Tetraspanins regulate cell-to-cell transmission of HIV-1
ABSTRACT The presence of the tetraspanins CD9, CD63, CD81 and CD82 at HIV-1 budding sites, at the virological synapse (VS), and their enrichment in HIV-1 virions has been well-documented, but it remained unclear if these proteins play a role in the late phase of the viral replication cycle. Here we used overexpression and knockdown approaches to address this question.
Neither ablation of CD9, CD63 and/or CD81, nor overexpression of these tetraspanins was found to affect the efficiency of virus release. However, confirming recently reported data, tetraspanin overexpression in virus-producing cells resulted in the release of virions with substantially reduced infectivity. We also investigated the roles of these tetraspanins in cell-to-cell transmission of HIV-1. Overexpression of CD9 and CD63 led to reduced cell-to-cell transmission of this virus. Interestingly, in knockdown experiments we found that ablation of CD63, CD9 and/or CD81 had no effect on cell-free infectivity. However, knockdown of CD81, but not CD9 and CD63, enhanced productive particle transmission to target cells, suggesting additional roles for tetraspanins in the transmission process. Finally, tetraspanins were found to be downregulated in HIV-1-infected T lymphocytes, suggesting that HIV-1 modulates the levels of these proteins in order to maximize the efficiency of its transmission within the host.
Altogether, these results establish an active role of tetraspanins in HIV-1 producer cells.
Full-textDOI: · Available from: Marie Lambele, Jun 02, 2015
[Show abstract] [Hide abstract]
ABSTRACT: Macrophages and CD4(+) T-cells are the major reservoirs for HIV-1 infection. CD63 is a tetraspanin transmembrane protein, which has been shown to play an essential role during HIV-1 replication in macrophages. In this study, we further confirm the requirement of CD63 in HIV-1 replication events in primary human CD4(+) T-cells, dendritic cells, and a CD4(+) cell line. Most interestingly, we also show the evidences for the co-localization and internalization of CD63 and HIV-1 major receptor CD4 in primary human macrophages and CD4(+) cell line by confocal microscopy and Co-Immunoprecipitation assay. Analysis revealed that CD63-depleted CD4(+) T-cells, dendritic cells, and a cell line showed significant decrease in HIV-1 production. Further analysis showed that CD63 down regulation reduced production of the early HIV protein Tat, and affected HIV protein Gag by CD63-Gag interaction. In agreement, CD63 silencing also inhibited production of the late protein p24. Furthermore, we revealed that CD63 silencing has no effect on HIV-1 replication with extensive viral challenge (MOI > 0.2). These findings suggest that CD63 plays a dual-role both in early and late HIV-1 life cycle with a range of HIV-1 infection (MOI < 0.2).International journal of clinical and experimental pathology 01/2015; 8(2):1184-98.
[Show abstract] [Hide abstract]
ABSTRACT: Tetraspanins constitute a family of cellular proteins that organize various membrane-based processes. Several members of this family, including CD81, are actively recruited by HIV-1 Gag to viral assembly and release sites. Despite their enrichment at viral exit sites, the overall levels of tetraspanins are decreased in HIV-1-infected cells. Here, we identify Vpu as the main viral determinant for tetraspanin downregulation. We also show that reduction of CD81 levels by Vpu is not a by-product of CD4 or BST-2/Tetherin elimination from the surface of infected cells and likely occurs through an interaction between Vpu and CD81. Finally, we document that Vpu-mediated downregulation of CD81 from the surface of infected T cells can contribute to preserving the infectiousness of viral particles, thus revealing a novel Vpu function that promotes virus propagation by modulating the host cell environment. The HIV-1 accessory protein Vpu has previously been shown to downregulate various host-cell factors, thus helping the virus to overcome restriction barriers, evade immune attack, and maintain the infectivity of viral particles. Our study identifies tetraspanins as an additional group of host factors whose expression at the surface of infected cells is lowered by Vpu. While the downregulation of these integral membrane proteins, including CD81 and CD82, likely affects more than one function of HIV-1-infected cells, we document that Vpu-mediated lowering of CD81 levels in viral particles can be critical to maintaining their infectiousness. Copyright © 2015, American Society for Microbiology. All Rights Reserved.Journal of Virology 01/2015; 89(6). DOI:10.1128/JVI.03719-14
[Show abstract] [Hide abstract]
ABSTRACT: THE INFECTION OF CELLS BY HUMAN IMMUNODEFICIENCY VIRUS - 1 (HIV-1) occurs most commonly by direct virion transfer via cell-cell contact.1 The particles passes through cell contact-induced structures which in analogy to the immunological synapses are called virological synapses.2 Interaction between infected and uninfected cells is the main route of viral spread in vitro and therefore it is likely that it plays also an important role in vivo, in the infected human. Contact with the target cells results in re-localization of HIV-1 to the site of interaction and release of the virus at this site. At the virological synapses the viral envelope (Env) protein, the cellular receptor CD4 and the co-receptors co-localizes. This requires cytoskeletal rearrangements, in addition lipid rafts domains are enriched at the synapse and cholesterol is critical. Tetraspanin proteins such as CD81 and CD63 were characterized by four transmembrane domains, and they were also found to accumulate at the virological synapse.3 Working in the field of cell-cell transfer of HIV-1 and protein-protein interactions at the virological synapse, we demonstrated by three dimensional (3D) confocal imaging that the tetraspanin CD63 is predominantly present at the virological synapse (Figure 1). Jurkat cells were nucleofected with the vector pHIV-1JR-FL Gag-iGFP expressing the Gag-internal green fluorescent protein (iGFP) and uninfected Jurkat cells were added. Endogenous expressed CD63 was immunostained and localized by confocal laser microscopy in order to obtain a 3D image from cells in contact. To note, we recently demonstrated that CD63 interacts with the transmembrane envelope protein gp41 of HIV-1.4 We conclude that HIV virions are routed through the virological synapse utilizing host proteins such like CD63.AIDS research and human retroviruses 04/2015; DOI:10.1089/AID.2014.0252