Lentiviral Nef Proteins Utilize PAK2-Mediated Deregulation of Cofilin as a General Strategy To Interfere with Actin Remodeling

Abstract

Nef is an accessory protein and pathogenicity factor of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) which elevates virus replication in vivo. We recently described for HIV type 1 SF2 (HIV-1 SF2 ) the potent interference of Nef with T-lymphocyte chemotaxis via its association with the cellular kinase PAK2. Mechanistic analysis revealed that this interaction results in deregulation of the actin-severing factor cofilin and thus blocks the chemokine-mediated actin remodeling required for cell motility. However, the efficiency of PAK2 association is highly variable among Nef proteins from different lentiviruses, prompting us to evaluate the conservation of this actin-remodeling/cofilin-deregulating mechanism. Based on the analysis of a total of 17 HIV-1, HIV-2, and SIV Nef proteins, we report here that inhibition of chemokine-induced actin remodeling as well as inactivation of cofilin are strongly conserved activities of lentiviral Nef proteins. Of note, even for Nef variants that display only marginal PAK2 association in vitro, these activities require the integrity of a PAK2 recruitment motif and the presence of endogenous PAK2. Thus, reduced in vitro affinity to PAK2 does not indicate limited functionality of Nef-PAK2 complexes in intact HIV-1 host cells. These results establish hijacking of PAK2 for deregulation of cofilin and inhibition of triggered actin remodeling as a highly conserved function of lentiviral Nef proteins, supporting the notion that PAK2 association may be critical for Nef's activity in vivo.

HIV-1 Nef interferes with T-lymphocyte circulation through confined environments in vivo

“…These results were mirrored in 3D matrices where Nef specifically inhibited T-lymphocyte motility in high-density collagen, although motility at low collagen density remained unaffected by Nef expression. It thus emerges that Nef specifically interferes with cell-motility modes that rely on the generation of contractile force to overcome physical constraints of the direct environment, including T-lymphocyte chemotaxis across transwell membranes (4,5,12,14) and diapedesis (present study). This specificity would also explain why Nef-mediated inhibition is relatively moderate on T-lymphocyte homing to spleen, which does not involve the crossing of an endothelial barrier, and motility in the lymph node parenchyma, where lymphocytes are embedded in a relatively wide-spaced extracellular matrix structure (19).…”

“…How these functions individually contribute to the prominent role of Nef in AIDS pathogenesis remains to be established. Inhibition of dynamic host cell actin remodeling, mediated via association with the cellular kinase PAK2 that induces inactivation of the actin-severing factor cofilin to reduce actin turnover, represents a conserved activity of lentiviral Nef proteins (2)(3)(4)(5)(6)(7)(8). Nef-PAK2 association depends on a critical phenylalanine at position 195 of Nef (or 191 depending on the HIV-1 nef allele analyzed) that is dispensable for other Nef activities (9).…”

Rho’ing in and out of cells

“…174 Furthermore, the Nef-PAK2 interaction potently inhibits motility of fibroblasts through accumulation of phosphorylated, inactive cofilin, a process that is highly conserved among different nef alleles of HIV-1, HIV-2, and SIV. 168,171,175,176 In addition, Nef has been reported to interact in a PAK2-dependent manner with components of the exocyst complex. 177 The exocyst complex is an octameric complex that tethers vesicles at the plasma membrane, regulates polarized exocytosis, and recruits membranes and proteins required for the formation of nanotubes that interconnect cells.…”

Functions of the Lentiviral Accessory Protein Nef During the Distinct Steps of HIV and SIV Replication Cycle

“…Then, Nef activates Rac by binding the DOCK2-ELMO1 complex, a key activator of Rac in antigen-and chemokine-initiated signaling pathways, and this interaction is linked to the abilities of Nef to inhibit chemotaxis and promote T cell activation (Janardhan et al, 2004). Finally, Nef association with Pak2 prevents actin remodeling to impair host cell motility by disregulation of cofilin, which is an actin-depolymerizing factor that promotes actin turnover and subsequent cell motility (Stolp et al, 2010). The cytoskeleton reorganization induced by Nef is associated with an impairment of cell movements combined with induction of long filopodium-like structures in T lymphocytes (Stolp et al, 2010).…”

“…Finally, Nef association with Pak2 prevents actin remodeling to impair host cell motility by disregulation of cofilin, which is an actin-depolymerizing factor that promotes actin turnover and subsequent cell motility (Stolp et al, 2010). The cytoskeleton reorganization induced by Nef is associated with an impairment of cell movements combined with induction of long filopodium-like structures in T lymphocytes (Stolp et al, 2010). In summary, Nef displays a variety of complex effects on the motility and cellular morphology of HIV-1-infectected T lymphocyte, thus resulting in a strategy to improve immune evasion and viral spread in the infected host.…”