Cofilin Activation in Peripheral CD4 T Cells of HIV-1 Infected Patients: A Pilot Study

Cofilin is an actin-depolymerizing factor that regulates actin dynamics critical for T cell migration and T cell activation. In unstimulated resting CD4 T cells, cofilin exists largely as a phosphorylated inactive form. Previously, we demonstrated that during HIV-1 infection of resting CD4 T cells, the viral envelope-CXCR4 signaling activates cofilin to overcome the static cortical actin restriction. In this pilot study, we have extended this in vitro observation and examined cofilin phosphorylation in resting CD4 T cells purified from the peripheral blood of HIV-1-infected patients. Here, we report that the resting T cells from infected patients carry significantly higher levels of active cofilin, suggesting that these resting cells have been primed in vivo in cofilin activity to facilitate HIV-1 infection. HIV-1-mediated aberrant activation of cofilin may also lead to abnormalities in T cell migration and activation that could contribute to viral pathogenesis.Department of Defense (National Defense Science and Engineering Fellowship); National Institute of Allergy and Infectious Diseases (AI069981

The HIV Envelope but Not VSV Glycoprotein Is Capable of Mediating HIV Latent Infection of Resting CD4 T Cells

HIV fusion and entry into CD4 T cells are mediated by two receptors, CD4 and CXCR4. This receptor requirement can be abrogated by pseudotyping the virion with the vesicular stomatitis virus glycoprotein (VSV-G) that mediates viral entry through endocytosis. The VSV-G-pseudotyped HIV is highly infectious for transformed cells, although the virus circumvents the viral receptors and the actin cortex. In HIV infection, gp120 binding to the receptors also transduces signals. Recently, we demonstrated a unique requirement for CXCR4 signaling in HIV latent infection of blood resting CD4 T cells. Thus, we performed parallel studies in which the VSV-G-pseudotyped HIV was used to infect both transformed and resting T cells in the absence of coreceptor signaling. Our results indicate that in transformed T cells, the VSV-G-pseudotyping results in lower viral DNA synthesis but a higher rate of nuclear migration. However, in resting CD4 T cells, only the HIV envelope-mediated entry, but not the VSV-G-mediated endocytosis, can lead to viral DNA synthesis and nuclear migration. The viral particles entering through the endocytotic pathway were destroyed within 1–2 days. These results indicate that the VSV-G-mediated endocytotic pathway, although active in transformed cells, is defective and is not a pathway that can establish HIV latent infection of primary resting T cells. Our results highlight the importance of the genuine HIV envelope and its signaling capacity in the latent infection of blood resting T cells. These results also call for caution on the endocytotic entry model of HIV-1, and on data interpretation where the VSV-G-pseudotyped HIV was used for identifying HIV restriction factors in resting T cells

Chemokine Coreceptor Signaling in HIV-1 Infection and Pathogenesis

Binding of the HIV-1 envelope to its chemokine coreceptors mediates two major biological events: membrane fusion and signaling transduction. The fusion process has been well studied, yet the role of chemokine coreceptor signaling in viral infection has remained elusive through the past decade. With the recent demonstration of the signaling requirement for HIV latent infection of resting CD4 T cells, the issue of coreceptor signaling needs to be thoroughly revisited. It is likely that virus-mediated signaling events may facilitate infection in various immunologic settings in vivo where cellular conditions need to be primed; in other words, HIV may exploit the chemokine signaling network shared among immune cells to gain access to downstream cellular components, which can then serve as effective tools to break cellular barriers. This virus-hijacked aberrant signaling process may in turn facilitate pathogenesis. In this review, we summarize past and present studies on HIV coreceptor signaling. We also discuss possible roles of coreceptor signaling in facilitating viral infection and pathogenesis

HIV -1 envelope signaling through CXCR4 activates cofilin to promote actin dynamics critical for HIV-1 infection of resting CD4 T cells

Binding of the HIV-1 envelope to chemokine coreceptors mediates two major biological events: membrane fusion and signal transduction. The fusion process has been well characterized. Yet the role of coreceptor signaling in HIV-1 infection remained elusive. In my dissertation, I present a summary of my investigation into the mechanism of how chemokine coreceptor signaling facilitates HIV-1 infection of human resting CD4 T cells. In human T cells, chemokine receptor signaling is directly linked to cytoskeletal remodeling, thus, I hypothesized that the actin dynamics driven through HIV envelopeCXCR4 signaling could be important for HIV-1 infection of T cells. My study revealed that inhibition of HIV-mediated actin change markedly diminishes viral latent infection of resting T cells, confirming a critical role of actin dynamics in HIV infection of resting T cells. Moreover, I discovered that HIV-1 uses CXCR4 signaling to activate a cellular actin depolymerizing factor, cofilin, to increase actin dynamics. Induction of cofilin activation in resting CD4 T cells greatly enhanced HIV-1 infection of resting CD4 T cells. Overall, these results provide a mechanism of how HIV-1 exploits the chemokine receptor signaling to alter the cellular environment in its favor. Given the critical role of cofilin in T cell activation and chemotaxis, my study of cofilin activation in HIV-1 infection has numerous implications for HIV-1 pathogenesis in T cells in vivo

HIV-1 envelope-CXCR4 signaling triggers cofilin activation to promote cortical actin dynamics and HIV nuclear migration.

<p>(A) HIV-1 envelope binding to CD4 and the chemokine coreceptor, CXCR4, triggers membrane fusion and signaling transduction. The initial viral contact with CD4 and then CXCR4 may trigger rapid actin polymerization to facilitate CD4/CXCR4 cocapping for fusion and entry. Following fusion, the viral preintegration complex (PIC) may be directly anchored onto F-actin to facilitate reverse transcription. Subsequent actin activity mediated by cofilin activation through CXCR4 promotes viral nuclear migration. (B) Model of HIV PIC migration along the cortical actin filaments. It is possible that cofilin activation increases actin treadmilling, which promotes the movement of the viral PIC across the cortical actin barrier, allowing PIC to gain access to the perinuclear or nuclear region. The number is arbitrarily assigned to an actin monomer to demonstrate the actin movement during treadmilling. (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000520#ppat-1000520-g003" target="_blank">Figure 3</a> is modified from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000520#ppat.1000520-Yoder1" target="_blank">[44]</a> with permission).</p

Components of the chemokine coreceptor signaling pathways activated by HIV-1 envelope.

<p>HIV-1 gp120 binding to CXCR4 or CCR5 activates a number of signaling molecules common to chemokine-mediated signaling pathways, including (A) PLC-γ-dependent calcium flux and NFAT nuclear translocation; (B) PI3K-dependent activation of FAK, PyK2, AKT, and ERK1/2; (C) the downstream targets of the Rho family GTPases such as LIMK1 and cofilin for actin rearrangement.</p

Chemokine receptor signaling pathways.

<p>SDF-1 binding to CXCR4 or RANTES/MIP-1α/MIP-1β binding to CCR5 activates G proteins (Gα particularly Gα_i, Gα_q, and Gβγ) and multiple downstream pathways. (A) Gα_q activates phospholipases such as phospholipase C-γ (PLC-γ), which hydrolyzes phosphatidylinositol-4,5-biphosphate (PIP2) to generate inositol triphosphate (IP3) and diacylglyerol (DAG), triggering calcium influx and the activation of kinases such as protein kinase C (PKC). (B) Gα_i activates phospholipases, phosphodiesterases, and the lipid kinase PI3K via Src-family kinases. Gβγ also activates PI3Kγ. PI3K activation stimulates downstream targets such as protein kinase B (PKB/Akt), NF-κB, mitogen/extracellular signal-regulated kinase (MEK-1), and extracellular signal-regulated kinase (ERK1/2). PI3K also triggers the tyrosine phosphorylation of focal adhesion complex components such as proline-rich tyrosine kinase (Pyk2), paxillin, Crk, and p130Cas. (C) GTP-bound Gβγ stimulates guanine nucleotide exchange factors (GEFs) such as TIAM1 and PREX1 specific for the Rho family GTPases (Rac/CDC42/RhoA). These GTPases activate pathways regulating cytoskeleton: Rac activates p21-activated kinase (PAK), which then activates LIM kinase (LIMK), leading to cofilin phosporylation and actin polymerization. CDC42 promotes actin assembly through the Wiskott-Aldrich Syndrome family protein (WASP) and actin-nucleating protein Arp2/3. RhoA activates Rho kinase (ROCK) , leading to myosin light-chain (MLC) phosporylation and microtubule rearrangement. (D) SDF-1 may also trigger Gα_i-independent activation of the JAK-STAT pathways.</p

Effects of Microtubule Modulators on HIV-1 Infection of Transformed and Resting CD4 T Cells ▿

Previous studies have observed fluorescently labeled HIV particles tracking along microtubule networks for nuclear localization. To provide direct evidence for the involvement of microtubules in early steps of HIV infection of human CD4 T cells, we used multiple microtubule modulators such as paclitaxel (originally called taxol; 1 μM), vinblastine (1 and 10 μM), colchicine (10 and 100 μM), and nocodazole (10 and 100 μM) to disturb microtubule networks in transformed and resting CD4 T cells. Although these drugs disrupted microtubule integrity, almost no inhibition of HIV-1 infection was observed. Our results do not appear to support an essential role for microtubules in the initiation of HIV infection of CD4 T cells