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 Gαq-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.

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 G␣ q and its downstream targets. Fusion and Rac-1 activation are mediated by G␣ q and phospholipase C (PLC), as shown by attenuation of fusion and Rac-1 activation in cells either expressing small interfering RNA (siRNA) targeting G␣ 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 Ca 2؉ 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 G␣ i or G␣ s signaling mediators had no effect on Env-mediated Rac-1 activation or cell fusion, indicating that the G␣ 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.Entry of human immunodeficiency virus type 1 (HIV-1) is mediated by sequential binding of the trimeric HIV envelope glycoprotein (Env) to CD4 and one of two primary chemokine coreceptors, CXCR4 or CCR5. This binding triggers a series of conformational changes in Env that expose the fusion peptide, which then induces the merger of viral or infected cell membranes with target membranes (14,24,53). The HIV-1 Env interaction with CD4 and a coreceptor also stimulates various intracellular signaling events similar to those initiated by their natural ligands, such as phosphorylation of Pyk2, Ca 2ϩ mobilization, activation of RhoGTPases, and actin cytoskeleton rearrangements (15,20,36,37,48,51,53). Actin cytoskeletal remodeling and RhoGTPases play a central role in regulating fusion of biological membranes (13,22). In the case of HIV-1-induced membrane fusion, activation of the RhoGTPase Rac-1 and subsequent actin cytoskeletal reorganizations are required for efficient virus entry and infection (29, 48). The exact mechanism of Env-induced Rac-1 activation that mediates actin cytoskeletal rearrangements and induces membrane fusion has not been investigated.Binding of both CD4 and the coreceptors elicits signaling pathways that result in Rac-1 activation. However, previous results suggest that Env-induced Rac-1 activation is mediated via the coreceptor rat...

c Paramyxoviruses initiate entry through the concerted action of the tetrameric attachment glycoprotein (HN, H, or G) and the trimeric fusion glycoprotein (F). The ectodomains of HN/H/G contain a stalk region important for oligomeric stability and for the F triggering resulting in membrane fusion. Paramyxovirus HN, H, and G form a dimer-of-dimers consisting of disulfidelinked dimers through their stalk domain cysteines. The G attachment protein stalk domain of the highly pathogenic Nipah virus (NiV) contains a distinct but uncharacterized cluster of three cysteine residues (C146, C158, C162). On the basis of a panoply of assays, we report that C158 and C162 of NiV-G likely mediate covalent subunit dimerization, while C146 mediates the stability of higher-order oligomers. For HN or H, mutation of stalk cysteines attenuates but does not abrogate the ability to trigger fusion. In contrast, the NiV-G stalk cysteine mutants were completely deficient in triggering fusion, even though they could still bind the ephrinB2 receptor and associate with F. Interestingly, all cysteine stalk mutants exhibited constitutive exposure of the Mab45 receptor binding-enhanced epitope, previously implicated in F triggering. The enhanced binding of Mab45 to the cysteine mutants relative to wild-type NiV-G, without the addition of the receptor, implicates the stalk cysteines in the stabilization of a pre-receptor-bound conformation and the regulation of F triggering. Sequence alignments revealed that the stalk cysteines were adjacent to a proline-rich microdomain unique to the Henipavirus genus. Our data propose that the cysteine cluster in the NiV-G stalk functions to maintain oligomeric stability but is more importantly involved in stabilizing a unique microdomain critical for triggering fusion.