OrfA downregulates feline immunodeficiency virus primary receptor CD134 on the host cell surface and is important in viral infection

Abstract

Feline immunodeficiency virus (FIV) OrfA is an accessory protein that is critical for productive viral replication and infection in T cells. Here, we show that OrfA acts to markedly reduce cell surface expression of the FIV primary binding receptor. Downregulation does not occur at the transcriptional or translational level in that the amounts of CD134 mRNA and protein in total cell lysates are not altered between parental 104-C1 T cells and the same cell line stably expressing OrfA (104-C1-OrfA). Analysis by confocal microscopy revealed significant accumulation of CD134 in the Golgi apparatus of 104-C1 cells expressing OrfA. OrfA does not cause a generalized disruption of membrane trafficking in that surface expression of CD9 is unaffected by OrfA overexpression. Consistent with the above observations, OrfA-negative FIV-34TF10 productively infects CrFK (CD134-negative) and 104-C1-OrfA (CD134 downregulated by OrfA) cells but fails to productively infect either 104-C1 (CD134-positive) cells or GFox (CrFK cells overexpressing CD134) cells. FIV-34TF10 in which the OrfA reading frame is open (OrfArep) productively infects CrFK, GFox, 104-C1, and 104-C1-OrfA cells. We hypothesize that reduced surface expression of the receptor, a hallmark of retrovirus infections, may facilitate an increase in virus release from the infected cell by minimizing receptor interactions with budding virus particles.

FIG. 1.

CrFK, GFox, and 104-C1 cell infection assay using FIV-34TF10 and OrfArep strains. Virus growth in cells was evaluated by a reverse transcriptase activity assay over time. (A) Both 34TF10 and OrfArep strains can productively infect CrFK cells, and the RT activity level is significantly above that of the cell-only control at 14 days postinfection. (B and C) 104-C1 and GFox cells can be productively infected with the OrfArep strain but not with the 34TF10 strain.

FIG. 2.

FACS assays for detection of cell surface receptors. (A) Detection of CD134 and CD9 receptors on 104-C1 and 104-C1-OrfA cells. (Left) Detection of CD134 using rabbit anti-CD134 antibody. Red, background control; blue, 104-C1-OrfA cells; green, 104-C1 cells. (Middle) Detection of CD134 using SU-Fc adhesion. Red, background control; blue, 104-C1-OrfA cells; green, 104-C1 cells. (Right) Detection of CD9 using mouse anti-CD9 antibody. Red, background control; blue, 104-C1-OrfA cells; green, 104-C1 cells. (B) FACS analysis of infected 104-C1 cells using rabbit anti-CD134 antibody. (Left) Detection of CD134 on FIV-PPR-infected 104-C1 cells. Red, background control; blue, FIV-PPR-infected 104-C1 cells; green, uninfected 104-C1cells. (Right) Detection of CD134 on 34TF10-OrfArep-infected 104-C1 cells. Red, background control; blue, OrfArep-infected 104-C1 cells; green, uninfected 104-C1 cells.

FIG. 3.

OrfA mRNA quantitation and FACS assays on CD134 surface levels for siRNA-treated OrfA knockdown cells. (A) Quantitative real-time PCR showed that OrfA mRNA level was greatly reduced in siRNA-transfected 104-C1-OrfA cells. Scrambled siRNA had no effect on the turnover rate of OrfA mRNA. (B) siRNA knockdown assay for confirming OrfA as a function of CD134 downregulation. FACS analysis for detecting CD134 on siRNA-treated OrfA transfected 104-C1 cells. The CD134 level on transfected cells is upregulated 2-fold compared to the level for 104-C1-OrfA. Scrambled siRNA had no effect on the regulation of CD134.

FIG. 4.

Quantitative real-time PCR and Western blot analysis on CD134 and CD9 expression. (A) Quantitative real-time PCR assessment on CD134 mRNA levels in various cell lines. The amount of CD134 mRNA in 104-C1-OrfA cells is approximately the same as that in 104-C1 cells. (B) Western blot against CD134 and CD9 in 104-C1 and 104-C1-OrfA cells. Lane 1, 104-C1 total cell lysate (7 μg); lane2, 104-C1-OrfA total cell lysate (7 μg); lane3, 104-C1 plasma membrane protein (2 μg); lane 4, 104-C1-OrfA plasma membrane protein (2 μg); lane 5, CrFK (upper panel) and 3201 (lower panel) plasma membrane protein (2 μg) as negative controls.

FIG. 5.

Infection assays on 104-C1-OrfA cells by FIV-34TF10 and OrfArep showing that the cells were infected by both viral strains.

FIG. 6.

β-Gal assay for measurement of the entry of FIV-PPR or FIV-34TF10 envelope-pseudotyped virons. (A) pCFIV vectors pseudotyped with FIV-PPR Env were used to assess the degree of viral entry. A β-Gal assay performed at 48 h posttransduction shows that pCFIV pseudotyped with PPR Env infected GFox and 104-C1 cells more efficiently than it did CrFK and 104-C1-OrfA cells. RLU, relative luminescence units. (B) pCFIV vectors pseudotyped with 34TF10 Env were used to assess the viral entry for the same cell lines as those used for the above-described assay. The results showed that the viron infected CrFK cells more readily than it did GFox cells. On the other hand, no significant difference was observed for the 34TF10 Env-mediated viral entry between 104-C1 and 104-C1-OrfA cells.

FIG. 7.

Confocal microscopy analysis showing CD134 subcellular localization in 104-C1 and 104-C1-OrfA cells. (A) Confocal imaging of permeabilized 104-C1 cells. (B) The same staining for permeabilized 104-C1-OrfA cells. The panels in the 1st column represent staining for CD134 receptors (red) The panels in the 2nd column represent staining with GM130-Alexa for Golgi complexes (intense red, represented by green). The panels in the 3rd column show nuclear stain with DAPI (4′,6-diamidino-2-phenylindole) (blue) and the colocalization (white) of the two fluorescently labeled reagents. The panels in the 4th column show colocalization only. The results show that significantly more CD134 colocalizes with Golgi complexes in OrfA-expressing cells (B) than in untreated 104-C1 cells (A). PE, phycoerythrin.