Article Figures & Data
Figures
- FIG. 1.
ELR1 remains on the surface of EIAV-infected cells. ED cells and eUVECs (A) and chronically EIAV-infected cells (B) were immunostained for surface expression of ELR1 using rabbit polyclonal antisera against the ectodomain of ELR1. EIAV-infected populations were greater than 90% positive for EIAV antigens. Allophycocyanin-conjugated goat-anti-rabbit was used as a secondary antibody. Flow cytometry of live, stained cells was used to determine the percentage of the population that was positive for ELR1 (solid area). Secondary antisera alone (black line) as well as rabbit control sera (gray line) were used as negative controls. A representative flow experiment is shown. The experiments were performed three independent times.
- FIG. 2.
EIAV SU blocks entry of wild-type strains of EIAV. ED cells (A) or eUVECs (B) were incubated with increasing amounts of 293T cell supernatants that had been either transfected with an empty vector or a codon-optimized EIAVSP19 SU expression vector. The cells were subsequently infected with EIAVMA-1 or EIAVvMA-1c or transduced with VSV-G-pseudotyped EIAV particles that express β-galactosidase. Cells were evaluated for infection or transduction at 40 h. Shown is the ratio of the number of infected or transduced cells in the presence of supernatant divided by the number of infected or transduced cells when no supernatant was added. Data represent the means and standard errors of the means from three separate experiments performed in triplicate. *, P < 0.05; **, P < 0.001.
- FIG. 3.
EIAVvMA-1c can enter ED cells in an ELR1-independent manner. ED cells (A) or eUVECs (B) were incubated with preimmune serum or polyclonal anti-ELR1 antiserum at 4°C for 30 min. The cells were subsequently infected with EIAVMA-1 or EIAVvMA-1c or transduced with VSV-G-pseudotyped EIAV particles. Cells were stained 40 h after infection. Shown is the ratio of the number of infected or transduced cells in the presence of antiserum divided by the number of infected or transduced cells when no antiserum was added. Data represent the means and standard errors of the means from three separate experiments performed in triplicate. *, P < 0.05; **, P < 0.001.
- FIG. 4.
siRNA knockdown of ELR1 decreases EIAV entry into ED cells. ED cells were transfected with a control siRNA or ELR1 siRNA. Forty-eight hours after transfection, cells were either harvested for immunoblotting or used in infection/transduction studies. Cell lysates were immunoblotted for ELR1 and a tubulin loading control (A). Cells were stained for EIAV antigens or β-Gal activity at 40 h after infection (88 h after transfection) (B). Data represent the means and standard errors of the means from three separate experiments performed in triplicate. *, P < 0.05.
- FIG. 5.
EIAVvMA-1c retains the ability to interact with ELR1, and this interaction inhibits entry. EIAVMA-1 or EIAVvMA-1c virions and VSV-G-pseudotyped EIAV particles (MOI, 0.005) were incubated with the soluble ectodomain of ELR1 for 30 min at 4°C. The viruses were evaluated for ED cell infection. Cells were stained 40 h after infection. Shown is the ratio of the number of infected or transduced cells in the presence of ELR1 ectodomain divided by the number of infected or transduced cells when no ectodomain was added. Data represent the means and standard errors of the means from three separate experiments performed in triplicate.
- FIG. 6.
Interference with ELR1-EIAV interactions does not inhibit EIAVvMA-1c superinfection-dependent cell killing. ED cells were treated with inhibitors of ELR1-dependent entry at concentrations previously shown to inhibit 90% of EIAVMA-1 infectivity. The treated cells were infected with EIAVvMA-1c, and 4 days after infection the cells were assayed for viability. Data represent the means and standard errors of the means from three separate experiments performed in triplicate. Anti-EIAV, 1:60 dilution of equine anti-EIAV serum 2085; control sups, 100 μl of supernatant from 293T cells; WT EIAV SU sups, 100 μl of supernatant from codon-optimized SU-transfected 293T cells; normal rabbit serum, 1:60 dilution of normal rabbit serum; anti-ELR1, 1:60 of rabbit anti-ELR1 antiserum; NH4Cl, 30 mM ammonium chloride; CPZ, 20 μg/ml of chlorpromazine. **, P < 0.001.
- FIG. 7.
EIAVvMA-1c has similar binding kinetics for ELR1 as EIAVMA-1. EIAVMA-1 or EIAVvMA-1c binding kinetics were evaluated in ED cells (A), in ED cells in the presence of preimmune serum or anti-ELR1 antiserum (diluted 1:60) (B), and eUVECs (C). Virus particles were added to cells and then removed at the time points indicated. Cells were stained 40 h after infection and compared to the number of infected cells when particles were not removed. Data represent the means and standard errors of the means from three separate experiments performed in triplicate. *, P < 0.05.
