18 Increased gIV levels in the parental lines, D1-1 and D1-4, h prior to labeling. Radiolabeled lysates were immunoprecipitated following 18 h of Cd2+ treatment did not alter the level of with an anti-gIV monoclonal antibody, separated on sodium dodecyl viral resistance significantly (data not shown). sulfate-polyacrylamide gels and fluorographed. The positions of HSV-1 plaque formation was also decreased by gIV molecular mass markers are indicated. expression (Table 1). This effect on HSV-1 plaque formation was less with a 2-, 3-, and 15-fold reduction seen in the D1-4 low, D1-4 high, and D1-1 cell lines, respectively. The effect of gIV expression on BHV-1 protein synthesis and PRV plaque formation was also decreased in gIV-express- cell protein shutdown. Viral proteins could be seen in control ing cells (Table 1). The D1-4 low- and high-expressing lines cells infected for 6 h (Fig. 5A, lane INF, BF), while had a 3- and 25-fold decrease in plaque formation. The D1-1 BHV-1-infected gIV-expressing cells had no viral proteins at cell line had a 1,000-fold reduction in PRV plaques (Table 1). 6 h (Fig. 5A, lane INF, gIV). The BHV-1-infected gIV- Although plaque numbers were reduced more than 1,000- expressing cells had two to three times fewer viral proteins fold in some experiments (Table 1), the sizes of the plaques present at 14 and 22 h (Fig. 5A, lane INF, gIV; 5B, lane INF, formed by all three viruses were equal on gIV-expressing gIV) than did normal cells (Fig. 5A, lane INF, BF; 5B, lane and normal cells (data not shown). As a control to show that INF, BF). At 30 h, viral proteins were present in gIV- gIV-expressing cells do not nonspecifically resist viral infec- expressing cells at levels comparable to BHV-1-infected tions, all cell lines were infected with vesicular stomatitis normal cells (Fig. 5B, lanes INF BF and INF gIV). Cellular virus. There was no significant difference in the number of proteins continued to be synthesized throughout the 30-h vesicular stomatitis virus plaques in the gIV-expressing and infection by gIV-expressing cells (Fig. 5A, lanes INF gIV; control cell lines (data not shown). A transfected bovine 5B, lanes INF, gIV), although their level of expression fibroblast cell line LB36CGBPV, containing the 11.3-kb significantly declined by the end of the infection (Fig. 5B, pCGBPVgAB5 (19), was also infected with BHV-1, HSV-1, lane INF, gIV). This cellular protein synthesis was in sharp and PRV. This was an important control to establish that contrast to the protein shutdown seen in the BHV-1-infected neither the shuttle vector nor the aminoglycoside G418 was normal cells at 14, 22, and 30 h (Fig. 5A, lane INF, BF; SB, responsible for herpesvirus resistance. Plaque numbers from lanes INF, BF). these cells were similar to those of the infected normal Resistance of gIV-expressing cells to alphaherpesviruses. fibroblasts (Table 1). The D1-1 and the four subclonal D1-4 cell lines were infected The percentage of gIV-expressing cells susceptible to with BHV-1 in a plaque assay. BHV-1 plaque numbers were BHV-1 was determined by an immunofluorescence assay reduced 10- to 1,000-fold in cells expressing gIV (Table 1). directed against another early glycoprotein, gI. Twelve 4870 CHASE ET AL. J. VIROL.

0-6 HRS 6-14 HRS 14-22 HRS 22-30 HRS A I l I 1 B I I I' I Ml INF Mi NF Ml INF Ml INF m-- r- 1 7 1 BF gIV BF gIV BF g9V BF gIV BF glV BF gIV BF gIV BE gIV 205 kD- _ _w_~~205 k D- 116kD- ~~~~~~116kD-

97 kD- _~~~~~~~~~97 kD-

66~~~kD- 66 kD-

45 kD-V45 kD-

FIG. 5. Proteins produced by mock-infected (MI) and BHV-1-infected (INF) bovine fibroblasts (BF) and gIV-expressing cells. (A) Cells labeled at 0 to 6 h and 6 to 14 h as described in Materials and Methods. (B) Cells labeled at 14 to 22 and 22 to 30 h, as described in Materials and Methods. The positions of molecular mass markers are indicated. hours after BHV-1 infection, 2% of the D1-1 cells, 13% of the DISCUSSION high-expressing D1-4 cells, and 27% of the low-expressing D1-4 cell lines were expressing gI, compared with 78% of the Our work showed that cellular expression of BHV-1 gIV BHV-1-infected normal fibroblasts. provides partial resistance to replication by three alpha- herpesvirus, BHV-1, PRV, and HSV-1 in bovine cells. Infection with the homologous virus was resisted to the TABLE 1. Effect of gIV expression on plaque formation of three highest degree, whereas resistance to PRV and HSV-1 was alphaherpesviruses in bovine fibroblastsa at lower levels. The resistance seen with gIV-expressing No. of plaques/well (t of control) cells against BHV-1 was very similar to the resistance Cell line Sub- following infection with: reported by Johnson and Spear (12) in gD-1-expressing cells clone infected with HSV-1. Our work differs in our ability to BHV-1 HSV-1 PRV demonstrate inhibition against two other alphaherpesvi- Control 130 (100) 160 (100) 150 (100) ruses, HSV-1 and PRV, whereas gD-1 cells were unable to inhibit HSV-2 infection (12). Our results were also similar to LB36CGBPV 2.1 155 (119) 171 (107) 135 (90) those reported by Petrovskis et al. (22), in which viral titers of PRV or HSV were reduced 10- to 30-fold from porcine and D1-4, low gIV 1 12 (7) 68 (43) 26 (38.5) human cells expressing PRV gpSO, but our study suggests a expression 2 14.5 (11) 91 (57) 39 (58) possible site of action. Our work then further extends and D1-4, high gIV 10 1 (0.8) 51 (32) 1 (2) expands the effect seen with gIV analogs gD-1 against expression 21 1 (0.8) 43.5 (27) 7 (10.5) HSV-1 (1, 12) and gpSO against PRV and HSV-1 (22). Three possible mechanisms of action might be envisioned D1-1 0 (<0.1)b 10.5 (6.5) 0 (0.1)c to explain gIV interference with herpesvirus infection. First, gIV existing in the cell at the time of infection might repress a Normal bovine fibroblasts and gIV-expressing bovine fibroblasts were grown to confluency in 24-well plates. Ten-fold-decreasing dilutions of each herpesvirus alpha genes, as has been shown for the HSV-1 virus were added to duplicate wells and adsorbed for 1 h. The wells were beta gene product ICP8 (9). The ability of BHV-1 to form overlaid with MEM containing 0.5% agarose and incubated at 37°C for 2 to 4 normal size plaques when it infects gIV-expressing cells days. Cells were fixed and stained with crystal violet. Then plaques were argues against this hypothesis. Second, gIV expression may counted. The number of plaques is the average of duplicate wells. b BHV-1 plaques could not be detected at the next more-concentrated trigger a generalized antiviral state when expressed in cells. dilution. However, this hypothesis is inconsistent with the ability of c Two PRV plaques were detected at the next more-concentrated dilution. vesicular stomatitis virus to form equal numbers of plaques VOL. 64, 1990 CELLS EXPRESSING BHV-1 gIV RESIST BHV-1, HSV-1, AND PRV 4871 on gIV-expressing and normal bovine fibroblasts. Third, gIV spread of virus while permitting transmission by direct may have an effect on the cell membrane events of adsorp- cell-to-cell contact. tion and/or penetration. Monoclonal antibodies directed Thus, it appears that BHV-1 gIV expression in bovine against gIV neutralize BHV-1 after virus adsorption to cells cells specifically blocks penetration of individual mature at 4°C with titers equivalent to preadsorptive neutralization herpesvirus virions through the membranes of otherwise (J. Dubuisson, B. A. Israel, and G. J. Letchworth III, susceptible cells. Whether this block is related to interaction submitted for publication), supporting the function of gIV in between the cellular gIV and the virion or between the membrane penetration. We have shown that gIV expression cellular gIV and some yet unidentified cellular protein has has no effect on the number of radiolabeled BHV-1 virions not been determined. The latter hypothesis is supported by adsorbed to cells (C. C. L. Chase, unpublished data). This is two findings. First, cells pretreated with as few as 5,000 consistent with the findings of Campadelli-Fiume et al. (4) UV-inactivated wild-type HSV-1 but not gD-deficient viri- that HSV could enter cells expressing gD but only by an ons become resistant to infection with live HSV-1 (11). This endocytic pathway that resulted in virion degradation rather suggests that gD-1 binds to a cellular molecule that is easily than infection. Thus, our attention has been focused on saturated and is required for HSV-1 to penetrate cell mem- penetration events. Herpes simplex virus gD has been branes. It is probable that the gD-1 homolog, gIV, would shown to be critical in viral penetration of cell membranes have a similar function. Second, we failed to detect cell (8, 10-12, 15, 23). Monoclonal antibodies directed against surface gIV in our immunofluorescence assay. This may HSV-1 gD prevent fusion of infected cells (21) and prevent preclude any direct gIV-virion effect on the cell membrane fusion between the virion and cellular membranes (8, 10). and suggests that the large accumulations of gIV in the Golgi Herpes simplex virions lacking gD are unable to penetrate apparatus may interact with a cellular molecule and interfere cells and thus are noninfectious (11, 15). However, HSV-1 with the processing and/or transport of this cellular protein. gD appears to have no function in virion attachment (11, 12). A similar depletion of the human immunodeficiency virus Cell membrane expression of HSV-1 gD does not interfere receptor CD4 has been described for cells expressing human with adsorption of HSV-1 to the cell, but it does prevent immunodeficiency virus gp160 (13). normal viral penetration and therefore prevents replication Expression of gIV in bovine cells did not result in cell (11, 12). Cell membrane expression of PRV gp5O, the ho- fusion. This is unlike the spontaneous fusion seen with molog of HSV-1 gD, not only prevents replication of PRV HSV-1 gD-l-expressing hamster cells (3). Although basal but also of HSV-1 (22). The ability of BHV-1 gIV expression expression of gIV is not harmful to cells, high levels of gIV to interfere with BHV-1, HSV-1, and PRV infection suggests after a 30-h Cd2" induction resulted in the death of gIV- that interference with infection of all three viruses hinges on expressing cells (data not shown). This is consistent with the a common cell membrane molecule. Interestingly, gIV- hypothesis that gIV is interacting with a cellular receptor and expressing cells appear to resist PRV infection at a level at high levels is interfering with normal functions of this comparable to BHV-1. molecule to the detriment of the cell. The 100- to 1,000-fold resistance to BHV-1 plaque devel- We have begun studies to produce gIV-expressing trans- opment in the gIV-expressing D1-1 cell line (Table 1) may genic mice as a means to evaluate the effectiveness of this seem inconsistent with the 10- to 30-fold reduction in viral gene as an in vivo herpesvirus defense mechanism. Our growth (Fig. 4) and the abundant production of viral proteins ultimate goal is to develop practical and useful methods to and observable shutoff of cellular protein synthesis (Fig. 5) prevent herpesvirus infections in livestock. in these same cells. One possible explanation is that only the initial interaction between BHV-1 and a population of cells is ACKNOWLEDGMENTS inhibited 100- to cellular 1,000-fold by gIV expression, We thank Molecular Genetics, Inc., and BIOSTAR, Inc., for leading to a dramatic decrease in viral plaque numbers. providing the gIV cDNA clone and sequences. We also thank Rick However, once inside any cell, the virus replicates and Gneiser and Ed Phillips for the photographic work. We also ac- spreads to adjacent cells via intracellular bridges, thus knowledge an anonymous reviewer for many helpful comments. bypassing the original route of infection; replicates abun- This work was supported by Hatch Grant WIS02802, U.S. De- dantly; and produces a normal number of infectious progeny partment of Agriculture Special Research Grant 86-CRSR-2-2902 virions (Fig. 4) and a normal amount of virion proteins and and a Shaw Scholarship to G.J.L. from the Milwaukee Foundation. the expected host protein shutoff (Fig. 5) in those cells that become infected. Cells that are not in direct contact with LITERATURE CITED infected cells do not become infected and continue to 1. Arsenakis, M., G. Campadeili-Fiume, and B. Roizman. 1988. produce normal cellular proteins. This view is supported by Regulation of glycoprotein D synthesis: does a-4, the major two observations. the number but not the size regulatory protein of herpes simplex virus 1, regulate late genes First, of both positively and negatively? J. Virol. 62:148-158. BHV-1 plaques on gIV-expressing cells was reduced, sug- 2. Birnboim, H. C., and J. Doly. 1979. A rapid alkaline extraction gesting that although the initial infection event may have procedure for screening recombinant DNA. Nucleic Acids Res. been suppressed, leading to the reduced number of plaques, 7:1513-1523. the subsequent spread of virus between cells was not sup- 3. Campadelli-Fiume, G., E. Avitabile, S. Fini, D. Stirpe, M. pressed, thus producing the observable plaques. This was Arsenakis, and B. Roizman. 1988. Herpes simplex virus glyco- not related to the selection of viruses with gIV mutations protein D is sufficient to induce spontaneous pH-independent because virions isolated from these plaques were no more fusion in a cell line that constitutively expresses the glycopro- able to form plaques on gIV-expressing cells than were the tein. Virology 166:598-602. viruses not BHV-1 formed 4. Campadeili-Fiume, G., M. Arsenakis, F. Farabegoli, and B. parental (data shown). Second, Roizman. 1988. Entry of herpes simplex virus 1 in BJ cells that plaques of the same size on gIV-expressing cells either under constitutively express viral glycoprotein D is by endocytosis an agarose overlay or in the presence of the neutralizing and results in degradation of the virus. J. Virol. 62:159-167. monoclonal antibody 4807 (17). Since this antibody is di- 5. Chase, C. C. L., K. Carter-Allen, and G. J. Letchworth III. 1989. rected against BHV-1 gI, it had no detectable effect on either The effect of bovine herpesvirus 1 glycoproteins gI and glll on viral or cellular gIV, but it did prevent the extracellular herpesvirus infections. J. Gen. Virol. 70:1561-1569. 4872 CHASE ET AL. J. VIROL.

6. Eiden, M., M. Newman, A. G. Fisher, D. L. Mann, P. M. Cold Spring Harbor, N.Y. Howley, and M. S. Reitz. 1985. Type 1 human T-cell leukemia 17. Marshall, R. L., B. A. Israel, and G. J. Letchworth III. 1988. virus small envelope protein expressed in mouse cells by using Monoclonal antibody analysis ofbovine herpesvirus-1 glycopro- a bovine papilloma virus-derived shuttle vector. Mol. Cell. Biol. tein antigenic areas relevant to natural infection. Virology 5:3320-3324. 165:338-347. 7. Freshney, R. I. 1983. Disaggregation of the tissue and primary 18. Marshall, R. L., L. L. Rodriguez, and G. J. Letchworth III. culture, p. 104-110. In Culture of animal cells: a manual of basic 1986. Characterization of envelope proteins of infectious bovine techniques. Alan R. Liss, New York. rhinotracheitis virus (bovine herpesvirus 1) by biochemical and 8. Fuiler, A. O., and P. G. Spear. 1987. Anti-glycoprotein D immunological methods. J. Virol. 57:745-753. antibodies that permit adsorption but block infection by herpes 19. Matthias, P. D., H. U. Bernard, A. Scott, G. Brady, T. Hashi- simplex virus 1 prevent virion-cell fusion at the cell surface. moto-Gotch, and G. Schutz. 1983. A bovine papilloma virus Proc. Natl. Acad. Sci. USA 84:5454-5458. vector with a dominant resistance marker replicates extrachro- 9. Godowski, P. J., and D. M. Knipe. 1986. Transcriptional control mosomally in mouse and E. coli cells. EMBO J. 2:1487-1492. of herpesvirus gene expression: gene functions required for 20. McGeoch, D. J., A. Dolan, S. Donald, and F. J. Rixon. 1985. positive and negative regulation. Proc. Natl. Acad. Sci. USA Sequence determination and genetic content of the short unique 83:256-260. region of the genome of herpes simplex virus type 1. J. Mol. 10. Highlander, S. L., S. L. Sutherland, P. J. Gage, D. C. Johnson, Biol. 181:1-13. M. Levine, and J. C. Glorioso. 1987. Neutralizing monoclonal 21. Noble, A. G., G. T.-Y. Lee, R. Sprague, M. L. Parish, and P. G. antibodies specific for herpes simplex virus glycoprotein D Spear. 1983. Anti-gD monoclonal antibodies inhibit cell fusion inhibit virus penetration. J. Virol. 61:3356-3364. induced by herpes simplex virus type 1. Virology 129:218-224. 11. Johnson, D. C., and M. W. Ligas. 1988. Herpes simplex viruses 22. Petrovskis, E. A., A. L. Meyer, and L. E. Post. 1988. Reduced lacking glycoprotein D are unable to inhibit virus penetration: yield of infectious pseudorabies virus and herpes simplex virus quantitative evidence for virus-specific cell surface receptors. J. from cell lines producing viral glycoprotein gpSO. J. Virol. Virol. 62:4605-4612. 62:2196-2199. 12. Johnson, R. M., and P. G. Spear. 1989. Herpes simplex virus 23. Spear, P. G. 1985. Glycoproteins specified by herpes simplex glycoprotein D mediates interference with herpes simplex virus viruses, p. 315-347. In B. Roizman (ed.), The herpesviruses, infection. J. Virol. 63:819-827. vol. 3. Plenum Publishing Corp., New York. 13. Kawamura, I., Y. Koga, N. Oh-Hori, K. Onodera, G. Kimura, 24. Van Drunen Little-Van den Hurk, S., J. V. Van den Hurk, and and K. Nomoto. 1989. Depletion of the surface CD4 molecule by L. A. Babiuk. 1985. Topographical analysis of bovine herpesvi- the envelope protein of human immunodeficiency virus ex- rus type-1 glycoproteins: use of monoclonal antibodies to iden- pressed in a human CD4+ monocytoid cell line. J. Virol. tify and characterize functional epitopes. Virology 144:216-227. 63:3748-3754. 25. Van Drunen Littel-Van den Hurk, S., J. V. Van den Hurk, J. E. 14. Laemmli, U. K. 1970. Cleavage of structural proteins during the Gilchrist, V. Misra, and L. A. Babiuk. 1984. Interactions of assembly of the head of bacteriophage T4. Nature (London) monoclonal antibodies and bovine herpesvirus type 1 (BHV-1) 227:680-685. glycoproteins: characterization of their biochemical and immu- 15. Ligas, M. W., and D. C. Johnson. 1988. A herpes simplex virus nological properties. Virology 135:466-479. mutant in which glycoprotein D sequences are replaced by 26. Wyler, R., M. Engels, and M. Schwyzer. 1989. Infectious bovine ,-galactosidase sequences binds to but is unable to penetrate rhinotracheitis/vulvovaginitis (BHV1), p. 1-72. In G. Wittman into cells. J. Virol. 62:1486-1494. (ed.), Developments in veterinary virology: herpesvirus dis- 16. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular eases of cattle, horses and pigs. Kluwer Academic Publishing, cloning: a laboratory manual. Cold Spring Harbor Laboratory, Boston.