Cells and viruses.
Cell lines previously described are SK6-A7 cells (A7), a clonal nonpermissive porcine kidney cell line (23
); HB1-9, an A7 cell line that constitutively expresses HVEM (25
); and Neo, vector-only A7 cells. All porcine cells were maintained in Dulbecco modified Eagle medium (DMEM) with 5% fetal bovine serum (Gemini). Those cells transformed with drug selection markers were periodically passaged in 400 μg/ml G418 (Bethesda Research Laboratories [BRL]). Other cells were HEL, a fibroblast-like cell line from human embryonic lung; 293, adenovirus-transformed primary human embryonic kidney cells; A549, a human lung carcinoma; HEp-2, epidermoid carcinoma from human larynx; HeLa, human cervical epithelial carcinoma; SJNB 7, a human neuroblastoma cell line; and mouse L cells. Wild-type HSV-1 strains are F, KOS, and HFEM. HSV-2 strains are G and 333. Mutant viruses that encode lacZ
(tkRid12) (a gift of P. Spear), and HSV-1(SC11)gH− lac
) were propagated in supporting cell lines. The tkRid12, complemented gH−/+
viruses enter cells to express lacZ
under a constitutive cytomegalovirus promoter.
Cells exposed to virus at 37°C for 70 min were treated briefly with citrate buffer (40 mM, pH 3.0) to inactivate extracellular virions, washed with phosphate-buffered saline (PBS), and incubated in DMEM at 37°C as previously described (33
). To detect infection of reporter viruses, monolayers were washed three times with cold PBS and lysed for β-galactosidase (β-Gal) enzyme-linked immunosorbent assay (ELISA) performed according to the manufacturer's instructions (Roche). For quantifying blue foci by light microscopy, cells were fixed in 2% formaldehyde-0.2% glutaraldehyde for 5 min at 20 h postinfection and stained with 5-bromo-4-chloro-3-indolyl-β-d-
galactopyranoside (X-Gal) (Roche) and blue foci were counted. The substrate o
-galactopyranoside (ONPG) (3 mg/ml ONPG and 0.5% NP-40 in PBS) was used in colorimetric determination at optical density at 450 nm of β-Gal enzymatic activity. The titers of HSV progeny were determined in duplicate on Vero cells as described previously (33
Screening of human cDNA library.
The human fetal lung cDNA library (catalog number A900; Invitrogen) contained 2 × 106 primary recombinants. Pools of cDNA plasmids were transiently transfected into A7 cells using Lipofectamine (Lifetech/BRL). After 24 h, cells were infected at 25 PFU/cell with ICP4−/+ or gH−/+ virus. Thirty-six hours later, monolayers were X-Gal stained to detect β-Gal in infected cells. Plasmid pools containing cDNAs that allowed highest infection above background were subdivided and tested in the same manner until individual cDNAs in bacterial colonies were identified that consistently transferred HSV susceptibility to A7 cells.
Nucleotide sequencing of cDNA clones and computer-assisted analyses.
DNA sequencing (Biomedical Research Core of the University of Michigan) of both strands of isolated cDNA clones used T7 or SP6 primer followed by nested internal primers. Lasergene software (DNASTAR) was used for computer-based analyses of nucleotide and predicted amino acid sequences. A BLAST program was used to search the GenBank database (NCBI).
Coiled-coil predictions were made with the COILS program (19
) on the basis of a prediction protocol proposed by Parry (22
). The amino acid sequence is compared to a database of known parallel two-stranded coiled coils to derive a similarity score. Sequences of B5 and published sequences of other HSV receptors and HSV-1 glycoproteins were analyzed.
Construction of B5 expression vectors.
The hfl-B5 open reading frame (ORF) was amplified with primer pair 5′-GAATTCGTCCGCTGTGC (5′ primer) and 5′-AAAACTCAGGTATCAGAA (3′ primer) with added restriction enzyme digestion sites. The PCR product was digested and ligated into a pcDNA3.1-Zeo-CAT (Invitrogen) to generate plasmid pB5-CAT that has the chloramphenicol acetyltransferase (CAT) gene placed downstream of an untagged B5 sequence under the control of the same promoter. For C-terminal epitope-tagged B5 (pB5-myc), the B5 sequence was placed into pcDNA3.1-myc-his (Invitrogen) to place the ORF in frame with a short sequence encoding a C-terminal myc epitope (9 amino acids [aa]) and a polyhistidine epitope (6 aa). An N-terminal epitope-tagged B5 plasmid (pHis-B5) contained the B5 ORF downstream of a polyhistidine epitope and an Xpress epitope in pcDNA3.1-His (Invitrogen). For tetracycline induction by doxycycline (DOX), hfl-B5 was placed into the BamHI site of pTRE (Clontech) to produce a vector with a hygromycin resistance gene.
Transient and stable expression of B5 protein.
A7 porcine cells were transiently transfected with vector only or with the indicated purified plasmid containing hfl-B5. At 24 h posttransfection, cells were harvested and analyzed for B5 expression with antitag antibody. For untagged B5, we monitored expression of the B5 ORF indirectly by CAT activity of transfected cells.
B5 polyclonal cell lines (B5-1 or B5-2) were made by cotransfecting pB5-CAT and pSV2Neo into A7 cells and double selection with zeocin (0.6 mg/ml) (Invitrogen) and G418 (400 μg/ml) (BRL). Multiple cell colonies selected in one flask after 14 days were trypsinized and grown as B5-1 or B5-2 polyclonal monolayers. Epitope-tagged clonal porcine cell lines were CB5 that expressed myc-tagged B5 (pB5myc) and NB5 that expressed N-tagged B5 (pHisB5). CB5-1 and NB5-1 polyclonal cell lines were made by transfection of pB5myc or pHisB5 and G418 selection for 7 days followed by harvesting all the cell colonies from one dish. Neo, Zeo, and Seo cell lines were A7 cells that contain vectors without inserts. HB1-9/Zeo is an HB1-9 cell line transfected with vector encoding zeocin resistance. For all cell lines, the presence of the transfected plasmid was confirmed by PCR. mRNA was detected by reverse transcription-PCR (RT-PCR), and tagged protein was detected by Western blot analyses of cell lysates with anti-myc (9E-10) or anti-Xpress antibody (Invitrogen).
Over 30 stable clonal porcine cells lines, such as 10-1G1, were made using A7 cells and untagged B5 (pDNA-B5) pcDNA3.1 (Invitrogen) (G. DeLassus and A. O. Fuller, unpublished). After selection in G418 (400 μg/ml) (BRL), individual cell colonies were isolated and propagated as cell lines.
Inducible B5 expression.
Transfected A7 cells were subjected to two cycles of drug selection. Cell colonies from transfection of pTet-ON plasmid (neomycin resistant) were propagated and screened by transient transfection with reporter plasmid pTRE-luc, which expresses luciferase under the control of the TRE promoter when 1 μg/ml of doxycycline (DOX) is in the medium. Two cell lines with a relatively high level of induction and minimum background without induction were used in a second transfection with pTRE-B5. Double selected (neomycin and hygromycin) clones were screened for B5 mRNA by RT-PCR. The presence of functional B5 upon DOX induction was confirmed by RT-PCR detection of mRNA and fluorescence-activated cell sorting (FACS) detection of HSV binding.
Immunostaining and FACS to detect epitope-tagged B5 protein.
Parallel wells containing about 5 × 105 cells or cells on coverslips were transiently transfected with pB5-myc using Lipofectamine (Lifetech/BRL). Forty-eight hours later, cells were left alone or permeabilized and fixed in methanol-acetone at −20°C for 10 min or fixed by treating the cells with 2% paraformaldehyde in PBS at room temperature for 20 min. Cells were incubated with anti-myc 9E-10 antibody and goat anti-mouse secondary antibody (1:100) (HyClone). For immunostaining, cells at about 100% confluency were fixed with 2% paraformaldehyde-0.2% glutaraldehyde and stained with anti-myc (1:1,000) followed by anti-mouse secondary antibody and diaminobenzidine substrate (Lifetech).
For FACS of transiently expressed CB5, A7 monolayers were transfected with pB5myc or vector only. At 48 h posttransfection, cells were gently removed from the monolayer, and unfixed cells were exposed to anti-myc 9E10 antibody and secondary fluorescein isothiocyanate (FITC)-conjugated anti-mouse secondary antibody (Sigma) and analyzed as described below. For FACS of NB5, HB1-9 or Neo stable cell lines, 1 × 106 cells harvested by gentle trypsinization were allowed to recover in growth medium without serum for 20 min at room temperature. Cells were gently pelleted, washed in cold PBS, and either left alone or fixed and permeabilized in cold methanol-acetone for 10 min. To cells on ice, we added anti-Xpress antibody (Invitrogen) (1:500) followed by a secondary FITC-conjugated antibody. Controls lacked the primary anti-Xpress antibody. Cells were washed with cold PBS between incubations and analyzed immediately with a FACScan (Becton Dickinson).
FACS of virus binding.
Virus binding was determined as described previously (23
) using PBS wash with or without heparin (100 μg/ml). Cell-bound virus was determined with a FACScan (Becton-Dickinson), a cocktail of monoclonal antibodies to HSV glycoproteins and FITC-conjugated anti-mouse secondary antibody.
Northern blotting and RT-PCR to detect mRNA.
Total RNA was extracted from cell lines using Trizol reagent (BRL) and electrophoresed in a gel containing 1% formaldehyde before transfer to a nylon membrane (MSI). A nonradioactive probe was generated by PCR incorporation of digoxigenin (DIG)-11-dUTP (Roche) with a primer pair that amplifies a fragment of 500 bp from the 3′ end of the B5 ORF. Membranes were prehybridized, washed, and incubated with alkaline phosphatase-conjugated anti-DIG-11-dUTP antibody according to the manufacturer's instructions (Roche). After serial washes, the nitroblue tetrazolium/5-bromo-4-chloro-3-indolylphosphate (NBT/BCIP) substrate was added to visualize hybridized bands.
RT-PCR was performed with a Titan One Tube kit (Roche). Oligo(dT) primer was used to initiate reverse transcription. A B5-specific upper primer (GAATTCGTCCGCTGTGC) amplified hfl-B5. β-Actin primers served as a control for RNA. The PCR products were analyzed by electrophoresis on 1% agarose gels and representative DNA bands eluted and sequenced using an automated sequencer ABI377 (Perkin-Elmer).
Immunoprecipitation and Western blots of biotinylated proteins.
Flasks (75 cm2
) of confluent NB5 or HB1-9 cells were trypsinized gently to detach cells and allowed to recover for 20 min at 37°C without serum. Cells were washed twice in PBS before biotin labeling (1
). Briefly, pelleted cells were resuspended in sulfosuccinimidyl-6-(biotinamido)hexanoate (Sigma) in PBS and rotated at room temperature for 30 min. After PBS washes, cells were lysed in detergent buffer (50 mM Tris, 100 mM NaCl, 1% Triton X-100, 0.5% deoxycholic acid, 1 mM phenylmethylsulfonyl fluoride) for 30 min at 4°C before centrifugation at 2,400 × g
for 5 min.
After preclearing by incubation for 3 h at 4°C with protein A beads, lysate supernatants were divided into three parallel samples and 100-μl samples incubated overnight at 4°C with preimmune serum or with anti-HVEM or anti-Xpress antibodies. After 50 μl of protein A beads were added for 3 hours at 4°C, protein A-bound precipitants were processed for sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Electrophoresed proteins were transferred to a nitrocellulose membrane (Bio-Rad) where biotinylated proteins were detected in Western blots with streptavidin-conjugated peroxidase (1:500) and visualized with enhanced chemiluminescence (Amersham). The same membrane was exposed to anti-Xpress monoclonal antibody (1:1,000) followed by alkaline phosphatase-conjugated anti-mouse secondary antibody at 1:1,000 and an NBT/BCIP substrate mix (Lifetech).
Inhibition by synthetic peptides.
Synthetic peptides were a B5 peptide corresponding to aa 344 to 374 (KQQWQQLYDTLNAWKQNLNKVKNSLLSLSD) that covered the C-terminal heptad repeat, a gL 28-mer peptide corresponding to a predicted α-helix region from aa 114 to 141 (ANTQETETRLALYKEIRQALDSRKQAAS), and a randomly chosen hydrophilic HVEM peptide corresponding to aa 41 to 60 (CKEDEYPVGSECCPKCSPG). They were synthesized and purified by the University of Michigan protein core facility. Peptides were acetylated at the N terminus, amidated at the C terminus, and dissolved in PBS containing 1% bovine serum albumin. For inhibition of binding or entry, cells were incubated with the indicated concentrations of B5 peptide for 1 hour at 37°C. Cells were washed twice with cold PBS supplemented with 1% bovine serum albumin and exposed to 10 PFU/cell for 1 hour at 4°C. After a peptide dose curve was performed, 42 mM solutions of the indicated peptide were used in inhibition experiments. In an alternative procedure, viruses were incubated with 42 mM of peptide for 1 hour at 4°C before infecting cells that had not been exposed to peptide.
The effects of peptides on virus binding were determined by FACS as described above and previously (23
). Peptide was added either to cells and washed out, or peptide was preincubated with virus and this material was added to cells. After infection, parallel cell samples were washed with heparin (100 μg/ml) (Sigma) or PBS followed by anti-HSV antibodies in FACS as described above.
For inhibition of HSV infection detected by ELISA, confluent cell monolayers in six-well plates were incubated with peptides (42 mM/1 × 106 cells) at 37°C for 1 hour. Unbound peptide was removed by washing with PBS. Alternatively, gH−/+ virus was pretreated for 1 hour with peptide. Peptide-treated or untreated virus at 10 PFU/cell was exposed to cells at 34°C for 90 min, followed by a 40 mM citrate buffer (pH 3.0). Monolayers were overlaid with DMEM, incubated at 37°C for 8 h, and analyzed for the level of β-Gal protein by ELISA (Roche) or by counting blue foci after X-Gal staining.
Transient transfection of B5 into human cells.
HEp-2 cells seeded to 30 to 40% confluency in six-well dishes or cells on coverslips in these dishes were transfected with either pcDNA3 or pB5-myc (5 μg) and incubated at 37°C. For peptide inhibition, at 3 hours posttransfection, 30-mer peptide in PBS was added to the medium to a final concentration of 42 mM. Forty-eight hours posttransfection, cells were washed and fixed with 0.25% glutaraldehyde on ice for 10 min. Anti-myc (9E-10) antibody was added (1:300) for 1 h at room temperature followed by FITC-conjugated anti-mouse secondary antibody (Sigma) (1:100) for 30 min. Cells were visualized by UV light microscopy. A fusion event was defined as a polykaryocyte with three or more nuclei stained with 4′,6′-diamidino-2-phenylindole (DAPI). The total numbers of polykaryons with three or more nuclei were counted, and the number of nuclei per polykaryon was scored directly from fluorescence microscopy of coverslips or from photographs of six randomly chosen fields.