Role of Abl kinase and the Wave2 signaling complex in HIV-1 entry at a post-hemifusion step

Abstract

Entry of human immunodeficiency virus type 1 (HIV-1) commences with binding of the envelope glycoprotein (Env) to the receptor CD4, and one of two coreceptors, CXCR4 or CCR5. Env-mediated signaling through coreceptor results in Galphaq-mediated Rac activation and actin cytoskeleton rearrangements necessary for fusion. Guanine nucleotide exchange factors (GEFs) activate Rac and regulate its downstream protein effectors. In this study we show that Env-induced Rac activation is mediated by the Rac GEF Tiam-1, which associates with the adaptor protein IRSp53 to link Rac to the Wave2 complex. Rac and the tyrosine kinase Abl then activate the Wave2 complex and promote Arp2/3-dependent actin polymerization. Env-mediated cell-cell fusion, virus-cell fusion and HIV-1 infection are dependent on Tiam-1, Abl, IRSp53, Wave2, and Arp3 as shown by attenuation of fusion and infection in cells expressing siRNA targeted to these signaling components. HIV-1 Env-dependent cell-cell fusion, virus-cell fusion and infection were also inhibited by Abl kinase inhibitors, imatinib, nilotinib, and dasatinib. Treatment of cells with Abl kinase inhibitors did not affect cell viability or surface expression of CD4 and CCR5. Similar results with inhibitors and siRNAs were obtained when Env-dependent cell-cell fusion, virus-cell fusion or infection was measured, and when cell lines or primary cells were the target. Using membrane curving agents and fluorescence microscopy, we showed that inhibition of Abl kinase activity arrests fusion at the hemifusion (lipid mixing) step, suggesting a role for Abl-mediated actin remodeling in pore formation and expansion. These results suggest a potential utility of Abl kinase inhibitors to treat HIV-1 infected patients.

Figure 1. Down regulation of Wave2 signaling complex with siRNA reduces HIV-1 Env-mediated cell-cell fusion and virus-cell fusion.

U87.CD4.CCR5 cells were transfected with control siRNA (control) or siRNA targeted against (A) Trio, Tiam-1, Abl, Rac, (B) IRSp53, Wave2, and Arp3. Cells were serum starved 24 h post-transfection (pt), infected with vCB21R alone or with vRacV12 48 h pt, and 72 h pt cells were incubated for 3 h with HIV_UNC (subtracted as background), HIV_ADA, HIV_YU2, HIV_89.6 or HIV_HXB2 Env-expressing cells and β-gal activity was measured (C) Each population of transfected cells was analyzed by Western blot with antibodies to the designated protein or actin. The relative reduction index (RI) is the quotient of the densitometry signal for the target band and that for actin, normalized by the ratio obtained with control siRNA. (D) U87.CD4.CCR5 cells engineered to express a siRNA resistant clone of Arp3 were transfected with control siRNA, or siRNA targeted against Arp3 or Rac. Cell fusion was measured by β-gal activity, and was normalized using control siRNA transfected cells incubated with HIV_ADA Env as 100%. (E) Western blots were performed on siRNA-resistant U87.CD4.CC5 cells expressing siRNA-resistant Arp3. (F) TZM-BL cells were transfected with 200 nM of targeted siRNA indicated and 48 h pt cells were incubated for 90 min with X4 HIV_HXB2 virus R5 HIV_ADA virus or HIV_YU2 virus. Fusion was stopped by adding lysis buffer with BlaM substrate. Cells were then incubated at rt overnight in the dark. OD values for no virus samples was subtracted as background and percent Blam activity was normalized using control siRNA transfected cells incubated with X4 HIV_HXB2 virus as 100%. All data are representative of results from three similar experiments performed in triplicate.

Figure 2. Abl kinase is required upstream and downstream of Rac for HIV-1 entry.

(A) U87.CD4.CCR5 cells were infected with vCB21R alone, or with (B) vRacV12 overnight, then treated with DMSO alone, TAK-779, IMB, NIL, or DAS for 1 h and the inhibitors were also present during 3 h incubation with HIV-1 Env-expressing cells and β-gal activity as measured. (C) U87.CD4.CCR5 cells were treated for 1 h with TAK-779, IMB, NIL, or DAS and during 30 min incubation with BSC40 cells expressing no Env (subtracted as background), HIV_ADA Env or HIV_HXB2 Env. Whole cell lysates were analyzed by Rac specific G-LISA activation assay. Average A490 of triplicate wells ± standard deviation are shown. (D) PBMCs were infected with vCB21R in complete media overnight, treated with DMSO, TAK-779, IMB, NIL, or DAS for 1 h prior to addition of HIV-1 Env-expressing cells and β-gal activity was measured. (E) U87.CD4.CCR5 cells engineered to express indicated clones of Bcr-Abl were treated with Abl inhibitors and HIV_UNC or HIV_ADA Env as described above or analyzed by Western blot with anti-Abl or anti-actin antibody (inset). (F) U87.CD4.CCR5 cells were infected overnight with vCB21R or vPT7-3, then mixed (1∶1) in triplicate wells, treated for 1 h with DMSO, TAK-779, T20, IMB, NIL, DAS, and with 100 ng of HIV_YU2 for 3 h at 37°C. β-gal activity was measured and cell fusion was normalized using DMSO treated cells mixed with HIV_YU2 as 100%. (G) TZM-BL cells were treated with DMSO, 1 µM TAK-779 or AMD3100, 10 µM IMB, 500 nM NIL, or 150 nM DAS for 1 h prior to 90 min incubation with indicated HIV viruses and BlaM activity was measured. Activity was normalized using DMSO treated cells mixed with HIV_HXB2 virus as 100%. All data are representative of results from three similar experiments performed in triplicate.

Figure 3. Infection with HIV-1 particles but not particles pseudotyped with MLV Env or VSV-G depends on the Abl and Wave2 signaling complex.

(A) TZM-BL cells were incubated for 1 h with DMSO, TAK-779, AMD3100, NH₄Cl, IMB, NIL, or DAS and 150 ng of HIV_HXB2, HIV_YU2, A-MLV-or VSV-G-HIV-1 per well was added for 3 h, washed, and cells were incubated with inhibitors overnight and luc activity was measured. (B) TZM-BL cells were transfected with control siRNA or siRNA directed against indicated target proteins and 48 h later infected with 150 ng of HIV_HXB2, HIV_YU2, A-MLV- or VSV-G-HIV-1 for 3 h, cells were washed and incubated overnight, and luc activity measured. Cell infection was normalized using (A) DMSO treated cells or (B) cells transfected with control siRNA as 100%. All data are representative of results from three similar experiments performed in triplicate.

Figure 4. Abl kinase inhibitors and expression of siRNA targeted to the Wave2 complex block HIV-1 Env-mediated infection at a post-hemifusion step.

(A) TZM-BL cells were treated with DMSO, TAK-779, AMD3100, IMB, NIL, or DAS for 1 h. HIV_YU2 HIV_HXB2, A-MLV-Env-HIV-1 or VSV-G-HIV-1 (150 ng) was added for 1 h then cells were then treated with indicated lipid analogs for 1–5 min. Cells were washed and incubated in inhibitor overnight and luc activities were measured. (B) TZM-BL cells were transfected with 200 nM control siRNA or siRNA targeted to Tiam-1, Abl, Rac, IRSp53, Wave2 or Arp3 and 48 h pt cells were incubated with 150 ng of HIV_YU2 or HIV_HXB2. After 3 h cells were washed and incubated at 37° in complete media overnight and luc activities were measured. Data are representative of results from three similar experiments performed in triplicate. Cell infection was normalized using DMSO treated cells or control siRNA transfected cells infected with HIV_YU2 or HIV_HXB2 as 100%.

Figure 5. Abl kinase inhibitors arrest HIV-1 Env-mediated fusion at the hemifusion step.

CHO-K1 cells that do not express GM1 were transfected with a GFP expressing plasmid (green), and 24 h later infected with WT vaccinia virus or vaccinia virus expressing HIV_ADA Env. After another 24 h, CHO-K1 cells were overlaid for 3 h with U87.CD4.CCR5 cells pre-treated for 1 h with DMSO, TAK-779, or IMB. Cells were fixed and stained with CTX-555 (red), and counterstained with TO-PRO 3 (blue). Images were collected using an oil objective (magnification X63). Images were cropped but relative cell size was maintained. The percentage of hemifused cells is listed.