Induction of the Galpha(q) signaling cascade by the human immunodeficiency virus envelope is required for virus entry

Abstract

Binding of human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) with the primary receptor CD4 and one of two coreceptors, CXCR4 or CCR5, activates a signaling cascade resulting in Rac-1 GTPase activation and stimulation of actin cytoskeletal reorganizations critical for HIV-1-mediated membrane fusion. The mechanism by which HIV-1 Env induces Rac-1 activation and subsequent actin cytoskeleton rearrangement is unknown. In this study, we show that Env-mediated Rac-1 activation is dependent on the activation of Galpha(q) and its downstream targets. Fusion and Rac-1 activation are mediated by Galpha(q) and phospholipase C (PLC), as shown by attenuation of fusion and Rac-1 activation in cells either expressing small interfering RNA (siRNA) targeting Galpha(q) or treated with the PLC inhibitor U73122. Rac-1 activation and fusion were also blocked by multiple protein kinase C inhibitors, by inhibitors of intracellular Ca2+ release, by Pyk2-targeted siRNA, and by the Ras inhibitor S-trans,trans-farnesylthiosalicylic acid (FTS). Fusion was blocked without altering cell viability or cell surface localization of CD4 and CCR5. Similar results were obtained when cell fusion was induced by Env expressed on viral and cellular membranes and when cell lines or primary cells were the target. Treatment with inhibitors and siRNA specific for Galpha(i) or Galpha(s) signaling mediators had no effect on Env-mediated Rac-1 activation or cell fusion, indicating that the Galpha(q) pathway alone is responsible. These results could provide a new focus for therapeutic intervention with drugs targeting host signaling mediators rather than viral molecules, a strategy which is less likely to result in resistance.

FIG. 1.

Model of signal transduction pathway elicited by Env interaction with CCR5 required for Rac-1 activation and membrane fusion. This study establishes the roles of various signaling molecules during HIV-1-induced membrane fusion by examining the effects of selective inhibitors and siRNA targeting the Gαq pathway using Rac-1 activation, virus entry, and infection assays. Signaling molecules previously shown to be activated by gp120 are shown in bold, and confirmed pathways are shown with a solid line (32, 35, 36, 48, 56). Suspected factors that may be involved are shown in lightface font, and potential pathways are shown with dotted lines. Ras is shown in black because it was shown to be activated by Env in this study. Open boxes represent siRNA or selective small-molecule inhibitors used to inhibit Gα_q and molecules downstream of Gα_q.

FIG. 2.

Env-mediated Rac activation and cell-cell fusion are PTX insensitive and PKA independent. (A) U87.CD4.CCR5 cells were treated with 200 ng/ml PTX for 18 h prior to and during a 30-min incubation with BSC40 cells expressing no Env (gray) or Env from HIV-1 strain ADA (black). Whole-cell lysates were then analyzed by Rac-1-specific G-LISA activation assay (average A₄₉₀ of duplicate wells [±standard deviation] is shown; data are representative of results from three similar experiments). (B) (Top) Actin-dependent cell fusion. Average fusion compared to untreated control reactions and detected by β-galactosidase activity (±standard deviation) is shown. U87.CD4.CCR5 cells were infected with vaccinia virus expressing β-galactosidase (vCB21R) overnight, and then these cells were pretreated with DMSO alone (black) or the PKA inhibitor H89 (DMSO soluble; white) or PKA-I(14-22) (grey) at the indicated concentrations (μM) for 1 h. A portion of these cells was mixed 1:1 with HIV_ADA or HIV_UNC Env-expressing cells (subtracted as background) for 3 h in the presence of inhibitors. Data are representative of results from three similar experiments performed in triplicate. (Bottom) Cell fusion was normalized using untreated cells incubated with HIV_ADA Env as 100%. The rest of the cells were lysed, and whole-cell lysates were analyzed for PKA activity in the presence of inhibitors. PKA was based on the standard curve of A₅₇₀ of various amounts of PKA. Results are representative of three independent experiments performed in duplicate. (C) U87.CD4.CCR5 cells were treated for 1 h with 1 μM TAK-779 or 10 μM H89 prior to and during a 30-min incubation with BSC40 cells expressing no Env (subtracted as background), HIV_ADA Env (open bars), or HIV_HXB2 Env (filled bars). Whole-cell lysates were then analyzed by Rac-1-specific G-LISA activation assay. Average A₄₉₀ of duplicate wells (±standard deviation) is shown; data are representative of results from three similar experiments (*, P < 0.01).

FIG. 3.

Gα_q downregulation with siRNA reduces Env-mediated cell-cell fusion. (A) Average fusion compared to untreated control reactions and detected by β-galactosidase activity (±standard deviation) is shown. U87.CD4.CCR5 cells were transfected with 100 nM control siRNA (control) or 100 nM siRNA targeted against Gα_q, Gα_i, or Gα_s. Twenty-four hours posttransfection, U87.CD4.CCR5 cells were serum starved. Forty-eight hours posttransfection, the cells were infected with vCB21R alone or with vRacV12 in complete media. Seventy-two hours posttransfection, the transfected U87.CD4.CCR5 cells were incubated with HIV_UNC (subtracted as background), HIV_ADA, HIV_YU2, HIV_89.6, or HIV_HXB2 Env-expressing cells for 3 h. Data are representative of results from three similar experiments performed in triplicate (**, P < 0.05). Cell fusion was normalized using control-siRNA-transfected cells incubated with HIV_ADA Env as 100%. (B) Each population of transfected cells (2 × 10⁵) was lysed, and whole-cell lysates were analyzed by Western blotting. The lysates from cells transfected with 100 nM control siRNA (lane 1) or 100 nM siRNA targeted to Gα_q (lane 2), Gα_i (lane 3), or Gα_s (lane 4) were loaded on each immunoblot (IB) and were initially probed with anti-Gα_q (left), anti-Gα_i (middle), or anti-Gα_s (right); then, all blots were stripped and probed with antiactin (bottom). The relative reduction index (RI) was calculated as the quotient of the densitometry signal for the Gα_q, Gα_i, or Gα_s band and that for actin and then normalized by the ratio obtained with control siRNA (considered 1). Data represent results from one of three experiments with similar results. (C) U87.CD4.CCR5 cells were transfected with 100 nM control siRNA or 100 nM Gα_q siRNA, Gα_i siRNA, or Gα_s siRNA as described above. Seventy-two hours posttransfection, cells were incubated for 30 min with BSC40 cells expressing no Env (subtracted as background) or HIV_ADA Env. Whole-cell lysates were then analyzed by Rac-1-specific G-LISA activation assay. Average A₄₉₀ of duplicate wells (±standard deviation) is shown; data are representative of results from three similar experiments (**, P < 0.05).

FIG. 4.

PLCβ is required for Env-mediated fusion upstream of Rac-1. Average fusion compared to untreated control reactions and detected by β-galactosidase activity (±standard deviation) is shown. (A) Serum-starved U87.CD4.CCR5 cells were infected with vCB21R alone or with vRacV12 in complete media overnight. These cells were then pretreated with DMSO alone or 3 μM of PLCβ inhibitor U73122 or its negative analog U73343 for 1 h, and the inhibitors were also present during the incubation with HIV_UNC (subtracted as background), HIV_ADA, HIV_YU2, HIV_89.6, or HIV_HXB2 Env-expressing cells for 3 h. Data are representative of results from three similar experiments performed in triplicate (*, P < 0.01; **, P < 0.05). Cell fusion was normalized using DMSO-treated cells incubated with HIV_ADA Env as 100%. (B) U87.CD4.CCR5 cells were treated for 1 h with 1 μM TAK-779, 3 μM U73122, 3 μM U73343, or DMSO alone (no inhibitor) prior to and during a 30-min incubation with BSC40 cells expressing no Env (subtracted as background) or HIV_ADA Env. Whole-cell lysates were then analyzed by Rac-1-specific G-LISA activation assay. Average A₄₉₀ of duplicate wells (±standard deviation) is shown; data are representative of results from three similar experiments (*, P < 0.01; **, P < 0.05). (C) Serum-starved PBLs were infected with vCB21R in complete media overnight. These cells were then pretreated with DMSO alone, 1 μM TAK-779, or 3 μM of PLCβ inhibitor U73122 or its negative analog U73343 for 1 h, and the inhibitors were also present during the incubation with HIV_UNC (subtracted as background), HIV_ADA, HIV_YU2, HIV_89.6, or HIV_HXB2 Env-expressing cells for 3 h. Data are representative of results from three similar experiments performed in triplicate (*, P < 0.01; **, P < 0.05). Cell fusion was normalized using DMSO-treated cells incubated with HIV_ADA Env as 100%.

FIG. 5.

Intracellular Ca²⁺ release is required for Env-mediated fusion upstream and downstream of Rac-1. Average fusion compared to untreated control reactions and detected by β-galactosidase activity (±standard deviation) is shown. (A) Serum-starved U87.CD4.CCR5 cells were infected with vCB21R alone or with vRacV12 in complete media overnight. These cells were then pretreated with DMSO alone or 100 μM dantrolene, 10 μM CPA, 2 μM TG, or 5 μM XC for 1 h, and the inhibitors were also present during the incubation with HIV_UNC (subtracted as background), HIV_ADA, HIV_YU2, HIV_89.6, or HIV_HXB2 Env-expressing cells for 3 h. Data are representative of results from three similar experiments performed in triplicate (*, P < 0.01). Cell fusion was normalized using DMSO-treated cells incubated with HIV_ADA Env as 100%. (B) U87.CD4.CCR5 cells were treated for 1 h with DMSO alone, 1 μM TAK-779, 100 μM dantrolene, 10 μM CPA, 2 μM TG, 5 μM XC, or 100 μM RacGEF inhibitor prior to and during a 30-min incubation with BSC40 cells expressing no Env (subtracted as background) or Env from HIV_ADA Env. Whole-cell lysates were then analyzed by Rac-1-specific G-LISA activation assay. Average A₄₉₀ of duplicate wells (±standard deviation) is shown; data are representative of results from three similar experiments (*, P < 0.01). (C) Serum-starved PBLs were infected with vCB21R in complete media overnight. These cells were then pretreated with DMSO alone, 100 μM dantrolene, 10 μM CPA, 2 μM TG, or 5 μM XC for 1 h, and the inhibitors were also present during the incubation with HIV_UNC (subtracted as background), HIV_ADA, HIV_YU2, HIV_89.6, or HIV_HXB2 Env-expressing cells for 3 h. Data are representative of results from three similar experiments performed in triplicate (*, P < 0.01). Cell fusion was normalized using DMSO-treated cells incubated with HIV_ADA Env as 100%.

FIG. 6.

PKC is required for Env-mediated fusion upstream of Rac-1. Average fusion compared to untreated control reactions and detected by β-galactosidase activity (±standard deviation) is shown. (A and B) Serum-starved U87.CD4.CCR5 cells were infected with vCB21R alone or with vRacV12 overnight in complete media. These cells were then pretreated with DMSO alone or the PKC inhibitor Bis I (10 μM), calphostin C (0.5 μM), or Ch Ch (50 μM) for 1 h (A) or pretreated with the specific PKC inhibitor Go-6976 (Ca²⁺ dependent, 1 μM), Ro-32-0432 (selective for PKCα and PKCβ, 10 μM), or PKC-I(20-28) (myristolated peptide inhibitor specific for PKCα and PKCβ, 100 μM) for 1 h (B). The inhibitors were also present during the incubation with HIV_UNC (subtracted as background), HIV_ADA, HIV_YU2, HIV_89.6, or HIV_HXB2 Env-expressing cells for 3 h. Data are representative of results from three similar experiments performed in triplicate (*, P < 0.01; **, P < 0.05). Cell fusion was normalized using DMSO-treated cells incubated with HIV_ADA Env as 100%. (C) U87.CD4.CCR5 cells were treated for 1 h with DMSO alone (no inhibitor), 1 μM TAK-779, 0.5 μM calphostin C, 50 μM Ch Ch, 1 μM Go-6976, 100 μM PKC-I(20-28), or 100 μM RacGEF inhibitor prior to and during a 30-min incubation with BSC40 cells expressing no Env (subtracted as background) or HIV_ADA Env. Whole-cell lysates were then analyzed by Rac-1-specific G-LISA activation assay. Average A₄₉₀ of duplicate wells (±standard deviation) is shown; data are representative of results from three similar experiments (*, P < 0.01). (D) Serum-starved PBLs were infected with vCB21R overnight in complete media. These cells were then pretreated with DMSO alone or the PKC inhibitor Ch Ch (50 μM) or Go-6976 for 1 h. The inhibitors were also present during the incubation with HIV_UNC (subtracted as background), HIV_ADA, HIV_YU2, HIV_89.6, or HIV_HXB2 Env-expressing cells for 3 h. Data are representative of results from three similar experiments performed in triplicate (*, P < 0.01). Cell fusion was normalized using DMSO-treated cells incubated with HIV_ADA Env as 100%.

FIG. 7.

Env-induced fusion and Rac-1 activation are dependent on Pyk2. (A) Average fusion compared to untreated control reactions and detected by β-galactosidase activity (±standard deviation) is shown. U87.CD4.CCR5 cells were transfected with 100 nM control siRNA (control) or 100 nM siRNA targeted against Pyk2 or Rac-1. Twenty-four hours posttransfection, U87.CD4.CCR5 cells were serum starved. Forty-eight hours posttransfection, the cells were infected with vCB21R alone or with vRacV12 in complete media. Seventy-two hours posttransfection, the transfected U87.CD4.CCR5 cells were incubated with HIV_UNC (subtracted as background), HIV_ADA, HIV_YU2, HIV_89.6, or HIV_HXB2 Env-expressing cells for 3 h. Data are representative of results from three similar experiments performed in triplicate (*, P < 0.01). Cell fusion was normalized using control-siRNA-transfected cells incubated with HIV_ADA Env as 100%. (B) Each population of transfected cells (2 × 10⁵) was lysed, and whole-cell lysates were analyzed by Western blotting. The lysates from cells transfected with 100 nM control siRNA (lane 1) or 100 nM siRNA targeted to Pyk2 (lane 2) or Rac-1 (lane 3) were loaded on each immunoblot (IB) and were initially probed with anti-Pyk2 (left) or anti-Rac-1 (right); then, all blots were stripped and probed with antiactin (bottom). The relative reduction index (RI) was calculated as the quotient of the densitometry signal for the Pyk2 or Rac-1 band and that for actin and then normalized by the ratio obtained with control siRNA (considered 1). Data represent results from one of three experiments with similar results. (C) U87.CD4.CCR5 cells were transfected with 100 nM control siRNA, 100 nM Pyk2 siRNA, or 100 nM Rac-1 siRNA as described above. Seventy-two hours posttransfection, cells were incubated for 30 min with BSC40 cells expressing no Env (subtracted as background) or HIV_ADA Env. Whole-cell lysates were then analyzed by Rac-1-specific G-LISA activation assay. Average A₄₉₀ of duplicate wells (±standard deviation) is shown; data are representative of results from three similar experiments (**, P < 0.05).

FIG. 8.

Ras is required for Env-mediated fusion upstream and downstream of Rac-1. Average fusion compared to untreated control reactions and detected by β-galactosidase activity (±standard deviation) is shown. (A) Serum-starved U87.CD4.CCR5 cells were infected with vCB21R alone or with vRacV12 in complete media overnight. These cells were then pretreated with DMSO alone or the indicated concentrations of the Ras inhibitor FTS for 1 h, and the inhibitor was also present during the incubation with HIV_UNC (subtracted as background), HIV_ADA, HIV_YU2, HIV_89.6, or HIV_HXB2 Env-expressing cells for 3 h. Data are representative of results from three similar experiments performed in triplicate (*, P < 0.01). Cell fusion was normalized using DMSO-treated cells incubated with HIV_ADA Env as 100%. (B) U87.CD4.CCR5 cells were treated for 1 h with DMSO alone (no inhibitor), 1 μM TAK-779, 50 μM FTS, or 100 μM RacGEF inhibitor prior to and during a 30-min incubation with BSC40 cells expressing no Env (subtracted as background) or Env from HIV_ADA Env. Whole-cell lysates were then analyzed by Rac-1-specific G-LISA activation assay. Average A₄₉₀ of duplicate wells (±standard deviation) is shown; data are representative of results from three similar experiments (*, P < 0.01; **, P < 0.05). (C) Serum-starved PBLs were infected with vCB21R in complete media overnight. These cells were then pretreated with DMSO alone, 50 μM Ras inhibitor FTS, or 100 μM RacGEF inhibitor for 1 h, and the inhibitor was also present during the incubation with HIV_UNC (subtracted as background), HIV_ADA, HIV_YU2, HIV_89.6, or HIV_HXB2 Env-expressing cells for 3 h. Data are representative of results from three similar experiments performed in triplicate (*, P < 0.01). Cell fusion was normalized using DMSO-treated cells incubated with HIV_ADA Env as 100%. (D) Ras activation assay. Western blot analysis of Raf-1 binding fractions from lysates of U87.CD4.CCR5 cells mixed with BSC40 cells expressing no Env (lane 3), HIV_HXB2 Env (lane 4), or HIV_ADA Env (lanes 5 to 9). TAK-779 (1 μM) was included to inhibit CCR5-Env binding (lane 5). Cells were pretreated with DMSO alone (lane 6) or the indicated concentrations of FTS to inhibit Ras (lanes 7 to 9) for 1 h prior to and during the 30-min incubation with Env-expressing cells. Positive (lane 1) and negative (lane 2) controls were generated by GTPγS and GDP loading of reaction lysates, respectively. Increases (n-fold) in the amount of Ras-GTP compared to that in lane 3 were determined by densitometry (normalized to lysate loading control) and are indicated. Data represent results from one of three experiments with similar results.

FIG. 9.

Effect of inhibitors on virus-dependent cell fusion with HIV-1 R5 virus and infection of TZM-BL cells with HIV-1 R5 virus or A-MLV-pseudotyped or VSV-G-pseudotyped HIV-1 virus. (A) Average fusion compared to untreated control reactions and detected by β-galactosidase activity (±standard deviation) is shown. One population of serum-starved U87.CD4.CCR5 cells was infected with vCB21R and the other population of U87.CD4.CCR5 cells was infected with vPT7-3 in complete media overnight. These cells were then mixed (1:1) in triplicate wells of a 96-well plate and pretreated with DMSO alone, TAK-779 (1 μM), U73122 (3 μM), U73343 (3 μM), Ch Ch (50 μM), Go-6976 (1 μM), PKC-I(20-28) (100 μM), dantrolene (100 μM), CPA (10 μM), TG (2 μM), XC (5 μM), or FTS (50 μM) for 1 h. Inhibitors were also present during incubation with 100 ng of HIV_YU2 per well for 3 h at 37°C. Data are representative of results from three similar experiments (*, P < 0.01). Cell fusion was normalized using DMSO-treated cells mixed with HIV_YU2 as 100%. VDFA, virus-dependent fusion assay. (B) JC53-BL cells were preincubated with DMSO alone, TAK-779 (1 μM), U73122 (3 μM), U73343 (3 μM), Ch Ch (50 μM), Go-6976 (1 μM), dantrolene (100 μM), CPA (10 μM), TG (2 μM), XC (5 μM), FTS (50 μM), NH₄Cl (50 mM), or OA (100 nM) for 1 h, and inhibitors were also present during the 3-h infection period with 150 ng of HIV_YU2, A-MLV-pseudotyped HIV-1, or VSV-G-pseudotyped HIV-1 per well. Virus and inhibitor were then washed off of the cells, and the cells were incubated in the same concentration of inhibitor at 37°C overnight. Luciferase activities in the infected cell lysates were measured 24 h postinfection and were used to calculate virus infectivity relative to that of the control. Data are representative of results from three similar experiments performed in triplicate (**, P < 0.05). Cell infection was normalized using DMSO-treated cells as 100%. (C) JC53-BL cells were transfected with 100 nM control siRNA (control) or 100 nM siRNA targeted against Gα_q, Gα_i, Gα_s, Pyk2, or Rac-1. Twenty-four hours posttransfection, JC53-BL cells were serum starved. Forty-eight hours posttransfection, the cells were infected with 150 ng of HIV_YU2 for 3 h. Virus was then washed off, and the cells were incubated at 37°C overnight. Luciferase activities in the infected cell lysates were measured at 24 h postinfection and were used to calculate virus infectivity relative to that of the control. Data are representative of results from three similar experiments performed in triplicate (**, P < 0.05). Cell infection was normalized using control siRNA-transfected calls infected with 150 ng of HIV_YU2 as 100%.

FIG. 10.

Gα_q inhibitors do not affect surface expression and localization of CD4 and CCR5 GFP. (A) Confocal micrographs of U87.CD4.CCR5 cells untreated or treated with DMSO alone, TAK-779 (1 μM), U73122 (3 μM), U73343 (3 μM), calphostin C (0.5 μM), Ch Ch (50 μM), PKC-I(20-28) (100 μM), dantrolene (100 μM), FTS (50 μM), or RacGEF inhibitor (100 μM) for 3 h or with cytochalasin D (1 μM) for 15 min, fixed, stained with anti-CD4-PE antibody (red), and counterstained with TO-PRO3 (blue). The green GFP signal and the red PE signal have been merged to show areas of colocalization (yellow). Data are representative of results from three experiments. Images were collected using an oil objective (magnification, ×63). (B) U87.CD4.CCR5 cells were incubated for 3 h with all inhibitors listed for panel A except cytochalasin D and detached by treatment with 5 mM EDTA. U87 cells and untreated and treated U87.CD4.CCR5 cells were labeled for surface expression of CD4 and CCR5 and analyzed by flow cytometry. Unlabeled U87.CD4.CCR5 cells were used to compensate for GFP. Data are expressed as percentages of surface expression based on DMSO-treated cells as 100%. Cal C, calphostin C.

FIG. 11.

Pretreatment with inhibitors results in specific effects on target molecules. (A) U87.CD4.CCR5 cells were treated with the PKA inhibitor H89 (10 μM), PLC inhibitor U73122 or its negative analog U73343 (10 μM), PKC inhibitor Ch Ch (50 μM), intracellular Ca²⁺ inhibitor dantrolene (100 μM), or Ras inhibitor FTS (50 μM) for 1 h. Whole-cell lysates were then analyzed for PKA activity in the presence of inhibitors. PKA was based on standard curves of densitometry of various amounts of PKA. Results are representative of two independent experiments performed in duplicate (*, P < 0.01; **, P < 0.05). (B) Western blot analysis of phosphorylated Pyk2 from lysates of U87.CD4.CCR5 cells mixed with BSC40 cells expressing no Env (lane 1) or Env from HIV-1_HXB2 (lane 3) or HIV-1_ADA (lanes 2 and 4 to 12) at 37°C for 5 min. Cells were pretreated with DMSO alone (no inhibitor [NI], lanes 1 to 3), 1 μM TAK-779 (lane 4), 3 μM U73122 (lane 5), 3 μM U73343 (lane 6), 0.5 μM calphostin C (lane 7), 50 μM Ch Ch (lane 8), 1 μM Go-6976 (lane 9), 100 μM PKC-I(20-28) (lane 10), 100 μM dantrolene (lane 11), or 50 μM FTS (lane 12) for 1 h prior to and during the 5-min incubation with Env-expressing cells. Cell lysates were resolved by 10% SDS-PAGE, transferred to a nitrocellulose membrane, and probed with anti-Pyk2 phosphospecific (pY579/580) antibody (P-Pyk2). The same blot was then stripped and reprobed with an antibody specific for total Pyk2. The fold indicated below the P-Pyk2 bands and above the total Pyk2 bands was calculated as the quotient of the densitometry signal for the P-Pyk2 band and that for total Pyk2 (shown below) and then normalized by the ratio obtained from cells treated with DMSO alone and mixed with BSC40 cells expressing no HIV-1 Env (considered 1). Blots shown are representative of three independent experiments.