Fusion Stage of HIV-1 Entry Depends on Virus-Induced Cell Surface Exposure of Phosphatidylserine

Abstract

HIV-1 entry into host cells starts with interactions between the viral envelope glycoprotein (Env) and cellular CD4 receptors and coreceptors. Previous work has suggested that efficient HIV entry also depends on intracellular signaling, but this remains controversial. Here we report that formation of the pre-fusion Env-CD4-coreceptor complexes triggers non-apoptotic cell surface exposure of the membrane lipid phosphatidylserine (PS). HIV-1-induced PS redistribution depends on Ca²⁺ signaling triggered by Env-coreceptor interactions and involves the lipid scramblase TMEM16F. Externalized PS strongly promotes Env-mediated membrane fusion and HIV-1 infection. Blocking externalized PS or suppressing TMEM16F inhibited Env-mediated fusion. Exogenously added PS promoted fusion, with fusion dependence on PS being especially strong for cells with low surface density of coreceptors. These findings suggest that cell-surface PS acts as an important cofactor that promotes the fusogenic restructuring of pre-fusion complexes and likely focuses the infection on cells conducive to PS signaling.

Keywords: HIV entry; TMEM16F activity; cell activation; cell signaling; gp120-CD4-coreceptor; hemifusion; lipid scramblase; membrane fusion; phosphatidylserine exposure; viral entry.

Figure 1. Binding of HIV-1 pseudovirus to the target cell induces co-receptor-dependent and TMEM16F-mediated PS exposure at the cell surface

A. JkT-CCR5 cells were incubated with GaG-Clover R5-tropic pseudovirus (JR-FL) (2,3,4) (38 ng p24/ml) or mock solution (1) at 22°C for 15 min; then, Ruby-LactC2 was added and 45 min later unbound viruses were removed and the cells were imaged. 1μM CCR5 antagonist TAK-779 (3) and 60 μM TMEM16-inhibitor A01 (4) were added as described in Methods. Top images show LactC2 in red and virus in green. Bottom images are bright field. B. PS exposure at the surface of JkT-CCR5 cells for R5-tropic (JR-FL) (38 ng p24/ml) and X4-tropic (HXB2) virions (27 ng p24/ml). C. PS exposure at the surface of HeLa45 cells with varied expression and function of TMEM16F (see also Figure S3). Cell surface PS after virus (JR-FL,39 ng p24/ml) application and without it for HeLa45 cells, which along with CD4 and CCR5 and endogenous TMEM16F express: control shRNA (1), TMEM16F-silencing shRNA (2), TMEM16F-silencing shRNA together with shRNA-resistant form of the TMEM16F (3), w.t. TMEM16F (4), and constitutively active mutant TMEM16F (5). B, C. In each experiment, LactC2 fluorescence was normalized to that in the control experiment without virus (B, C, bars 1). Data are presented as means with 95 % confidence intervals.

Figure 2. HIV-1 Env-mediated cell–cell fusion depends on PS

A. Blocking of accessible PS with LactC2 inhibits, and adding exogenous PS promotes fusion. Fluorescence microscopy images of co-plated mCherry-labeled Env-cells (red) and eGFP-labeled TZM-bl cells (green). The cells were treated with 4 or 12 μM LactC2, exogenous PS or left untreated. Fused cells are seen as co-labeled (yellow) cells. B. PS-binding LactC2 and full-length lactadherin inhibit fusion. C, D Exogenous PS (C, D) and PG (D), in contrast to PC (D), promote fusion and increase fusion-inhibiting concentrations of TAK-779 (C) and C52L (D). B, C, D. Fusion extents measured by fusion-per-contact assay were normalized to those observed for untreated cells. All results are means± SEM (n≥3).

Figure 3. Interfering with cell signaling pathways involved in PS exposure inhibits Env-mediated cell–cell fusion

A. Fusion inhibition by blocking the elevation of intracellular calcium with BAPTA-AM (10μM), thapsigargin (TG, 2μM), cyclopiazonic acid (CPA, 10μM) and dantrolene (100μM) was partially rescued by exogenous PS. B. Fusion inhibition by suppressing PS externalization with A01 was partially lifted by PS. A, B. Fusion between mCherry-labeled Env-cells (red) and eGFP-labeled TZM-bl cells (green) was measured by the fusion-per-contact assay and fusion extents were normalized to those in the experiments with the untreated cells. C. Fluorescence microscopy images of the contacting Env-cells and TZM-bl cells illustrate inhibition of cell fusion (a decrease in the number of yellow cells) by 60 μM A01 (image 2 vs. image 1) and partial recovery of the fusion for the A01-treated cells after PS application (image 3 vs. 2). D. Fusion between Env-cells and HeLa45 cells with modified expression of TMEM16F was assayed with the fusion-per-target-cell assay. Fusion extents for the target cells expressing (2) TMEM16F-silencing shRNA, (3) TMEM16F-silencing shRNA together with shRNA-resistant TMEM16F, (4) w.t. TMEM16F, and (5) constitutively active mutant TMEM16F (5) were normalized to those in the experiments with the HeLa45 cells expressing control shRNA (bar 1). (6) HeLa4 cells expressing CD4 and constitutively active mutant TMEM16F but not expressing CCR5. All results are means± SEM (n≥3). Levels of significance relative to controls (bars 1) are shown as * for P < 0.05; ** for P<0.01 and *** for P < 0.001.

Figure 4. PS-dependent fusion stage follows gp120–coreceptor interactions and precedes hemifusion

A, B. An inhibitor of PS externalization, A01 (60 μM), suppresses both content- and lipid- mixing and thus blocks Env-mediated cell fusion upstream of hemifusion. A. Fluorescence microscopy images of a few Env-cells labeled with both RFP (red, content probe) and Vybrant DiI (green, membrane probe) bound to the adherent TZM-bl cells labeled with Hoechst 33342 (blue). Arrows mark a cell after completed fusion event (an adherent cell labeled with both membrane and content probes). As illustrated in the cartoon on the right, fusion arrest at a stage between hemifusion and fusion completion would produce adherent cells that acquired green membrane probe but not red content probe. B. Content and lipid mixing extents quantified as described in Methods were normalized to the number of Env-cells. C. LactC2 inhibits fusion, if present throughout cell interactions and fusion (bars 2 and 4). It does not inhibit fusion if applied to the cells accumulated for 3 h at the temperature-arrested stage (TAS) at the time of raising the temperature to 37°C (bars 3 and 5). D. Downstream of the PS-dependent stage, fusion progression does not require additional gp120–coreceptor engagements. The fusion block for 60 μM A01-treated cells was (still in the presence of A01) lifted by application of exogenous PS with (6) or without (5) 1 μM TAK-779. Controls in which the cells were untreated (1), treated with A01 (2), treated with TAK-779 (3), and treated with TAK-779 and PS (4). C, D. Fusion extents measured by fusion-per-contact assay were normalized to those observed for untreated cells. All results are means± SEM (n≥3). NS – stands for ‘no significant difference’. E. Our data place the PS-dependent fusion stage blocked by LactC2 and A01 downstream of gp120 interactions with coreceptors blocked by coreceptor antagonists and upstream of the temperature-dependent transition from assembled gp120–CD4-coreceptor to gp41 restructuring blocked at the TAS.

Figure 5. The dependence of HIV-1 virus-cell fusion on TMEM16F activity

A, B Effects of A01 on virus-cell fusion (A) and cell viability (B) for the pseudotyped HIV-1 particles HXB2pp or JR-CSFpp (red and blue circles, respectively) or for VSV G-pseudotyped particles (VSVpp, green) measured as BlaM activity. Pseudoviruses at MOI of ~1 (5 ng p24/ml for HXB2 and VSV-G, and 6 ng p24/ml for JR-CSF) were bound to adherent TZM-bl cells in the cold, and fusion was allowed to proceed for 90 min at 37°C. C, D. The knockdown of TMEM16F using shRNAs in JkT-CCR5 cells significantly inhibits HIV-1– and VSV–cell fusion (C) but does not lower cell viability (D). For panel C, the pseudoviruses were added to cells at MOI of ~3 (15 ng p24/ml). C52L (1 μM) and acidification inhibitor NH₄Cl (70 mM), were used as controls for HIV-1 and VSV fusion. Data points are means ± SEM of two independent triplicate experiments. ***P < 0.001.

Figure 6. Single-round infection with HIV-1 pseudovirus depends on PS externalization in the target cells

A, B. Blocking accessible PS with LactC2 inhibits infection of JkT-CCR5 cells by RFP-encoding HIV-1 pseudoviruses (JR-FL) at MOI of 0.5 (0.4 ng p24/ml). A. The fluorescence microscopy images of infected cells (seen in A as red) were taken and analyzed (B) 72 h post-infection. C. The effects of A01 pretreatment of Jurkat cells on the infection at MOI of 0.5 by RFP-encoding pseudoviruses carrying Env of three HIV-1 strains: X4-tropic HXB2 (2.7 ng p24/ml), and R5-tropic JR-FL (0.4 ng p24/ml) and BaL.01 (0.44 ng p24/ml). D, E. Inhibition of infection of TZM-bl cells with JR-CSF HIV-1 pseudovirus at MOI of 0.5 (6 ng p24/ml) with LactC2 (D) and with A01 pretreatment of the cells (E). F. The efficiency of RFP-encoding JR-FL virus infection (MOI of 0.5; 0.42 ng p24/ml) of the cells with modified expression of TMEM16F. Percentages of infected (=RFP-labeled) cells were measured for the target HeLa45 cells expressing (2) control siRNA, (3) TMEM16F-silencing shRNA, (4) TMEM16F-silencing shRNA together with shRNA-resistant TMEM16F, (5) w.t. TMEM16F, and (6) constitutively active mutant TMEM16F. The data were normalized to those in the control experiment with HeLa45 cells with unmodified expression of TMEM16F (1). B, C, D, E, F. The average numbers of infected cells per field of view at 72h post-infection were normalized to those in the control experiment with neither LactC2 (B, D) nor A01 (C, E) applied or to the control experiment with HeLa45 cells with unmodified expression of TMEM16F ((1), F). All results are means± SEM (n=4). F. Levels of significance relative to controls (bars 1) are shown as *** for P < 0.001.

Figure 7. Productive infection with HIV-1 depends on TMEM16F activity

A–F. Jurkat cells expressing either TMEM16F shRNA (A901 cells A, B, C, D, E, F upper panel) or control shRNA (C112 cells A, B, C, D, E, F lower panel) were inoculated with HIV_LAI.04 at 5ng (A, D), 0.5ng (B, E) or 0.05ng of p24 (C, F) per 10⁵ cells in 200 μl. Infection was evaluated from intracellular staining for p24 at days 3 (A–C) and 7 post-infection (D–F). G. Comparison between the percentages of infected A901cells and C112 cells at 7th day post-infection. Means± SEM of two experiments. H. Suppression of TMEM16F activity inhibits HIV_LAI.04 infection of human lymphoid tissue ex vivo. Human tonsillar tissue blocks were inoculated with HIV_LAI.04 with 5 μl of HIV_LAI.04 viral stock (50ng of p24/ml) per tissue block; DMSO or A01 was applied at 30 or 60μM. Tissue cultures were monitored for 12 days, medium was collected and replaced every 3 days, with replenishment of fresh A01. Virus production was evaluated from measurement of p24 accumulated in the culture medium. Infection was normalized to that in untreated tissue. I. The effects of A01 on the viability of the target cells were characterized by comparing the percentages of viable CD45+/CD3+/CD8− cells without virus (bars 1, 2) and after infection (bars 3–5). G, H, I. Means± SEM (n=2). * denotes statistical significant difference (P< 0.05) relative to controls (bars 1).