Involvement of the Rac1-IRSp53-Wave2-Arp2/3 Signaling Pathway in HIV-1 Gag Particle Release in CD4 T Cells

ABSTRACTDuring HIV-1 assembly, the Gag viral proteins are targeted and assemble at the inner leaflet of the cell plasma membrane. This process could modulate the cortical actin cytoskeleton, located underneath the plasma membrane, since actin dynamics are able to promote localized membrane reorganization. In addition, activated small Rho GTPases are known for regulating actin dynamics and membrane remodeling. Therefore, the modulation of such Rho GTPase activity and of F-actin by the Gag protein during virus particle formation was considered. Here, we studied the implication of the main Rac1, Cdc42, and RhoA small GTPases, and some of their effectors, in this process. The effect of small interfering RNA (siRNA)-mediated Rho GTPases and silencing of their effectors on Gag localization, Gag membrane attachment, and virus-like particle production was analyzed by immunofluorescence coupled to confocal microscopy, membrane flotation assays, and immunoblot assays, respectively. In parallel, the effect of Gag expression on the Rac1 activation level was monitored by G-LISA, and the intracellular F-actin content in T cells was monitored by flow cytometry and fluorescence microscopy. Our results revealed the involvement of activated Rac1 and of the IRSp53-Wave2-Arp2/3 signaling pathway in HIV-1 Gag membrane localization and particle release in T cells as well as a role for actin branching and polymerization, and this was solely dependent on the Gag viral protein. In conclusion, our results highlight a new role for the Rac1-IRSp53-Wave2-Arp2/3 signaling pathway in the late steps of HIV-1 replication in CD4 T lymphocytes.IMPORTANCEDuring HIV-1 assembly, the Gag proteins are targeted and assembled at the inner leaflet of the host cell plasma membrane. Gag interacts with specific membrane phospholipids that can also modulate the regulation of cortical actin cytoskeleton dynamics. Actin dynamics can promote localized membrane reorganization and thus can be involved in facilitating Gag assembly and particle formation. Activated small Rho GTPases and effectors are regulators of actin dynamics and membrane remodeling. We thus studied the effects of the Rac1, Cdc42, and RhoA GTPases and their specific effectors on HIV-1 Gag membrane localization and viral particle release in T cells. Our results show that activated Rac1 and the IRSp53-Wave2-Arp2/3 signaling pathway are involved in Gag plasma membrane localization and viral particle production. This work uncovers a role for cortical actin through the activation of Rac1 and the IRSp53/Wave2 signaling pathway in HIV-1 particle formation in CD4 T lymphocytes.

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A Dichotomy in Cortical Actin and Chemotactic Actin Activity between Human Memory and Naive T Cells Contributes to Their Differential Susceptibility to HIV-1 Infection

Journal of Biological Chemistry ◽

10.1074/jbc.m112.362400 ◽

2012 ◽

Vol 287 (42) ◽

pp. 35455-35469 ◽

Cited By ~ 21

Author(s):

Weifeng Wang ◽

Jia Guo ◽

Dongyang Yu ◽

Paul J. Vorster ◽

WanJun Chen ◽

...

Keyword(s):

T Cells ◽

Cd4 T Cells ◽

Differential Susceptibility ◽

Human Memory ◽

Actin Dynamics ◽

Cortical Actin ◽

Naive T Cells ◽

Naïve T Cells ◽

Memory Cd4 T Cells ◽

Hiv 1

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.

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Role of the Gag Matrix Domain in Targeting Human Immunodeficiency Virus Type 1 Assembly

Journal of Virology ◽

10.1128/jvi.74.6.2855-2866.2000 ◽

2000 ◽

Vol 74 (6) ◽

pp. 2855-2866 ◽

Cited By ~ 185

Author(s):

Akira Ono ◽

Jan M. Orenstein ◽

Eric O. Freed

Keyword(s):

Human Immunodeficiency Virus ◽

Plasma Membrane ◽

Virus Particle ◽

Particle Formation ◽

Membrane Targeting ◽

Basic Domain ◽

Golgi Vesicles ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) particle formation and the subsequent initiation of protease-mediated maturation occur predominantly on the plasma membrane. However, the mechanism by which HIV-1 assembly is targeted specifically to the plasma membrane versus intracellular membranes is largely unknown. Previously, we observed that mutations between residues 84 and 88 of the matrix (MA) domain of HIV-1 Gag cause a retargeting of virus particle formation to an intracellular site. In this study, we demonstrate that the mutant virus assembly occurs in the Golgi or in post-Golgi vesicles. These particles undergo core condensation in a protease-dependent manner, indicating that virus maturation can occur not only on the plasma membrane but also in the Golgi or post-Golgi vesicles. The intracellular assembly of mutant particles is dependent on Gag myristylation but is not influenced by p6Gag or envelope glycoprotein expression. Previous characterization of viral revertants suggested a functional relationship between the highly basic domain of MA (amino acids 17 to 31) and residues 84 to 88. We now demonstrate that mutations in the highly basic domain also retarget virus particle formation to the Golgi or post-Golgi vesicles. Although the basic domain has been implicated in Gag membrane binding, no correlation was observed between the impact of mutations on membrane binding and Gag targeting, indicating that these two functions of MA are genetically separable. Plasma membrane targeting of Gag proteins with mutations in either the basic domain or between residues 84 and 88 was rescued by coexpression with wild-type Gag; however, the two groups of MA mutants could not rescue each other. We propose that the highly basic domain of MA contains a major determinant of HIV-1 Gag plasma membrane targeting and that mutations between residues 84 and 88 disrupt plasma membrane targeting through an effect on the basic domain.

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Requirement for an Intact T-Cell Actin and Tubulin Cytoskeleton for Efficient Assembly and Spread of Human Immunodeficiency Virus Type 1

Journal of Virology ◽

10.1128/jvi.01469-06 ◽

2007 ◽

Vol 81 (11) ◽

pp. 5547-5560 ◽

Cited By ~ 134

Author(s):

Clare Jolly ◽

Ivonne Mitar ◽

Quentin J. Sattentau

Keyword(s):

Human Immunodeficiency Virus ◽

T Cells ◽

Plasma Membrane ◽

T Cell ◽

Immunodeficiency Virus ◽

Fixed Cell ◽

Cell Spread ◽

Hiv 1 ◽

Cell Cell

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) infection of CD4+ T cells leads to the production of new virions that assemble at the plasma membrane. Gag and Env accumulate in the context of lipid rafts at the inner and outer leaflets of the plasma membrane, respectively, forming polarized domains from which HIV-1 buds. HIV-1 budding can result in either release of cell-free virions or direct cell-cell spread via a virological synapse (VS). The recruitment of Gag and Env to these plasma membrane caps in T cells is poorly understood but may require elements of the T-cell secretory apparatus coordinated by the cytoskeleton. Using fixed-cell immunofluorescence labeling and confocal microscopy, we observed a high percentage of HIV-1-infected T cells with polarized Env and Gag in capped, lipid raft-like assembly domains. Treatment of infected T cells with inhibitors of actin or tubulin remodeling disrupted Gag and Env compartmentalization within the polarized raft-like domains. Depolymerization of the actin cytoskeleton reduced Gag release and viral infectivity, and actin and tubulin inhibitors reduced Env incorporation into virions. Live- and fixed-cell confocal imaging and assay of de novo DNA synthesis by real-time PCR allowed quantification of HIV-1 cell-cell transfer. Inhibition of actin and tubulin remodeling in infected cells interfered with cell-cell spread across a VS and reduced new viral DNA synthesis. Based on these data, we propose that HIV-1 requires both actin and tubulin components of the T-cell cytoskeleton to direct its assembly and budding and to elaborate a functional VS.

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Control of cortical cytoskeleton-membrane interaction by RhoA regulates peripheral nerve myelination

10.1101/2021.11.09.467948 ◽

2021 ◽

Author(s):

Ana I. Seixas ◽

Miguel R. G. Morais ◽

Cord Brakebusch ◽

Jo&atildeo B. Relvas

Keyword(s):

Schwann Cell ◽

Schwann Cells ◽

Rho Gtpases ◽

Intracellular Signaling ◽

Cell Interaction ◽

Actin Dynamics ◽

Specific Gene ◽

Cortical Actin ◽

Membrane Attachment ◽

Cytoskeleton Dynamics

Bidirectional transmission of mechanical and biochemical signals is integral to cell-environment communication and underlies the function of Schwann cells, the myelinating glia of the peripheral nervous system. As major integrators of "outside-in" signaling, Rho GTPases link actin cytoskeleton dynamics with cellular architecture to regulate adhesion and cell deformation. Using Schwann cell-specific gene inactivation, we discovered that RhoA promotes the initiation of myelination, axonal wrapping and axial spreading of Schwann cells, and is later required to restrict myelin growth in peripheral nerves. These effects are mediated by modulation of actomyosin contractility, actin dynamics and cortical actin-membrane attachment, which collectively couple tensional forces to intracellular signaling that regulate axon-Schwann cell interaction and myelin synthesis. This work establishes RhoA as an intrinsic regulator of a biomechanical response that controls the switch of Schwann cells towards the myelinating and the homeostatic states.

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Identification of SERINC5-001 as the Predominant Spliced Isoform for HIV-1 Restriction

Journal of Virology ◽

10.1128/jvi.00137-17 ◽

2017 ◽

Vol 91 (10) ◽

Cited By ~ 23

Author(s):

Xianfeng Zhang ◽

Tao Zhou ◽

Jie Yang ◽

Yumei Lin ◽

Jing Shi ◽

...

Keyword(s):

Plasma Membrane ◽

Transmembrane Domain ◽

The Other ◽

Stable Expression ◽

Membrane Localization ◽

Plasma Membrane Localization ◽

Immunodeficiency Virus ◽

Alternatively Spliced ◽

Anti Hiv ◽

Hiv 1

ABSTRACT Among the five serine incorporator (SERINC) family members, SERINC5 (Ser5) was reported to strongly inhibit HIV-1 replication, which is counteracted by Nef. Ser5 produces 5 alternatively spliced isoforms: Ser5-001 has 10 putative transmembrane domains, whereas Ser5-004, -005, -008a, and -008b do not have the last one. Here, we confirmed the strong Ser5 anti-HIV-1 activity and investigated its isoforms' expression and antiviral activities. It was found that Ser5-001 transcripts were detected at least 10-fold more than the other isoforms by real-time quantitative PCR. When Ser5-001 and its two isoforms Ser5-005 and Ser5-008a were expressed from the same mammalian expression vector, only Ser5-001 was stably expressed, whereas the others were poorly expressed due to rapid degradation. In addition, unlike the other isoforms, which are located mainly in the cytoplasm, Ser5-001 is localized primarily to the plasma membrane. To map the critical determinant, Ser5 mutants bearing C-terminal deletions were created. It was found that the 10th transmembrane domain is required for Ser5 stable expression and plasma membrane localization. As expected, only Ser5-001 strongly inhibits HIV-1 infectivity, whereas the other Ser5 isoforms and mutants that do not have the 10th transmembrane domain show very poor activity. It was also observed that the Nef counteractive activity could be easily saturated by Ser5 overexpression. Thus, we conclude that Ser5-001 is the predominant antiviral isoform that restricts HIV-1, and the 10th transmembrane domain plays a critical role in this process by regulating its protein stability and plasma membrane targeting. IMPORTANCE Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) express a small protein, Nef, to enhance viral pathogenesis in vivo. Nef has an important in vitro function, which is to make virus particles more infectious, but the mechanism has been unclear. Recently, Nef was reported to counteract a novel anti-HIV host protein, SERINC5 (Ser5). Ser5 has five alternatively spliced isoforms, Ser5-001, -004, -005, -008a, and -008b, and only Ser5-001 has an extra C-terminal transmembrane domain. We now show that the Ser5-001 transcripts are produced at least 10-fold more than the others, and only Ser5-001 produces stable proteins that are targeted to the plasma membrane. Importantly, only Ser5-001 shows strong anti-HIV-1 activity. We further demonstrate that the extra transmembrane domain is required for Ser5 stable expression and plasma membrane localization. These results suggest that plasma membrane localization is required for Ser5 antiviral activity, and Ser5-001 is the predominant isoform that contributes to the activity.

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3pM_K3Non-invasive Live-imaging of Plasma Membrane and Cortical Actin Dynamics in Endocytic Process by High-speed Atomic Force Microscopy

Microscopy ◽

10.1093/jmicro/dfy089 ◽

2018 ◽

Vol 67 (suppl_2) ◽

pp. i10-i10

Author(s):

Aiko Yoshida ◽

Yoshitsuna Itagaki ◽

Yuki Suzuki ◽

Shige H. Yoshimura

Keyword(s):

Atomic Force Microscopy ◽

Plasma Membrane ◽

High Speed ◽

Live Imaging ◽

Actin Dynamics ◽

Cortical Actin ◽

Force Microscopy ◽

Atomic Force

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MMP3 activity rather than cortical stiffness determines NHE1-dependent invasiveness of melanoma cells

Cancer Cell International ◽

10.1186/s12935-019-1015-7 ◽

2019 ◽

Vol 19 (1) ◽

Author(s):

Dennis Keurhorst ◽

Ivan Liashkovich ◽

Fabian Frontzek ◽

Svenja Nitzlaff ◽

Verena Hofschröer ◽

...

Keyword(s):

Plasma Membrane ◽

Cell Invasion ◽

Time Lapse ◽

Actin Dynamics ◽

Cell Stiffness ◽

Migration And Invasion ◽

Boyden Chamber ◽

Cortical Actin ◽

Structural Element ◽

Mmp Inhibitor

Abstract Background Both cell adhesion and matrix metalloproteinase (MMP) activity depend on pH at the cell surface. By regulating extracellular juxtamembrane pH, the Na+/H+ exchanger NHE1 plays a significant part in human melanoma (MV3) cell migration and invasion. Because NHE1, besides its pH-regulatory transport function, also serves as a structural element tying the cortical actin cytoskeleton to the plasma membrane, we investigated whether NHE1 affects cortical stiffness of MV3 cells, and how this makes an impact on their invasiveness. Methods NHE1 overexpressing MV3 cells were compared to the corresponding mock-transfected control cells. NHE1 expression was verified by Western blotting, cariporide (HOE642) was used to inhibit NHE1 activity, cell stiffness was determined by atomic force microscopy, and F-actin was visualized by phalloidin-staining. Migration on, and invasion of, native and glutaraldehyde-fixed collagen I substrates were analyzed using time-lapse video microscopy and Boyden-chamber assays, respectively. MMP secretion and activity were detected by Western blot and zymography, respectively. MMP activity was inhibited with NNGH. Results The cortical, but not the bulk stiffness, was significantly higher in NHE1 overexpressing cells. This increase in cortical stiffness was accompanied by a reorganization of the cortical cytoskeleton, i.e. a condensation of F-actin underneath and along the plasma membrane. However, it was not affected by NHE1 inhibition. Nevertheless, actin dynamics is required for cell invasion as demonstrated with the application of cytochalasin D. NHE1 overexpression was associated with an elevated MMP3 secretion and an increase in the invasion of a native matrix. This increase in invasiveness could be antagonized by the MMP inhibitor NNGH. Transmigration through a glutaraldehyde-fixed, indigestible substrate was not affected by NHE1 overexpression. Conclusion NHE1, as a structural element and independently of its transport activity, contributes to the organization of the cortical F-actin meshwork and thus impacts cortical stiffness. Since NHE1 overexpression stimulates MMP3 secretion but does not change transmigration through a fixed substrate, MV3 cell invasion of a native substrate depends on MMP activity rather than on a modifiable cortical stiffness.

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HIV-1 Nef and Vpu Interfere with L-Selectin (CD62L) Cell Surface Expression To Inhibit Adhesion and Signaling in Infected CD4+T Lymphocytes

Journal of Virology ◽

10.1128/jvi.00611-15 ◽

2015 ◽

Vol 89 (10) ◽

pp. 5687-5700 ◽

Cited By ~ 24

Author(s):

Lia Vassena ◽

Erica Giuliani ◽

Herwig Koppensteiner ◽

Sebastian Bolduan ◽

Michael Schindler ◽

...

Keyword(s):

T Cells ◽

Plasma Membrane ◽

T Lymphocytes ◽

Cell Surface ◽

Immune Responses ◽

Lymph Nodes ◽

Viral Proteins ◽

Peripheral Lymph ◽

Leukocyte Homing ◽

Hiv 1

ABSTRACTLeukocyte recirculation between blood and lymphoid tissues is required for the generation and maintenance of immune responses against pathogens and is crucially controlled by the L-selectin (CD62L) leukocyte homing receptor. CD62L has adhesion and signaling functions and initiates the capture and rolling on the vascular endothelium of cells entering peripheral lymph nodes. This study reveals that CD62L is strongly downregulated on primary CD4+T lymphocytes upon infection with human immunodeficiency virus type 1 (HIV-1). Reduced cell surface CD62L expression was attributable to the Nef and Vpu viral proteins and not due to increased shedding via matrix metalloproteases. Both Nef and Vpu associated with and sequestered CD62L in perinuclear compartments, thereby impeding CD62L transport to the plasma membrane. In addition, Nef decreased total CD62L protein levels. Importantly, infection with wild-type, but not Nef- and Vpu-deficient, HIV-1 inhibited the capacity of primary CD4+T lymphocytes to adhere to immobilized fibronectin in response to CD62L ligation. Moreover, HIV-1 infection impaired the signaling pathways and costimulatory signals triggered in primary CD4+T cells by CD62L ligation. We propose that HIV-1 dysregulates CD62L expression to interfere with the trafficking and activation of infected T cells. Altogether, this novel HIV-1 function could contribute to virus dissemination and evasion of host immune responses.IMPORTANCEL-selectin (CD62L) is an adhesion molecule that mediates the first steps of leukocyte homing to peripheral lymph nodes, thus crucially controlling the initiation and maintenance of immune responses to pathogens. Here, we report that CD62L is downmodulated on the surfaces of HIV-1-infected T cells through the activities of two viral proteins, Nef and Vpu, that prevent newly synthesized CD62L molecules from reaching the plasma membrane. We provide evidence that CD62L downregulation on HIV-1-infected primary T cells results in impaired adhesion and signaling functions upon CD62L triggering. Removal of cell surface CD62L may predictably keep HIV-1-infected cells away from lymph nodes, the privileged sites of both viral replication and immune response activation, with important consequences, such as systemic viral spread and evasion of host immune surveillance. Altogether, we propose that Nef- and Vpu-mediated subversion of CD62L function could represent a novel determinant of HIV-1 pathogenesis.

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The TRE17 Oncogene Encodes a Component of a Novel Effector Pathway for Rho GTPases Cdc42 and Rac1 and Stimulates Actin Remodeling

Molecular and Cellular Biology ◽

10.1128/mcb.23.6.2151-2161.2003 ◽

2003 ◽

Vol 23 (6) ◽

pp. 2151-2161 ◽

Cited By ~ 36

Author(s):

Jeffrey M. Masuda-Robens ◽

Sara N. Kutney ◽

Hongwei Qi ◽

Margaret M. Chou

Keyword(s):

Plasma Membrane ◽

Rho Gtpases ◽

Actin Polymerization ◽

Dependent Manner ◽

Cortical Actin ◽

Actin Remodeling ◽

Membrane Recruitment ◽

Truncation Mutant ◽

Effector Pathway

ABSTRACT The Rho family GTPases Cdc42 and Rac1 play fundamental roles in transformation and actin remodeling. Here, we demonstrate that the TRE17 oncogene encodes a component of a novel effector pathway for these GTPases. TRE17 coprecipitated specifically with the active forms of Cdc42 and Rac1 in vivo. Furthermore, the subcellular localization of TRE17 was dramatically regulated by these GTPases and mitogens. Under serum-starved conditions, TRE17 localized predominantly to filamentous structures within the cell. Epidermal growth factor (EGF) induced relocalization of TRE17 to the plasma membrane in a Cdc42-/Rac1-dependent manner. Coexpression of activated alleles of Cdc42 or Rac1 also caused complete redistribution of TRE17 to the plasma membrane, where it partially colocalized with the GTPases in filopodia and ruffles, respectively. Membrane recruitment of TRE17 by EGF or the GTPases was dependent on actin polymerization. Finally, we found that a C-terminal truncation mutant of TRE17 induced the accumulation of cortical actin, mimicking the effects of activated Cdc42. Together, these results identify TRE17 as part of a novel effector complex for Cdc42 and Rac1, potentially contributing to their effects on actin remodeling. The present study provides insights into the regulation and cellular function of this previously uncharacterized oncogene.

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