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Actin cytoskeletal reorganizations and coreceptor-mediated activation of rac during human immunodeficiency virus-induced cell fusion.

ABSTRACT: The membrane fusion events which initiate human immunodeficiency virus type 1 (HIV-1) infection and promote cytopathic syncytium formation in infected cells commence with the binding of the HIV envelope glycoprotein (Env) to CD4 and an appropriate coreceptor. Here, we show that HIV Env-coreceptor interactions activate Rac-1 GTPase and stimulate the actin filament network reorganizations that are requisite components of the cell fusion process. Disrupting actin filament dynamics with jasplakinolide or latrunculin A arrested fusion at a late step in the formation of Env-CD4-coreceptor complexes. Time-lapse confocal microscopy of living cells revealed vigorous activity of actin-based, target cell membrane extensions at the target cell-Env-expressing cell interface. The expression of dominant-negative forms of actin-regulating Rho-family GTPases established that HIV Env-mediated syncytium formation relies on Rac-1 but not on Cdc42 or Rho activation in target cells. Similar dependencies were found when cell fusion was induced by Env expressed on viral or cellular membranes. Additionally, Rac activity was specifically upregulated in a coreceptor-dependent manner in fusion reaction cell lysates. These results define a role for HIV Env-coreceptor interactions in activating the cellular factors essential for virus-cell and cell-cell fusion and provide evidence for the participation of pertussis toxin-insensitive signaling pathways in HIV-induced membrane fusion.

SUBMITTER: Pontow SE

PROVIDER: S-EPMC421652 | biostudies-literature | 2004 Jul

REPOSITORIES: biostudies-literature

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Actin cytoskeletal reorganizations and coreceptor-mediated activation of rac during human immunodeficiency virus-induced cell fusion.

Pontow S E SE Heyden N Vander NV Wei S S Ratner L L

Journal of virology 20040701 13

The membrane fusion events which initiate human immunodeficiency virus type 1 (HIV-1) infection and promote cytopathic syncytium formation in infected cells commence with the binding of the HIV envelope glycoprotein (Env) to CD4 and an appropriate coreceptor. Here, we show that HIV Env-coreceptor interactions activate Rac-1 GTPase and stimulate the actin filament network reorganizations that are requisite components of the cell fusion process. Disrupting actin filament dynamics with jasplakinoli ...[more]

PMID: 15194790

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Project description:CCR5, a chemokine receptor central for orchestrating lymphocyte/cell migration to the sites of inflammation and to the immunosurveillance, is involved in the pathogenesis of a wide spectrum of health conditions, including inflammatory diseases, viral infections, cancers and autoimmune diseases. CCR5 is also the primary coreceptor for the human immunodeficiency viruses (HIVs), supporting its entry into CD4+ T lymphocytes upon transmission and in the early stages of infection in humans. A natural loss-of-function mutation CCR5-Δ32, preventing the mutated protein expression on the cell surface, renders homozygous carriers of the null allele resistant to HIV-1 infection. This phenomenon was leveraged in the development of therapies and cure strategies for AIDS. Meanwhile, over 40 African nonhuman primate species are long-term hosts of simian immunodeficiency virus (SIV), an ancestral family of viruses that give rise to the pandemic CCR5 (R5)-tropic HIV-1. Many natural hosts typically do not progress to immunodeficiency upon the SIV infection. They have developed various strategies to minimize the SIV-related pathogenesis and disease progression, including an array of mechanisms employing modulation of the CCR5 receptor activity: (i) deletion mutations abrogating the CCR5 surface expression and conferring resistance to infection in null homozygotes; (ii) downregulation of CCR5 expression on CD4+ T cells, particularly memory cells and cells at the mucosal sites, preventing SIV from infecting and killing cells important for the maintenance of immune homeostasis, (iii) delayed onset of CCR5 expression on the CD4+ T cells during ontogenetic development that protects the offspring from vertical transmission of the virus. These host adaptations, aimed at lowering the availability of target CCR5+ CD4+ T cells through CCR5 downregulation, were countered by SIV, which evolved to alter the entry coreceptor usage toward infecting different CD4+ T-cell subpopulations that support viral replication yet without disruption of host immune homeostasis. These natural strategies against SIV/HIV-1 infection, involving control of CCR5 function, inspired therapeutic approaches against HIV-1 disease, employing CCR5 coreceptor blocking as well as gene editing and silencing of CCR5. Given the pleiotropic role of CCR5 in health beyond immune disease, the precision as well as costs and benefits of such interventions needs to be carefully considered.

Project description:UnlabelledHIV-1 Env glycoprotein-mediated fusion is initiated upon sequential binding of Env to CD4 and the coreceptor CXCR4 or CCR5. Whereas these interactions are thought to be necessary and sufficient to promote HIV-1 fusion, other host factors can modulate this process. Previous studies reported potent inhibition of HIV-1 fusion by selective P2X1 receptor antagonists, including NF279, and suggested that these receptors play a role in HIV-1 entry. Here we investigated the mechanism of antiviral activity of NF279 and found that this compound does not inhibit HIV-1 fusion by preventing the activation of P2X1 channels but effectively blocks the binding of the virus to CXCR4 or CCR5. The notion of an off-target effect of NF279 on HIV-1 fusion is supported by the lack of detectable expression of P2X1 receptors in cells used in fusion experiments and by the fact that the addition of ATP or the enzymatic depletion of ATP in culture medium does not modulate viral fusion. Importantly, NF279 fails to inhibit HIV-1 fusion with cell lines and primary macrophages when added at an intermediate stage downstream of Env-CD4-coreceptor engagement. Conversely, in the presence of NF279, HIV-1 fusion is arrested downstream of CD4 binding but prior to coreceptor engagement. NF279 also antagonizes the signaling function of CCR5, CXCR4, and another chemokine receptor, as evidenced by the suppression of calcium responses elicited by specific ligands and by recombinant gp120. Collectively, our results demonstrate that NF279 is a dual HIV-1 coreceptor inhibitor that interferes with the functional engagement of CCR5 and CXCR4 by Env.ImportanceInhibition of P2X receptor activity suppresses HIV-1 fusion and replication, suggesting that P2X signaling is involved in HIV-1 entry. However, mechanistic experiments conducted in this study imply that P2X1 receptor is not expressed in target cells or involved in viral fusion. Instead, we found that inhibition of HIV-1 fusion by a specific P2X1 receptor antagonist, NF279, is due to the blocking of virus interactions with both the CXCR4 and CCR5 coreceptors. The ability of NF279 to abrogate cellular calcium signaling induced by the respective chemokines showed that this compound acts as a dual-coreceptor antagonist. P2X1 receptor antagonists could thus represent a new class of dual-coreceptor inhibitors with a structure and a mechanism of action that are distinct from those of known HIV-1 coreceptor antagonists.

Dataset Information

Actin cytoskeletal reorganizations and coreceptor-mediated activation of rac during human immunodeficiency virus-induced cell fusion.

Publications

Actin cytoskeletal reorganizations and coreceptor-mediated activation of rac during human immunodeficiency virus-induced cell fusion.

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