Bovine Cells Expressing Bovine Herpesvirus 1 (BHV-1) Glycoprotein IV Resist Infection by BHV-1, Herpes Simplex Virus, and Pseudorabies Virus CHRISTOPHER C
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JOURNAL OF VIROLOGY, 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, Herpes Simplex Virus, 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 protein 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, herpes simplex virus, 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 viruses. 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 proteins. 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 genome 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 viral protein 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.