PMC

(A) 293T cells (left panels) and HeLa cells (right panels) were trans-fected with a plasmid expressing HA-tagged Ebola virus VP40, and HIV-1 Vpu as indicated. Cell and virus-like particle lysates were analyzed by western blotting.
(B) Same as (A) except that 293T cells were transfected with a plasmid expressing HA-tagged Ebola virus VP40, in the presence or absence of coexpressed HIV-1 Vpu and IFNα treatment, as indicated.

293T cells (A), Jurkat cells (B),orprimary PBMCs (C) were infected with VSV-G-pseudotyped, envelope-defective HIV-1(WT) or HIV-1(del Vpu) and cultured in the presence or absence of 1000 U/ml IFNα. Cell lysates and extracellular particles that were either constitutively released into the culture supernatant, or released following buffer or subtilis in stripping, were pelleted and analyzed by western blotting.

CD4+ Jurkat T cells (A) or PBMCs from two donors (B) were infected with HIV-1(WT) or HIV-1(del Vpu) and treated with 100 U/ml IFNα 24 hr after infection. Supernatant sampleswere harvested at the indicated times thereafter, and infectious virus yield was measured using HeLa-TZM cells and expressed in relative light units (RLU).

(A) 293T cells (left panels) or COS-7 cells (right panels) were infected with VSV-G-pseudotyped HIV-1(WT) or HIV-1(del Vpu) at an moi of 0.2 and then treated with the indicated dose of IFNα. Extracellular particles and cell lysates were analyzed by western blotting using an anti-p24CA monoclonal antibody.
(B) Same as (A), except that infectious virions in culture supernatants were measured using HeLa-TZM indicator cells, as in . Error bars indicate the standard deviations of the mean.

(A) 293T or HeLa cells were infected with VSV-G-pseudotyped HIV-1(WT) or HIV-1(del Vpu) at an moi of 0.2 and then treated with the indicated dose of IFNα. Extracellular particles and cell lysates were harvested and analyzed by western blotting using an anti-p24CA monoclonal antibody.
(B) Same as (A) except that infectious virions in culture supernatants were measured using HeLa-TZM indicator cells and a chemiluminescent assay and expressed in relative light units (RLU). Error bars indicate the standard deviations of the mean.
(C) Jurkat T cells were infected as in (A) with VSV-G-pseudotyped, envelope-defective mutants of HIV-1(WT) or HIV-1(del Vpu) and were left untreated (−) or were treated with 1000 U/ml IFNα (+).

(A) 293T cells were infected with VSV-G-pseudotyped HIV-1/MA-YFP(WT) or HIV-1/MA-YFP(del Vpu) encoding a modified matrix protein with YFP inserted into the stalk region. Cells were treated with 1000 U/ml IFNα where indicated and examined by deconvolution microcopy. Two examples are shown for each condition.
(B) A single representative example of HeLa cells infected with VSV-G-pseudotyped HIV-1/MA-YFP(WT) or HIV-1/MA-YFP(del Vpu), as indicated.
(C) The proportion of cells with intense MA-YFP accumulation at the plasma membrane (PM) only, or both at internal sites and plasma membrane (Internal + PM), was quantified for 293T cells or HeLa cells, as indicated following infection with HIV-1/MA-YFP(WT) or HIV-1/MA-YFP(del Vpu). Cells were otherwise untreated, or were treated with 1000 U/ml IFNα. For each data point, between 60 and 100 individual cells were evaluated.
(D and E) HIV-1/MA-YFP(del Vpu)-infected, IFNα-treated (1000 U/ml) 293T cells were immunostained using antibodies against human CD63 (D) or EEA-1 (E). Arrows indicate occasional MA-YFP puncta apparently colocalizing with EEA-1+ early endosomes. Scale bars represent 10 µm, except in the lower panels of (E), where they indicate 1 µm.

(A) Overview of a COS cell transfected with the HIV-1(del Vpu) proviral plasmid, but otherwise untreated. Arrows indicate examples of cell-associated virions (expanded view is shown in inset).
(B) Accumulations of mature HIV-1(WT) virions apparently adhered to the surface of COS-7 cells upon treatment with 500 U/ml IFNα. Note that analogous clusters were not seen in the absence of IFNα treatment. Arrows indicate virions within the lumen of endosomal compartments.
(C) Expanded image of HIV-1(WT) particles within the endosomal compartments of an IFNα-treated cell. Individual mature viral particles are highlighted.
(D) Image showing surface accumulations of mature HIV-1(del Vpu) virions apparently adhered to the surface of COS-7 cells treated with 500 U/ml IFNα.
(E) Image showing HIV-1(del Vpu) particles within the endosomal compartments of an IFNα-treated cell. Individual mature viral particles are highlighted with arrows.
(F) Surface accumulations of HIV-1(WT) mature virions adhered to the cell surface or to each other on an IFNα-treated cell. Note that the accumulation of virus within endosomal compartments was not observed in cells expressing the endocytosis inhibitor Rab5a(S34N).
(G and H) Expanded images of surface-associated HIV-1(WT) virions (G) or HIV-1(del Vpu) virions (H) in cells treated with IFNα and expressing Rab5a(S34N). Arrows highlight sites where virions appear to be adhered to the cell surface or to one another via external ‘‘tethers.’’ Scale bars in all panels represent 100 nm.