Transfer of HSV susceptibility to porcine cells and computer predictions from the hfl-B5 sequence. A7 cells exposed to lacZ⁺ reporter virus HSV-1(KOS) ICP4^−/+ after transient transfection with pools of individual human cDNAs were X-Gal stained to detect blue cell foci. (A) HEp-2 cells infected with 3 PFU/cell for 8 h. (B-D) A7 cells infected at 48 h posttransfection with 50 PFU/cell stained at 24 h postinfection. (B) Vector only or (C and D) two different pools of human cDNAs were used to transfect the cells. (E) Number of blue foci in a representative experiment after transfection with individual plasmids. pBEC10 encodes HVEM. Control is vector only. Relative susceptibility transfer for the plasmids was similar in multiple experiments where the overall transfection efficiencies varied. (F-I) Computer predictions from the hfl-B5 cDNA sequence (AY769947, NCBI). (F) Predicted amino acid sequence from the hfl-B5 nucleotide sequence. A putative membrane-spanning region is underlined, and amino acids that compose a C-terminal heptad repeat motif are shown in bold type. (G) A Kyte-Doolittle hydropathy plot generated from the B5 amino acid sequence. The predicted transmembrane domain is indicated. (H) A helix-wheel diagram of the predicted coiled-coil domain in the carboxy terminus shows leucine and valine alignment to one side of the helix. (I) COILS prediction plots for the indicated proteins. The y axis shows the COILS prediction score, and the amino acid residue position is shown on the x axis. Green lines represent the likelihood of a given region forming a two-helix bundle, blue lines a three-helix bundle and red lines a four-helix bundle.

Cell surface expression of B5 in porcine cells. (A) Cartoon of the predicted structure of B5 as an integral membrane protein with a predicted C-terminal α-helix. The C terminus and N terminus are labeled. (B) Cells transiently transfected with pB5-myc or vector only (pcDNA-myc) fixed at 48 h posttransfection and stained with anti-myc antibody, a secondary, peroxidase-conjugated antibody and diaminobenzidine substrate. (C) FACS of SK6-A7 cells transiently transfected with pcDNA vector (left) or pB5-myc (right) and stained with anti-myc (9E10) and a secondary, FITC-conjugated antibody (gray lines). Black peaks are cells in the absence of primary anti-myc antibody. (D) FACS of the indicated stable porcine cell line exposed to anti-Xpress and a secondary, FITC-conjugated antibody. Cells were permeabilized (P) with methanol-acetone or not permeabilized (unpermeabilized [U]) before staining. Neo is a vector-only-transformed cell line. NB5 is a stable cell line made with an N-terminal Xpress epitope-tagged B5. HB1-9 is a stable untagged HVEM-expressing porcine cell line. (E and F) NB5 and HB1-9 cells surface labeled with biotin and lysed as described in Materials and Methods. Lysates normalized for protein concentration were immunoprecipitated with preimmune serum (Pre) or with anti-HVEM polyclonal serum (HVEM), or anti-Xpress monoclonal antibody (XPRS). Proteins visualized in Western blots hybridized with streptavidin-conjugated peroxidase and enhanced chemiluminescence substrate (Amersham) (E) or anti-Xpress monoclonal antibody and alkaline phosphatase-conjugated anti-mouse secondary antibody and NBT/BCIP (F). The positions of molecular mass markers (in kilodaltons) are shown to the left of the gels. The arrow points to B5 at about 43 kDa.

Detection of B5 mRNA. Northern blot analyses to detect the presence of hfl-B5 mRNA. (A) Total RNA from the indicated cell line or (B) commercially available polyadenylated RNA from primary human fetal tissues or adult lung tissue probed with a DIG-11-dUTP-labeled hfl-B5 or β-actin-specific sequence. In panel A, the porcine cells are Seo vector only or CB5 that expresses myc-tagged B5. CB5 mRNA runs slightly larger due to the added epitope tags. The presence and quality of mRNA from all the cell lines were monitored by RT-PCR amplification of β-actin (not shown). In panel B, probes of the same primary tissue blots are shown with a B5-specific sequence (top) or a β-actin sequence (bottom).

Transfer of susceptibility to HSV infection by expression of B5. (A-C) Polyclonal porcine cell lines express untagged B5 (B5-1 and B5-2) or epitope-tagged B5 (NB5-1 and CB5-1). Clonal cell lines express HVEM (HB1-9) or vector only (Neo). HEL is a highly susceptible human cell line. HSV-1 strains ICP4^−/+ and gH^−/+ constitutively express lacZ. (A) Cell monolayers were exposed to 25 PFU/cell of ICP4^−/+ or 3 PFU/cell of HSV-2(G) at 37°C for 90 min followed by citrate buffer inactivation of extracellular virus. Infection was determined by X-Gal staining of infected blue cell foci (ICP4^−/+) or by progeny yields (G). (B) Infection at 25 PFU/cell to detect β-Gal protein by ELISA of lysates harvested at the time of infection (T₀) or 20 h postinfection (T₂₀). OD 405, optical density at 405 nm. (C) Cells infected with 10 PFU/cell of gH^−/+ for 8 h were harvested for ONPG detection of β-Gal enzymatic activity. (D and E) Infection of porcine cells that transiently express B5. The indicated plasmids were transfected into porcine A7 cells and 48 h later infected with gH^−/+ at 10 PFU/cell for 90 min followed by citrate buffer wash. After another 8 h, monolayers were X-Gal stained for infected blue foci. Plasmids are tagged B5 (NB5 or pcDNA3/His-B5; CB5 or pcDNA3/myc-His-B5), untagged B5 (pcDNA/B5), and vector only (pcDNA/myc). For inducible expression, a Tet-ON porcine cell line was transfected with pTRE-B5 and incubated for 24 h before induction with 1 μg/ml DOX or were left uninduced (unind). Forty-eight hours after induction, cells were infected and blue foci determined. Controls are the susceptible HB1-9 cell line infected at 3 PFU for 8 h and vector-only (Neo) porcine cells. (D) Photographs at a magnification of ×40 as viewed with an inverted microscope. (E) The average number of blue foci counted in four randomly chosen fields from transiently transfected cell monolayers. (F) HSV binding to cells. Profiles of virus binding to cells after exposure to 10 PFU/cell of purified HSV-1(F) at 4°C for 60 min. 10-1G1 cells constitutively express untagged B5. Transient B5 is A7 cells transfected with pcDNA/B5 for 48 h. Cells are as described above. Parallel cell samples were washed with either PBS to determine total bound virus (light gray line) or with buffer containing 100 μg/ml of heparin for heparin-resistant stable binding (thick black line) or were mock infected (black area). Virus was detected by FACS with a monoclonal antibody cocktail to HSV glycoproteins and FITC-conjugated anti-mouse secondary antibody.

Inhibition of HSV entry by synthetic peptides. A7 and Neo cells are parental or vector-only porcine cells. B5-1 is a polyclonal cell line that expresses untagged B5. HB1-9 constitutively expresses HVEM. (A-C) Cells were preincubated with equimolar amounts of peptide for 30 min before exposure to gH^−/+ at 25 PFU/cell in the presence of peptide for 70 min at 37°C. At 24 h postinfection, β-Gal was detected by either blue foci (A) or ELISA of B-Gal protein (B and C). Peptides (described in Materials and Methods) are a B5 30-mer, an HVEM 26-mer, a gL 28-mer, or a control peptide, UOM-59, composed of randomly chosen 26 amino acids (B and C). In panel B, 100% infectivity shows the infection without peptide measured by ELISA for the amount of β-Gal protein in the indicated cells. In panel C, B5 peptide was added after citrate buffer wash (B5 peptide post C.T.). (D and E) Effects of a 30-mer B5 peptide on HSV stable binding detected by FACS. The filled histograms in panels D and E are a no-virus control. (D) 10-1G1 clonal B5 cells were incubated without peptide or with 42 mM of 30-mer B5 peptide before adding to virus. Stably bound virus is indicated by the thick light gray line. (E) Effects of 42 mM of the 30-mer B5 peptide on HSV stable binding to susceptible HB1-9, Neo porcine cells, or human HEp-2 cells. Thick white lines show stable virus binding in the absence of peptide. The thick gray lines show stable binding in the presence of peptide. (F) Virus preincubated with 42 mM of the indicated peptides or no peptide before adding to the indicated cells. O.D. 405 nm, optical density at 405 nm; B5pep, B5 peptide.

Effects in human cells of synthetic peptides and of overexpression of hfl-B5. (A) HEp-2 cells transiently transfected with C-terminal tagged B5 or vector were stained at 20 h with anti-myc and FITC-conjugated antibody (α-myc FITC) and DAPI for cellular DNA. The lower bar represents 60 μM. (B) The average number of nuclei in each polykaryocyte for those in four dishes of transfected cells. N.T., no transfection. (C) The total number of cell nuclei found in polykaryons (# nuclei fused) and the total number of fused polykaryons (# fusion) are defined as FITC- and DAPI-stained cells foci with three or more nuclei. (D) HEp-2 cells grown on coverslips were transiently transfected as described above. Three hours posttransfection, monolayers were incubated in PBS with or without 42 mM of the 30-mer B5 peptide. Forty-five hours later, the cells were washed, fixed, stained, and scored as described above. Data are expressed as percentages of the totals counted, where 100% is set as the level of polykaryons in vector-transfected cells without peptide.

Model of minimum required receptors for HSV entry. A cartoon model to simplify use of multiple receptors into two minimum types of required receptor interactions to mediate HSV entry. The working model proposes that at least two types of viral glycoprotein and cellular receptor interactions are required to trigger pH-independent virus-cell membrane fusion. Ligands I and II on the virion are in an entry-incompetent state until ligand I (gD) encounters any one of its cell receptors (HVEM, nectin 1, nectin 2, or 3-OS-HS). It then undergoes conformational changes to an entry-active form. These changes would induce rearrangements in viral ligand II to an entry-competent form that can interact with host cell receptor II (B5 and possibly others). Interactions between ligand II and receptor II drive changes in the attachment-fusion complex to eventually trigger localized fusion of the viral envelope and host membrane. Similar to SNARE interactions or interactions in class I viral fusion proteins that contain α-helices, the putative coiled coils in B5 (a type of receptor II) and partner coils in its viral ligand(s) could mediate the interactions required to trigger membrane fusion at entry.