SUMMARY Binding of the HIV envelope to the chemokine coreceptors triggers membrane fusion and signal transduction. The fusion process has been well characterized, yet the role of coreceptor signaling remains elusive. Here we describe a critical function of the chemokine coreceptor signaling in facilitating HIV infection of resting CD4 T cells. We find that static cortical actin in resting T cells represents a restriction, and HIV utilizes the Gαi-dependent signaling from the chemokine coreceptor CXCR4 to activate a cellular actin depolymerizing factor, cofilin, to overcome this restriction. HIV envelope-mediated cofilin activation and actin dynamics are important for a post entry process that leads to viral nuclear localization. Inhibition of HIV-mediated actin rearrangement markedly diminishes viral latent infection of resting T cells. Conversely, induction of active cofilin greatly facilitates it. These findings shed new light on viral exploitation of cellular machinery in resting T cells, where chemokine receptor signaling becomes obligatory.

Almost all viral pathogens utilize a cytoskeleton for their entry and intracellular transport. In HIV-1 infection, binding of the virus to blood resting CD4 T cells initiates a temporal course of cortical actin polymerization and depolymerization, a process mimicking the chemotactic response initiated from chemokine receptors. The actin depolymerization has been suggested to promote viral intracellular migration through cofilin-mediated actin treadmilling. However, the role of the virus-mediated actin polymerization in HIV infection is unknown, and the signaling molecules involved remain unidentified. Here we describe a pathogenic mechanism for triggering early actin polymerization through HIV-1 envelope-mediated transient activation of the LIM domain kinase (LIMK), a protein that phosphorylates cofilin. We demonstrate that HIV-mediated LIMK activation is through gp120-triggered transient activation of the Rack-PAK-LIMK pathway, and that knockdown of LIMK through siRNA decreases filamentous actin, increases CXCR4 trafficking, and diminishes viral DNA synthesis. These results suggest that HIV-mediated early actin polymerization may directly regulate the CXCR4 receptor during viral entry and is involved in viral DNA synthesis. Furthermore, we also demonstrate that in resting CD4 T cells, actin polymerization can be triggered through transient treatment with a pharmacological agent, okadaic acid, that activates LIMK and promotes HIV latent infection of resting CD4 T cells. Taken together, our results suggest that HIV hijacks LIMK to control the cortical actin dynamics for the initiation of viral infection of CD4 T cells.Infection by the human immunodeficiency virus (HIV) causes severe depletion of blood CD4 T cells (1, 2). The early interaction between HIV and T cells, particularly virus binding to its receptors, plays an important role in viral infection and pathogenesis. This interaction mediates viral fusion and entry (3, 4). It also initiates intracellular signaling cascades that are important for the early steps of the HIV life cycle (5-9). For example, it has recently been shown that at the earliest time of HIV infection, viral binding to the chemokine coreceptor, CXCR4, activates an actin depolymerization factor cofilin to increase the cortical actin dynamics in resting T cells, facilitating viral intracellular migration (9).The cortical actin is a common structure that is targeted by most viruses for entry and intracellular transport (10, 11). In HIV-1 infection, the direct involvement of the cortical actin in early stages of viral infection has been suggested in HIV-mediated CD4-CXCR4 receptor clustering (5-7, 12-14), subsequent viral DNA synthesis (9, 15), and intracellular migration (9). It has been shown that the initial binding of gp120 to surface CD4 promotes localized aggregation of the CD4 and CXCR4 receptor, which appears to be dependent on the actin-crosslinking protein filamin (7) and the ezrin-radixin-moesin protein moesin (5, 6). Filamin-A interacts directly with the cortical actin and with bot...

BackgroundIndividual differences in initial sensitivity to ethanol are strongly related to the heritable risk of alcoholism in humans. To elucidate key molecular networks that modulate ethanol sensitivity we performed the first systems genetics analysis of ethanol-responsive gene expression in brain regions of the mesocorticolimbic reward circuit (prefrontal cortex, nucleus accumbens, and ventral midbrain) across a highly diverse family of 27 isogenic mouse strains (BXD panel) before and after treatment with ethanol.ResultsAcute ethanol altered the expression of ∼2,750 genes in one or more regions and 400 transcripts were jointly modulated in all three. Ethanol-responsive gene networks were extracted with a powerful graph theoretical method that efficiently summarized ethanol's effects. These networks correlated with acute behavioral responses to ethanol and other drugs of abuse. As predicted, networks were heavily populated by genes controlling synaptic transmission and neuroplasticity.Several of the most densely interconnected network hubs, including Kcnma1 and Gsk3β, are known to influence behavioral or physiological responses to ethanol, validating our overall approach. Other major hub genes like Grm3, Pten and Nrg3 represent novel targets of ethanol effects. Networks were under strong genetic control by variants that we mapped to a small number of chromosomal loci. Using a novel combination of genetic, bioinformatic and network-based approaches, we identified high priority cis-regulatory candidate genes, including Scn1b, Gria1, Sncb and Nell2.ConclusionsThe ethanol-responsive gene networks identified here represent a previously uncharacterized intermediate phenotype between DNA variation and ethanol sensitivity in mice. Networks involved in synaptic transmission were strongly regulated by ethanol and could contribute to behavioral plasticity seen with chronic ethanol. Our novel finding that hub genes and a small number of loci exert major influence over the ethanol response of gene networks could have important implications for future studies regarding the mechanisms and treatment of alcohol use disorders.

Human memory and naive CD4 T cells can mainly be identified by the reciprocal expression of the CD45RO or CD45RA isoforms. In HIV-1 infection, blood CD45RO memory CD4 T cells are preferentially infected and serve as a major viral reservoir. The molecular mechanism dictating this differential susceptibility to HIV-1 remains largely obscure. Here, we report that the different susceptibility of memory and naive T cells to HIV is not determined by restriction factors such as Apobec3G or BST2. However, we observed a phenotypic distinction between human CD45RO and CD45RA resting CD4 T cells in their cortical actin density and actin dynamics. CD45RO CD4 T cells possess a higher cortical actin density and can be distinguished as CD45RO+Actinhigh. In contrast, CD45RA T cells are phenotypically CD45RA+Actinlow. In addition, the cortical actin in CD45RO memory CD4 T cells is more dynamic and can respond to low dosages of chemotactic induction by SDF-1, whereas that of naive cells cannot, despite a similar level of the chemokine receptor CXCR4 present on both cells. We further demonstrate that this difference in the cortical actin contributes to their differential susceptibility to HIV-1; resting memory but not naive T cells are highly responsive to HIV-mediated actin dynamics that promote higher levels of viral entry and early DNA synthesis in resting memory CD4 T cells. Furthermore, transient induction of actin dynamics in resting naive T cells rescues HIV latent infection following CD3/CD28 stimulation. These results suggest a key role of chemotactic actin activity in facilitating HIV-1 latent infection of these T cell subsets.

The analysis of low abundance and low molecular weight biomolecules is challenging due to their labile nature and the presence of high abundance, high molecular weight species such as serum albumin, which can hinder their detection. Functionalized hydrogel particles have proven to be ideally suited for this application. We here report the synthesis of hydrogel core and core-shell particles with incorporated Cibacron Blue F3G-A, and analysis of their harvesting properties. Hydrogel particle scaffolds consisting of cross-linked N-isopropylacrylamide and allylamine copolymers were synthesized via surfactant-free precipitation polymerization, with the blue dye subsequently affixed via a nucleophilic substitution reaction. The dye-functionalized core and coreshell particles were found to efficiently harvest and sequester dilute low molecular weight peptides and proteins from solution, with the core-shell particles more effectively excluding larger proteins. Moreover, proteins bound by core and core-shell particles containing blue dye were protected from tryptic degradation. These findings suggest that core and core-shell hydrogel particles containing Cibacron Blue F3G-A constitute promising new tools for peptide/protein biomarker harvesting applications.