Association with PAK2 Enables Functional Interactions of Lentiviral Nef Proteins with the Exocyst Complex

Abstract

Human immunodeficiency virus type 1 (HIV-1) Nef enhances virus replication and contributes to immune evasion in vivo, but the underlying molecular mechanisms remain incompletely defined. Nef interferes with host cell actin dynamics to restrict T lymphocyte responses to chemokine stimulation and T cell receptor engagement. This relies on the assembly of a labile multiprotein complex including the host kinase PAK2 that Nef usurps to phosphorylate and inactivate the actin-severing factor cofilin. Components of the exocyst complex (EXOC), an octameric protein complex involved in vesicular transport and actin remodeling, were recently reported to interact with Nef via the same molecular surface that mediates PAK2 association. Exploring the functional relevance of EXOC in Nef-PAK2 complex assembly/function, we found Nef-EXOC interactions to be specifically mediated by the PAK2 interface of Nef, to occur in infected human T lymphocytes, and to be conserved among lentiviral Nef proteins. In turn, EXOC was dispensable for direct downstream effector functions of Nef-associated PAK2. Surprisingly, PAK2 was essential for Nef-EXOC association, which required a functional Rac1/Cdc42 binding site but not the catalytic activity of PAK2. EXOC was dispensable for Nef functions in vesicular transport but critical for inhibition of actin remodeling and proximal signaling upon T cell receptor engagement. Thus, Nef exploits PAK2 in a stepwise mechanism in which its kinase activity cooperates with an adaptor function for EXOC to inhibit host cell actin dynamics.

HIV-1 Balances the Fitness Costs and Benefits of Disrupting the Host Cell Actin Cytoskeleton Early after Mucosal Transmission

“…The actin cytoskeleton facilitates the cellular entry of HIV particles (Liu et al, 2009), and modulation of its activity via Nef-mediated inhibition of cofilin (Stolp et al, 2009) or through PAK2-dependent association with the exocyst complex (Imle et al, 2015; Mukerji et al, 2012) could either promote entry during initial cellular infection, or subsequently inhibit it to minimize super-infection, similar to what down-regulation of CD4 and the HIV co-receptors CXCR4 and CCR5 is thought to achieve (Michel et al, 2005; Venzke et al, 2006). Effects on viral entry would require either highly localized activity of the small quantities of Nef protein contained in entering viral particles, or trans -effects of Nef released from infected cells on uninfected cells.…”

“…Nef also disrupts actin-cytoskeletal turnover, necessary to establish stable cell polarity required for fast cellular migration. This can be achieved through its association with p21-activated kinase (PAK) 2, enabling the formation of a large multiprotein complex containing Nef, PAK2, Rac, Cdc42, Vav, and members of the exocyst complex (Imle et al, 2015; Mukerji et al, 2012). Formation of this complex requires a patch of hydrophobic residues of Nef to facilitate activation of PAK2, which in turn phosphorylates and inactivates the actin-severing protein cofilin to disrupt chemotaxis-driven actin-cytoskeletal turnover (Nobile et al, 2010; Stolp et al, 2012; 2009).…”

“…Formation of this complex requires a patch of hydrophobic residues of Nef to facilitate activation of PAK2, which in turn phosphorylates and inactivates the actin-severing protein cofilin to disrupt chemotaxis-driven actin-cytoskeletal turnover (Nobile et al, 2010; Stolp et al, 2012; 2009). Nef also attenuates T cell receptor (TCR)-driven signaling and actin polymerization through a mechanism that is independent of PAK2’s kinase activity and instead depends on PAK2-mediated association of Nef with exocyst proteins (Imle et al, 2015). Another HIV-1 accessory protein, Vpu, has also been reported to inhibit T cell chemotaxis in response to the LN chemokines CCL19 and CCL21 by downregulating their common receptor CCR7 from the surface of infected cells (Ramirez et al, 2014).…”

HIV-1 Nef Disrupts CD4+ T Lymphocyte Polarity, Extravasation, and Homing to Lymph Nodes via Its Nef-Associated Kinase Complex Interface

“…To this end, we tested a panel of HIV-1 SF2 Nef.GFP proteins in which individual functional motifs are disrupted (Fig. 3A, 3B (40,45,68) only moderately reduced the disruption of polarization as the activity of these mutants was not statistically significant from WT Nef. As for most Nef activities (2), myristoylation of glycine 2, and thus efficient interaction with cellular membranes, was essential for T cell polarity disruption (G2A).…”

“…Consistently, ex vivo analyses of T lymphocyte extravasation across an endothelial cell monolayer under physiological shear stress revealed that Nef potently disrupts T lymphocyte polarization and interfered specifically with the diapedesis step. The use of an Nef mutant (F195A) that is specifically deficient in PAK2 association, and thus inhibiting actin remodeling, but exerts all other known Nef activities (37,(40)(41)(42)(43) demonstrated that Nef affects extravasation via a dual mechanism: although inhibition of subendothelial migration appears to be achieved via the known deregulation of actin dynamics, this mechanism was dispensable for the effects of Nef on T cell polarization during diapedesis (37). Because it remains unknown how the viral protein induces these effects, the goal of this study was to gain insight into the mechanisms and relevance of Nef-mediated disruption of T cell polarization and diapedesis.…”