Role of Abl Kinase and the Wave2 Signaling Complex in HIV-1 Entry at a Post-Hemifusion Step

Brooke Harmon,Alexandra Trkola,Lee Ratner,Nancy Campbell

doi:10.1371/journal.ppat.1000956

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 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.

Highlights

human immunodeficiency virus type 1 (HIV-1) enters cells in a pH-independent manner by fusion at the plasma membrane or from within endosomes [1,2,3]
Patients infected with HIV-1 are currently treated with highly active antiretroviral therapy (HAART) that efficiently suppresses the virus but does not cure the infection
HIV-1 envelope activates Rac-mediated actin cytoskeleton rearrangements in the target cell that promote membrane fusion and entry. We discovered that these rearrangements require activation of the actin polymerization machinery including the tyrosine kinase Abl

Summary

Introduction

HIV-1 enters cells in a pH-independent manner by fusion at the plasma membrane or from within endosomes [1,2,3]. HIV-1 entry requires multiple conformational changes in the HIV-1 glycoprotein, and rearrangement of the actin cytoskeleton These events are triggered by binding of the viral envelope (Env) surface subunit gp120 to the primary receptor CD4 and one of two chemokine coreceptors, CCR5 or CXCR4 [1,4]. This interaction activates signaling events in the cell, similar to those initiated by natural ligands, such as Ca2+ mobilization, activation of RhoGTPases, and phosphorylation of tyrosine kinases, pyk, Zap and p56lck [4,5,6]. In the current study we identified the fusionspecific effectors of Rac required for actin cytoskeleton rearrangements that mediate membrane fusion and entry

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Discussion

Conclusion