HIV-1 Nef is a key factor in AIDS pathogenesis. Here, we report that Nef potently inhibits motility of fibroblasts and chemotaxis of HIV-1-infected primary human T lymphocytes toward the chemokines SDF-1alpha, CCL-19, and CCL-21 ex vivo. Furthermore, Nef inhibits guided motility of zebrafish primordial germ cells toward endogenous SDF-1a in vivo. These migration defects result from Nef-mediated inhibition of the actin remodeling normally triggered by migratory stimuli. Nef strongly induces phosphorylation of cofilin, inactivating this evolutionarily conserved actin-depolymerizing factor that promotes cell motility when unphosphorylated. Nef-dependent cofilin deregulation requires association of Nef with the cellular kinase Pak2. Disruption of Nef-Pak2 association restores the cofilin phosphorylation levels and actin remodeling that facilitate cell motility. We conclude that HIV-1 Nef alters Pak2 function, which directly or indirectly inactivates cofilin, thereby restricting migration of infected T lymphocytes as part of a strategy to optimize immune evasion and HIV-1 replication.

When B cells encounter antigens on the surface of an antigen-presenting cell (APC), B cell receptors (BCRs) are gathered into microclusters that recruit signaling enzymes. These microclusters then move centripetally and coalesce into the central supramolecular activation cluster of an immune synapse. The mechanisms controlling BCR organization during immune synapse formation, and how this impacts BCR signaling, are not fully understood. We show that this coalescence of BCR microclusters depends on the actin-related protein 2/3 (Arp2/3) complex, which nucleates branched actin networks. Moreover, in murine B cells, this dynamic spatial reorganization of BCR microclusters amplifies proximal BCR signaling reactions and enhances the ability of membrane-associated antigens to induce transcriptional responses and proliferation. Our finding that Arp2/3 complex activity is important for B cell responses to spatially restricted membrane-bound antigens, but not for soluble antigens, highlights a critical role for Arp2/3 complex-dependent actin remodeling in B cell responses to APC-bound antigens.

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 ...

Integrating signals from multiple receptors allows cells to interpret the physiological context in which a signal is received. Here we describe a mechanism for receptor crosstalk in which receptor-induced increases in actin dynamics lower the threshold for signalling by another receptor. We show that the Toll-like receptor ligands lipopolysaccharide and CpG DNA, which are conserved microbial molecules, enhance signalling by the B-cell antigen receptor (BCR) by activating the actin-severing protein cofilin. Single-particle tracking reveals that increased severing of actin filaments reduces the spatial confinement of the BCR within the plasma membrane and increases BCR mobility. This allows more frequent collisions between BCRs and greater signalling in response to low densities of membrane-bound antigen. These findings implicate actin dynamics as a means of tuning receptor signalling and as a mechanism by which B cells distinguish inert antigens from those that are accompanied by indicators of microbial infection.