IntroductionReplication of HIV-1 in primary human T lymphocytes is tightly coupled to their activation state. Whereas HIV-1 undergoes early replication events in quiescent CD4 ϩ T lymphocytes, subsequent steps in the viral life cycle require cell activation. 1 T lymphocyte activation is primarily governed by signaling through the TCR complex after engagement in a tight contact with APCs; this is referred to as the immunological synapse (IS).TCR engagement by specific MHC-presented peptides launches highly dynamic and coordinated transport events that recruit specific factors to the IS and exclude others from it. This signal initiation triggers a broad cascade of downstream signaling that include dynamic F-actin remodeling at the IS, tyrosine phosphorylation, release of calcium flux, and activation of transcription. These events increase production of the T-cell survival cytokine IL-2 and are coordinated by the TCR proximal tyrosine kinase Lck, a master switch of TCR signaling. Immediately after TCR engagement, active Lck is recruited to the IS. 2-4 Whereas signal diversification and enhancement occur at the plasma membrane (PM), subsequent TCR signaling is compartmentalized and also occurs at intracellular membranes. An important intracellular arm of the TCR response is regulated by the N-Ras GTPase that is activated at Golgi membranes downstream of Lck. [5][6][7][8][9] T-cell activation is thought to be beneficial to HIV-1 because it allows transcriptional activation of latent provirus and progression of the life cycle. However, activation-induced cell death after TCR engagement runs the risk of limiting the lifespan of productively infected cells and thus the amount of viral progeny produced.Consequently, HIV-1 encodes gene products such as Nef to fine-tune the activation states of infected T lymphocytes. 10,11 Nef is a 25-to 34-kDa myristoylated accessory protein encoded by HIV-1, HIV-2, and SIV. Ex vivo, Nef enhances the single-round infectivity of virus particles and moderately accelerates virus spread over multiple rounds. 12 In vivo, Nef strongly boosts virus replication, particularly during primary infection, when the presence of Nef can elevate virus titers by more than 2 logs, and is critical for rapid disease progression. [13][14][15] This role of Nef as a pathogenicity factor is also revealed in transgenic mice, in which Nef expression induces AIDS-like depletion of CD4 ϩ T lymphocytes. 16 Delineating the mechanisms of Nef action has been hampered by the multitude of interactions with host T-cell proteins suggested to modulate various intracellular transport and signaling pathways. 17,18 This includes modulating exposure of cell-surface receptors such as MHC-I and II, CD4, and chemokine receptors to evade immune recognition and to prevent superinfection of infected cells, respectively (reviewed in Laguette et al 12 ). In addition, Nef affects the basal states of T-cell activation and the responsiveness of T lymphocytes to TCR signaling. [19][20][21][22] Initial studies with overexpression strategies ...

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.

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