Endocytosis is a critical step in entry of subgroup B avian leukosis viruses

Abstract

The avian leukosis virus (ALV) entry mechanism is controversial, with evidence for and against a low-pH requirement for viral fusion. To further address this question, we tested the entry of human immunodeficiency virus type 1 (HIV-1) pseudotyped with the envelope protein of subgroup B ALV (ALV-B) in the presence of three different lysosomotropic agents. These lysosomotropic agents were able to block the entry of wild-type and pseudotyped ALV-B in two different cell lines, strongly suggesting that ALV-B requires a low-pH step for entry. ALV-B and pH-dependent Semliki Forest virus (SFV) entered cells with slower uptake kinetics than HIV-1, which is pH independent. These slow uptake rates support the theory that ALV-B utilizes endocytic pathways to enter cells. Using immunofluorescence and electron microscopy analysis, we visualized the colocalization of virus particles with the endosomal marker transferrin and demonstrated virus particles in clathrin-coated vesicles and endosome-like structures. Surprisingly, a low-pH treatment did not overcome the inhibition of ALV-B entry by lysosomotropic agents. This indicates that, in contrast to SFV, ALV-B is unable to fuse at the cellular surface, even at a low pH. Taken together, our findings suggest that endocytosis and a subsequent low-pH step are critical for successful ALV-B infection.

FIG. 1.

Entry assay of an HIV-1-based vector pseudotyped with the envelope protein of ALV-B. (A) Astrocytoma cells (U-251) and HEK 293 cells were stably transfected with TVB^S3-expressing vector. Extracts from chicken embryonic fibroblasts (DF-1) and human cell lines transfected with or without TVB^S3 were prepared, and 10 μg of protein was analyzed by Western blot analysis to detect TVB^S3 expression. (B) HIV-1 vector (NLluc⁺env⁻) was pseudotyped with the envelope protein of ALV-B. Parental and HEK 293-TVB^S3 cells were infected for 3 h with pseudotyped ALV-B viruses (10 ng of p24). Supernatant was removed and replaced with fresh medium, and luciferase activity was measured 48 h postinfection. (C) Parental and U-251-TVB^S3 were infected with pseudotyped ALV-B (10 ng of p24) as described above for panel B. Experiments were performed in triplicates, and standard deviations are indicated for panels B and C.

FIG. 2.

Entry of wild-type and pseudotyped ALV-B is inhibited by lysosomotropic agents. HEK 293-TVB^S3 cells were incubated with pseudotyped ALV-B, SFV, a-MLV, or HTLV-1 for 4 h in the presence or absence of 50 mM NH₄Cl (A), 100 μg of chloroquine/ml (B), or 50 nM bafilomycin A₁ (C). Cells were challenged with pseudotyped ALV-B, SFV, a-MLV, and HTLV-1 with similar amounts of p24.Luciferase activity was measured from cellular extracts 48 h postinfection. (D) HEK 293-TVB^S3 cells were incubated with wild-type ALV-B that expresses GFP (MOI of 10) in the presence or absence of 50 mM NH₄Cl, 100 μg of chloroquine/ml, or 50 nM of bafilomycin A₁ for 4 h. Infection was determined by flow cytometry analysis by counting GFP-positive cells 72 h postinfection. Experiments were performed in triplicate, and standard deviations are indicated.

FIG. 3.

Entry kinetics of pseudotyped ALV-B. Pseudotyped ALV-B (10 ng of p24) was prebound to HEK 293-TVB^S3 (A) or U-251-TVB^S3 (B) cells at 4°C for 1 h. Infection was initiated by shifting the temperature to 37°C. At the indicated time point, cells were incubated for 2 to 3 h with 50 mM NH₄Cl to stop viral entry. Subsequently, supernatants were replaced with fresh medium and luciferase activity was determined 48 h postinfection. Pseudotyped SFV was prebound to HEK 293-TVB^S3 cells, and entry kinetics were determined as described above (C). Experiments were performed in triplicate, and standard deviations are shown.

FIG. 4.

Pseudotyped ALV-B particles colocalize with an endosomal marker. (A) Pseudotyped ALV-B particles (10 ng of p24) were prebound to U-251-TVB^S3-3 cells, and infection was started by shifting cells to 37°C. Cells were processed for immunofluorescence, stained using the anti-p24 antibodies, and developed with a Cy3-labeled antibody (arrows). (B) Pseudotyped ALV-B and human transferrin-Alexa 488 (green) were prebound to U-251-TVB^S3-3 cells at 4°C for 1 h. Viral infection and transferrin uptake were initiated by shifting the temperature to 37°C. After 30 min, cells were processed for immunofluorescence with an anti-p24 antibody and developed with a Cy3-labeled antibody (red). Z sections were obtained in both filters (Alexa 488 and Cy3) of a wide-field fluorescence microscope. Images were deconvolved as described in Materials and Methods. The 3-D projection of an infected cell (30 sections) was done in Imaris version 3.0, showing pseudotyped ALV-B particles (red), endosomes (green), and colocalization (yellow). (C) Individual Z sections are shown, and the distance from the cell surface is indicated in micrometers.

FIG. 4.

Pseudotyped ALV-B particles colocalize with an endosomal marker. (A) Pseudotyped ALV-B particles (10 ng of p24) were prebound to U-251-TVB^S3-3 cells, and infection was started by shifting cells to 37°C. Cells were processed for immunofluorescence, stained using the anti-p24 antibodies, and developed with a Cy3-labeled antibody (arrows). (B) Pseudotyped ALV-B and human transferrin-Alexa 488 (green) were prebound to U-251-TVB^S3-3 cells at 4°C for 1 h. Viral infection and transferrin uptake were initiated by shifting the temperature to 37°C. After 30 min, cells were processed for immunofluorescence with an anti-p24 antibody and developed with a Cy3-labeled antibody (red). Z sections were obtained in both filters (Alexa 488 and Cy3) of a wide-field fluorescence microscope. Images were deconvolved as described in Materials and Methods. The 3-D projection of an infected cell (30 sections) was done in Imaris version 3.0, showing pseudotyped ALV-B particles (red), endosomes (green), and colocalization (yellow). (C) Individual Z sections are shown, and the distance from the cell surface is indicated in micrometers.

FIG. 5.

Wild-type ALV-B is in clathrin-coated vesicles and endosome-like structures. Wild-type ALV-B viruses were prebound to DF-1 cells at 4°C for 1 h. Infection was initiated by shifting the temperature to 37°C. Thirty minutes after infection was initiated, samples were processed for ultrastructural analysis. Virus particles were observed (arrows) at a magnification of ×12,000 at the surface of DF-1 cells (A and B), in clathrin-coated vesicles (C and D), and in vesicles (E) while fusing to the vesicle membrane (F).

FIG. 6.

Entry of pseudotyped ALV-B is inhibited by chlorpromazine. HEK 293-TVB ^S3 cells were incubated with pseudotyped ALV-B, SFV, or a-MLV for 3 h in the presence or absence of 50 μM chlorpromazine. Cells were challenged with pseudotyped ALV-B, SFV, or a-MLV by using similar amounts of p24. Luciferase activity was measured from cellular extracts 48 h postinfection. Experiments were performed in triplicate, and standard deviations are shown.

FIG.7.

Wild-type and pseudotyped ALV-B were unable to fuse at the cell surface upon low-pH treatment. Pseudotyped SFV (10 ng of p24) (A), pseudotyped ALV-B (10 ng of p24) (B), and wild-type ALV-B (MOI = 10) (C) were prebound to HEK 293-TVB^S3 cells at 4°C for 1 h. Cells were treated with a medium, pH 5.3 or 7.0, for 2 min at 37°C. Subsequently, cells were incubated in fresh medium for 4 h in the presence or absence of 50 mM NH₄Cl. Supernatants were replaced with fresh medium, and luciferase activity was measured 48 h postinfection (A and B). Flow cytometry analysis for GFP-positive cells was used to determine infection by wild-type ALV-B (C). Pseudotyped ALV-B (10 ng of p24) was prebound to HEK 293-TVB^S3 cells at 4°C for 1 h. Cells were treated with a medium, pH 5.3, containing or lacking 50 mM NH₄Cl for 5, 15, or 30 min at 37°C. Subsequently, cells were incubated with medium containing 50 mM NH₄Cl for 4 h. Cells were grown in fresh medium, and extracts were prepared 48 h postinfection to measure luciferase activity (D). Experiments were performed in triplicate, and standard deviations are shown.