Vera Rocha-Perugini

The CD81 Partner EWI-2wint Inhibits Hepatitis C Virus Entry

Two to three percent of the worlds population is chronically infected with hepatitis C virus (HCV) and thus at risk of developing liver cancer. Although precise mechanisms regulating HCV entry into hepatic cells are still unknown, several cell surface proteins have been identified as entry factors for this virus. Among these molecules, the tetraspanin CD81 is essential for HCV entry. Here, we have identified a partner of CD81, EWI-2wint, which is expressed in several cell lines but not in hepatocytes. Ectopic expression of EWI-2wint in a hepatoma cell line susceptible to HCV infection blocked viral entry by inhibiting the interaction between the HCV envelope glycoproteins and CD81. This finding suggests that, in addition to the presence of specific entry factors in the hepatocytes, the lack of a specific inhibitor can contribute to the hepatotropism of HCV. This is the first example of a pathogen gaining entry into host cells that lack a specific inhibitory factor.

Interacting Regions of CD81 and Two of Its Partners, EWI-2 and EWI-2wint, and Their Effect on Hepatitis C Virus Infection

CD81 is a tetraspanin protein that is involved in several essential cellular functions, as well as in the hepatitis C virus (HCV) infection. CD81 interacts with a high stoichiometry with its partner proteins EWI-2, EWI-2wint, and EWI-F. These latter proteins modify the functions of CD81 and can thereby potentially inhibit infection or modulate cell migration. Here, we characterized the cleavage of EWI-2 leading to the production of EWI-2wint, which has been shown to inhibit HCV infection. We determined the regions of EWI-2/EWI-2wint and CD81 that are important for their interaction and their functionality. More precisely, we identified a glycine zipper motif in the transmembrane domain of EWI-2/EWI-2wint that is essential for the interaction with CD81. In addition, we found that palmitoylation on two juxtamembranous cysteines in the cytosolic tail of EWI-2/EWI-2wint is required for their interaction with CD81 as well as with CD9, another tetraspanin. Thus, we have shown that palmitoylation of a tetraspanin partner protein can influence the interaction with a tetraspanin. We therefore propose that palmitoylation not only of tetraspanins, but also of their partner proteins is important in regulating the composition of complexes in tetraspanin networks. Finally, we identified the regions in CD81 that are necessary for its functionality in HCV entry and we demonstrated that EWI-2wint needs to interact with CD81 to exert its inhibitory effect on HCV infection.

CD81 regulates cell migration through its association with Rac GTPase

Data presented here provide evidence for a new direct interaction of the GTPase Rac with the C-terminal cytoplasmic domain of tetraspanin CD81. Tetraspanin-enriched, microdomain-dependent compartmentalization is a novel regulatory mechanism of Rac activity turnover, which provides a novel mechanism for regulation of cell motility by tetraspanins.

The association of CD81 with tetraspanin-enriched microdomains is not essential for Hepatitis C virus entry.

BackgroundThree percent of the worlds population is chronically infected with hepatitis C virus (HCV) and thus at risk of developing liver cancer. Although precise mechanisms regulating HCV entry into hepatic cells are still unknown, several cell surface proteins have been identified as entry factors for this virus. Among these molecules, the tetraspanin CD81 is essential for HCV entry. Interestingly, CD81 is also required for Plasmodium infection. A major characteristic of tetraspanins is their ability to interact with each other and other transmembrane proteins to build tetraspanin-enriched microdomains (TEM).ResultsIn our study, we describe a human hepatoma Huh-7 cell clone (Huh-7w7) which has lost CD81 expression and can be infected by HCV when human CD81 (hCD81) or mouse CD81 (mCD81) is ectopically expressed. We took advantage of these permissive cells expressing mCD81 and the previously described MT81/MT81w mAbs to analyze the role of TEM-associated CD81 in HCV infection. Importantly, MT81w antibody, which only recognizes TEM-associated mCD81, did not strongly affect HCV infection. Furthermore, cholesterol depletion, which inhibits HCV infection and reduces total cell surface expression of CD81, did not affect TEM-associated CD81 levels. In addition, sphingomyelinase treatment, which also reduces HCV infection and cell surface expression of total CD81, raised TEM-associated CD81 levels.ConclusionIn contrast to Plasmodium infection, our data show that association of CD81 with TEM is not essential for the early steps of HCV life cycle, indicating that these two pathogens, while using the same molecules, invade their host by different mechanisms.

CD81 Controls Sustained T Cell Activation Signaling and Defines the Maturation Stages of Cognate Immunological Synapses

ABSTRACT In this study, we investigated the dynamics of the molecular interactions of tetraspanin CD81 in T lymphocytes, and we show that CD81 controls the organization of the immune synapse (IS) and T cell activation. Using quantitative microscopy, including fluorescence recovery after photobleaching (FRAP), phasor fluorescence lifetime imaging microscopy-Föster resonance energy transfer (phasorFLIM-FRET), and total internal reflection fluorescence microscopy (TIRFM), we demonstrate that CD81 interacts with ICAM-1 and CD3 during conjugation between T cells and antigen-presenting cells (APCs). CD81 and ICAM-1 exhibit distinct mobilities in central and peripheral areas of early and late T cell-APC contacts. Moreover, CD81–ICAM-1 and CD81-CD3 dynamic interactions increase over the time course of IS formation, as these molecules redistribute throughout the contact area. Therefore, CD81 associations unexpectedly define novel sequential steps of IS maturation. Our results indicate that CD81 controls the temporal progression of the IS and the permanence of CD3 in the membrane contact area, contributing to sustained T cell receptor (TCR)-CD3-mediated signaling. Accordingly, we find that CD81 is required for proper T cell activation, regulating CD3ζ, ZAP-70, LAT, and extracellular signal-regulated kinase (ERK) phosphorylation; CD69 surface expression; and interleukin-2 (IL-2) secretion. Our data demonstrate the important role of CD81 in the molecular organization and dynamics of the IS architecture that sets the signaling threshold in T cell activation.

EWI-2 Association with α-Actinin Regulates T Cell Immune Synapses and HIV Viral Infection

EWI motif-containing protein 2 (EWI-2) is a member of the Ig superfamily that links tetraspanin-enriched microdomains to the actin cytoskeleton. We found that EWI-2 colocalizes with CD3 and CD81 at the central supramolecular activation cluster of the T cell immune synapse. Silencing of the endogenous expression or overexpression of a cytoplasmic truncated mutant of EWI-2 in T cells increases IL-2 secretion upon Ag stimulation. Mass spectrometry experiments of pull-downs with the C-term intracellular domain of EWI-2 revealed the specific association of EWI-2 with the actin-binding protein α-actinin; this association was regulated by PIP2. α-Actinin regulates the immune synapse formation and is required for efficient T cell activation. We extended these observations to virological synapses induced by HIV and found that silencing of either EWI-2 or α-actinin-4 increased cell infectivity. Our data suggest that the EWI-2–α-actinin complex is involved in the regulation of the actin cytoskeleton at T cell immune and virological synapses, providing a link between membrane microdomains and the formation of polarized membrane structures involved in T cell recognition.

Nuclear Envelope Lamin-A Couples Actin Dynamics with Immunological Synapse Architecture and T Cell Activation

Communication between the nuclear lamina protein lamin-A and the actin cytoskeleton is required for optimal T cell responses. Nuclear Support for T Cell Activation A-type lamins are filamentous proteins that form the nuclear lamina inside the inner nuclear membrane. As well as maintaining the mechanical integrity of the nucleus, A-type lamins physically connect the nucleus and the actin cytoskeleton by associating with the linker of nucleoskeleton and cytoskeleton (LINC) complex. González-Granado et al. found that the abundance of lamin-A was rapidly and transiently increased in mouse and human CD4+ T cells in response to stimulation of the T cell receptor. Lamin-A enhanced F-actin polymerization at the immunological synapse between the T cells and antigen-presenting cells, thereby boosting T cell activation. Disruption of the interaction between lamin-A and the LINC complex also decreased T cell activation in culture, and mice lacking lamin-A in immune cells exhibited reduced T cell activation in response to antigen. These results suggest that the nuclear skeleton communicates with the cytoskeleton to enable optimal T cell activation. In many cell types, nuclear A-type lamins regulate multiple cellular functions, including higher-order genome organization, DNA replication and repair, gene transcription, and signal transduction; however, their role in specialized immune cells remains largely unexplored. We showed that the abundance of A-type lamins was almost negligible in resting naïve T lymphocytes, but was increased upon activation of the T cell receptor (TCR). The increase in lamin-A was an early event that accelerated formation of the immunological synapse between T cells and antigen-presenting cells. Polymerization of F-actin in T cells is a critical step for immunological synapse formation, and lamin-A interacted with the linker of nucleoskeleton and cytoskeleton (LINC) complex to promote F-actin polymerization. We also showed that lamin-A expression accelerated TCR clustering and led to enhanced downstream signaling, including extracellular signal–regulated kinase 1/2 (ERK1/2) signaling, as well as increased target gene expression. Pharmacological inhibition of the ERK pathway reduced lamin-A–dependent T cell activation. Moreover, mice lacking lamin-A in immune cells exhibited impaired T cell responses in vivo. These findings underscore the importance of A-type lamins for TCR activation and identify lamin-A as a previously unappreciated regulator of the immune response.

The PDZ-adaptor protein syntenin-1 regulates HIV-1 entry

Syntenin-1 is recruited to the human immunodeficiency virus (HIV)-induced capping area but vanishes once the viral particles have entered the cell. Syntenin-1 limits HIV-1 infection. Moreover, syntenin-1 depletion specifically increases the HIV-1 entry step without affecting viral attachment to the cell surface. Silencing of syntenin-1 expression blocks actin polymerization triggered by HIV-1 contact and enhances phosphatidylinositol 4,5-bisphosphate production.

Actin-binding Protein Drebrin Regulates HIV-1-triggered Actin Polymerization and Viral Infection

Background: Drebrin binds to F-actin and CXCR4 in T cells. Thus, it is a potential candidate for the modulation of HIV-1 infection. Results: Drebrin and CXCR4 accumulate at viral attachment areas. Drebrin knockdown decreases F-actin polymerization, and increases local profilin accumulation and HIV-1 infection. Conclusion: Drebrin inhibits HIV-1 entry by stabilizing HIV-1-triggered F-actin polymerization. Significance: Modulation of actin dynamics differentially regulates each viral step for an effective viral infection. HIV-1 contact with target cells triggers F-actin rearrangements that are essential for several steps of the viral cycle. Successful HIV entry into CD4+ T cells requires actin reorganization induced by the interaction of the cellular receptor/co-receptor complex CD4/CXCR4 with the viral envelope complex gp120/gp41 (Env). In this report, we analyze the role of the actin modulator drebrin in HIV-1 viral infection and cell to cell fusion. We show that drebrin associates with CXCR4 before and during HIV infection. Drebrin is actively recruited toward cell-virus and Env-driven cell to cell contacts. After viral internalization, drebrin clustering is retained in a fraction of the internalized particles. Through a combination of RNAi-based inhibition of endogenous drebrin and GFP-tagged expression of wild-type and mutant forms, we establish drebrin as a negative regulator of HIV entry and HIV-mediated cell fusion. Down-regulation of drebrin expression promotes HIV-1 entry, decreases F-actin polymerization, and enhances profilin local accumulation in response to HIV-1. These data underscore the negative role of drebrin in HIV infection by modulating viral entry, mainly through the control of actin cytoskeleton polymerization in response to HIV-1.

Tetraspanins CD9 and CD151 at the immune synapse support T-cell integrin signaling

Understanding how the immune response is activated and amplified requires detailed knowledge of the stages in the formation of the immunological synapse (IS) between T lymphocytes and antigen‐presenting cells (APCs). We show that tetraspanins CD9 and CD151 congregate at the T‐cell side of the IS. Silencing of CD9 or CD151 blunts the IL‐2 secretion and expression of the activation marker CD69 by APC‐conjugated T lymphocytes, but does not affect the accumulation of CD3 or actin to the IS, or the translocation of the microtubule‐organizing center toward the T‐B contact area. CD9 or CD151 silencing diminishes the relocalization of α4β1 integrin to the IS and reduces the accumulation of high‐affinity β1 integrins at the cell–cell contact. These changes are accompanied by diminished phosphorylation of the integrin downstream targets FAK and ERK1/2. Our results suggest that CD9 and CD151 support integrin‐mediated signaling at the IS.