Equine infectious anemia virus entry occurs through clathrin-mediated endocytosis

Abstract

Entry of wild-type lentivirus equine infectious anemia virus (EIAV) into cells requires a low-pH step. This low-pH constraint implicates endocytosis in EIAV entry. To identify the endocytic pathway involved in EIAV entry, we examined the entry requirements for EIAV into two different cells: equine dermal (ED) cells and primary equine endothelial cells. We investigated the entry mechanism of several strains of EIAV and found that both macrophage-tropic and tissue culture-adapted strains utilize clathrin-coated pits for entry. In contrast, a superinfecting strain of EIAV, EIAV(vMA-1c), utilizes two mechanisms of entry. In cells such as ED cells that EIAV(vMA-1c) is able to superinfect, viral entry is pH independent and appears to be mediated by plasma membrane fusion, whereas in cells where no detectable superinfection occurs, EIAV(vMA-1c) entry that is low-pH dependent occurs through clathrin-coated pits in a manner similar to wild-type virus. Regardless of the mechanism of entry being utilized, the internalization kinetics of EIAV is rapid with 50% of cell-associated virions internalizing within 60 to 90 min. Cathepsin inhibitors did not prevent EIAV entry, suggesting that the low-pH step required by wild-type EIAV is not required to activate cellular cathepsins.

FIG. 1.

Virion internalization kinetics. EIAV_MA-1, EIAV_vMA-1c, or VSV-G-pseudotyped EIAV particles were bound to ED cells at 4°C for 1 h. The virus was removed, and medium was refreshed. Cells were washed with citric acid buffer at the times indicated to inactivate all virions that had yet to be internalized. Cells were stained 40 h after infection and compared to the number of infected cells when no citric acid wash was performed. Data represent the averages and standard errors of three experiments performed in triplicate.

FIG. 2.

EIAV entry is dynamin dependent. ED cells were transduced with an adenoviral vector that expressed either GFP or DN dynamin 1. Twenty-four hours after transduction, cells were infected with EIAV_MA-1 or EIAV_vMA-1c or transduced with VSV-G-pseudotyped EIAV particles. Cells were stained 40 h after infection and compared to the number of infected cells transduced with Ad-GFP. Data represent the averages and standard errors of three experiments performed in triplicate. *, P < 0.05.

FIG. 3.

EIAV_vMA-1c requires dynamic actin rearrangement for efficient entry. CytoD- (A) or GEN- (B) treated cells were infected with EIAV_MA-1 or EIAV_vMA-1c or transduced with VSV-G- or EBOV-pseudotyped particles. Cells were stained 40 h after infection and compared to the number of infected cells treated with the DMSO control. Data represent the averages and standard errors of three experiments performed in triplicate. *, P < 0.05.

FIG. 4.

Cholesterol in the target cell is not required for EIAV infectivity. ED cells were treated with MβCD for 1 h and infected with EIAV_MA-1 or EIAV_vMA-1c or transduced with VSV-G- or EBOV-pseudotyped EIAV particles (A). EIAV_MA-1 and EIAV_vMA-1c viral particles or VSV-G-transducing particles were incubated with MβCD for 1 h. The particles were diluted into ED medium and used to infect ED cells (B). Cells were stained 40 h after infection and compared to the number of infected cells when particles were treated with the control. Data represent the averages and standard errors of three experiments performed in triplicate. *, P < 0.05; **, P < 0.001.

FIG. 5.

ELR1 is not found in lipid rafts. ED cells (A), ED cells bound with EIAV_MA-1 particles (B), or EIAV_MA-1 viral particles alone (C) were lysed and run on a Nycodenz gradient. Detergent-resistant membranes (DRM) floated to the top fractions (1 to 5), whereas solubilized protein can be seen in the bottom fractions (6 to 11). The fractions were analyzed for ELR1, EIAV capsid, and caveolin 1 by Western blotting. EIAV_MA-1-bound ED cells were shifted to 37°C for 20, 40, or 60 min, and fractions were Western blotted for ELR1 (D).

FIG. 6.

EIAV_MA-1 enters ED cells through clathrin-mediated endocytosis. ED cells were treated with CPZ for 1 h and infected with EIAV_MA-1 or EIAV_vMA-1c or transduced with VSV-G-pseudotyped EIAV particles (A). ED cells were transfected with GFP, Eps15D3Δ2-GFP, or DNEps15Δ95-295-GFP. Twenty-four hours after transfection, the cells were infected with EIAV_MA-1 or EIAV_vMA-1c or transduced with VSV-G-pseudotyped EIAV particles (B). ED cells were treated with CPZ for 1 h and infected with EIAV_SP19, EIAV_Th.1, EIAV_WSU5, or EIAV_UK (C). Cells were stained 40 h after infection and compared to the number of infected cells when particles were treated with the control. Data represent the averages and standard errors of three experiments performed in triplicate. *, P < 0.05.

FIG. 7.

EIAV_vMA-1c enters eUVEC through clathrin-mediated endocytosis. eUVEC were treated with MβCD for 1 h and infected with EIAV_MA-1 or EIAV_vMA-1c or transduced with VSV-G- or EBOV-pseudotyped particles (A). eUVEC were treated with CPZ for 1 h and infected with EIAV_MA-1 or EIAV_vMA-1c or transduced with VSV-G-pseudotyped EIAV particles (B). Cells were stained 40 h after infection and compared to the number of infected cells when particles were treated with the control. Data represent the averages and standard errors of three experiments performed in triplicate. *, P < 0.05; **, P < 0.001.

FIG. 8.

Cathepsin inhibitors do not inhibit EIAV entry. ED cells were treated with a cathepsin L inhibitor (A) or a pan-cathepsin inhibitor (B) and infected with EIAV_MA-1 or EIAV_vMA-1c or transduced with VSV-G-pseudotyped EIAV particles. Cells were stained 40 h after infection and compared to the number of infected cells when particles were treated with the control. Data represent the averages and standard errors of three experiments performed in triplicate. Viral particles were incubated with cathepsin L protease for 1 h, and Western analysis was performed to examine cleavage of the viral glycoprotein (C). **, P < 0.001.