Super-Resolution STED Microscopy-Based Mobility Studies of the Viral Env Protein at HIV-1 Assembly Sites of Fully Infected T-Cells

The ongoing threat of human immunodeficiency virus (HIV-1) requires continued, detailed investigations of its replication cycle, especially when combined with the most physiologically relevant, fully infectious model systems. Here, we demonstrate the application of the combination of stimulated emission depletion (STED) super-resolution microscopy with beam-scanning fluorescence correlation spectroscopy (sSTED-FCS) as a powerful tool for the interrogation of the molecular dynamics of HIV-1 virus assembly on the cell plasma membrane in the context of a fully infectious virus. In this process, HIV-1 envelope glycoprotein (Env) becomes incorporated into the assembling virus by interacting with the nascent Gag structural protein lattice. Molecular dynamics measurements at these distinct cell surface sites require a guiding strategy, for which we have used a two-colour implementation of sSTED-FCS to simultaneously target individual HIV-1 assembly sites via the aggregated Gag signal. We then compare the molecular mobility of Env proteins at the inside and outside of the virus assembly area. Env mobility was shown to be highly reduced at the assembly sites, highlighting the distinct trapping of Env as well as the usefulness of our methodological approach to study the molecular mobility of specifically targeted sites at the plasma membrane, even under high-biosafety conditions

Zooming in on virus surface protein mobility

[no abstract available

Immune mobilising T cell receptors redirect polyclonal CD8+ T cells in chronic HIV infection to form immunological synapses

T cell exhaustion develops in human immunodeficiency virus (HIV) infection due to chronic viral antigenic stimulation. This adaptive response primarily affects virus-specific CD8+ T cells, which may remain dysfunctional despite viral load-reducing antiretroviral therapy; however, abnormalities may also be evident in non-HIV-specific populations. Both could limit the efficacy of cell therapies against viral reservoirs. Here, we show that bulk (polyclonal) CD8+ T cells from people living with HIV (PLWH) express proposed markers of dysfunctional HIV-specific T cells at high levels yet form lytic immunological synapses (IS) and eliminate primary resting infected (HIV Gaglo) CD4+ T cells, when redirected by potent bispecific T cell-retargeting molecules, Immune mobilising monoclonal T cell receptors (TCR) Against Virus (ImmTAV). While PLWH CD8+ T cells are functionally impaired when compared to CD8+ T cells from HIV-naïve donors, ImmTAV redirection enables them to eliminate Gaglo CD4+ T cells that are insensitive to autologous HIV-specific cytolytic T cells. ImmTAV molecules may therefore be able to target HIV reservoirs, which represent a major barrier to a cure

HIV-1 Gag specifically restricts PI(4,5)P2 and cholesterol mobility in living cells creating a nanodomain platform for virus assembly

HIV-1 Gag protein assembles at the plasma membrane of infected cells for viral particle formation. Gag targets lipids, mainly PI(4,5)P2, at the inner leaflet of this membrane. Here, we address the question whether Gag is able to trap specifically PI(4,5)P2 or other lipids during HIV-1 assembly in the host CD4+ T lymphocytes. Lipid dynamics within and away from HIV-1 assembly sites were determined using super-resolution microscopy coupled with scanning fluorescence correlation spectroscopy in living cells. Analysis of HIV-1–infected cells revealed that, upon assembly, HIV-1 is able to specifically trap PI(4,5)P2 and cholesterol, but not phosphatidylethanolamine or sphingomyelin. Furthermore, our data showed that Gag is the main driving force to restrict the mobility of PI(4,5)P2 and cholesterol at the cell plasma membrane. This is the first direct evidence highlighting that HIV-1 creates its own specific lipid environment by selectively recruiting PI(4,5)P2 and cholesterol as a membrane nanoplatform for virus assembly

Molecular recognition of the native HIV-1 MPER revealed by STED microscopy of single virions

Antibodies against the Membrane-Proximal External Region (MPER) of the Env gp41 subunit neutralize HIV-1 with exceptional breadth and potency. Due to the lack of knowledge on the MPER native structure and accessibility, different and exclusive models have been proposed for the molecular mechanism of MPER recognition by broadly neutralizing antibodies. Here, accessibility of antibodies to the native Env MPER on single virions has been addressed through STED microscopy. STED imaging of fluorescently labeled Fabs reveals a common pattern of native Env recognition for HIV-1 antibodies targeting MPER or the surface subunit gp120. In the case of anti-MPER antibodies, the process evolves with extra contribution of interactions with the viral lipid membrane to binding specificity. Our data provide biophysical insights into the recognition of the potent and broadly neutralizing MPER epitope on HIV virions, and as such is of importance for the design of therapeutic interventions

Cholesterol in the Viral Membrane is a Molecular Switch Governing HIV-1 Env Clustering

HIV-1 entry requires the redistribution of envelope glycoproteins (Env) into a cluster and the presence of cholesterol (chol) in the viral membrane. However, the molecular mechanisms underlying the specific role of chol in infectivity and the driving force behind Env clustering remain unknown. Here, gp41 is demonstrated to directly interact with chol in the viral membrane via residues 751-854 in the cytoplasmic tail (CT751-854). Super-resolution stimulated emission depletion (STED) nanoscopy analysis of Env distribution further demonstrates that both truncation of gp41 CT751-854 and depletion of chol leads to dispersion of Env clusters in the viral membrane and inhibition of virus entry. This work reveals a direct interaction of gp41 CT with chol and indicates that this interaction is an important orchestrator of Env clustering.The authors are grateful to Barbara Müller, N. Landau, and Tom Hope for providing the plasmids pCHIV and pCAGGS NL4-3 Env, pMM310, and peGFP-Vpr, respectively. Proteomic analysis was performed by the SGIKER service of the University of the Basque Country. The authors would like to thank Advanced Light Microscopy Unit at the Centre for Genomic Regulation (CRG), Barcelona, Spain for the access to Leica STED microscope. The following reagents were obtained through the NIH AIDS Reagent Program (Division of NIAID, NIH): Anti-HIV-1 gp41 Hybridome (Chessie 8) (Cat# 526) from Dr. George Lewis; Antiviral bicyclam JM-2987 (hydrobromide salt of AMD-3100) from NIAID, DAIDS (cat# 8128). This project was supported by the Basque Government (grant number IT1264-19 to M.L. and F.-X.C.) and the Spanish Ministry of Science, Innovation, and Universities (BFU-2015-68981-P). This work was supported in part by the Fundación Biofísica Bizkaia and the Basque Excellence Research Centre (BERC) program of the Basque Government. J.A.N.-G. was supported by a FI predoctoral fellowship from the Basque Government and currently by Fundación Biofísica Bizkaia. A.A. was supported by Fundación Biofísica Bizkaia. S.O. was supported by an IKASIKER fellowship from the Basque Government. J.C. was supported by European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 793830. H.G.-K. was supported by a grant from the Deutsche Forschungsgemeischaft within TRR86

Molecular Recognition of the Native HIV-1 MPER Revealed by STED Microscopy of Single Virions

Antibodies against the Membrane-Proximal External Region (MPER) of the Env gp41 subunit neutralize HIV-1 with exceptional breadth and potency. Due to the lack of knowledge on the MPER native structure and accessibility, different and exclusive models have been proposed for the molecular mechanism of MPER recognition by broadly neutralizing antibodies. Here, accessibility of antibodies to the native Env MPER on single virions has been addressed through STED microscopy. STED imaging of fluorescently labeled Fabs reveals a common pattern of native Env recognition for HIV-1 antibodies targeting MPER or the surface subunit gp120. In the case of anti-MPER antibodies, the process evolves with extra contribution of interactions with the viral lipid membrane to binding specificity. Our data provide biophysical insights into the recognition of the potent and broadly neutralizing MPER epitope on HIV virions, and as such is of importance for the design of therapeutic interventions.This study was supported by the Spanish MINECO (BIO2015-64421-R (MINECO/ FEDER UE) to J.L.N.) and the Basque Government (IT838-13 to J.L.N.). P.C., E.R., and S. I. received pre-doctoral fellowships from the Basque Government. P.C. would like to acknowledge the European Biophysical Societies’ Association (EBSA) for receiving an EBSA Bursary for a working visit to a laboratory in an EBSA country. J.C., D.W., and C. E. greatly acknowledge support by the MRC (grant number MC_UU_12010/unit programs G0902418 and MC_UU_12025), the Wellcome Trust (grant 104924/14/Z/14 and Strategic Award 091911 (Micron)), MRC/BBSRC/EPSRC (grant MR/K01577X/1), BBSRC (Deutsche Forschungsgemeinschaft (Research unit 1905 “Structure and function of the peroxisomal translocon”)), the Wolfson Foundation (for initial funding of the Wolfson Imaging Centre Oxford), the EPA Cephalosporin Fund and the John Fell Fund. T.S. is a recipient of a Canada Graduate Scholarship Master’s Award and a Vanier Canada Graduate Scholarship from the Canadian Institutes of Health Research. This work was supported by operating grant NIH-150414 (J.-P.J.) from the Canadian Institutes of Health Research. This research was undertaken, in part, thanks to funding from the Canada Research Chairs program (J.-P.J.). We acknowledge valuable technical assistance from Miguel García-Porra

Three-dimensional imaging in myotonic dystrophy type 1

Altres ajuts: The research of G. Nogales-Gadea, A. Ramos-Fransi, and A. Lucia is funded by Instituto de Salud Carlos III and cofinanced by Fondos FEDER. G. Nogales-Gadea is supported by a Miguel Servet research contract and by a Trampoline Grant #21108 from AFM Telethon. A. Ballester-Lopez is funded by an FI Agaur fellowship and Generalitat de Catalunya. E. Koehorst is funded by the La Caixa Foundation (ID 100010434), fellowship code LCF/BQ/IN18/11660019, cofunded by the European Union's Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement no. 713673. I. Linares-Pardo is funded by CP14/00032 and SGR 1520 (GRC) Generalitat de Catalunya. J. Núñez-Manchón was funded by AFM Telethon Trampoline Grant #21108. G. Lucente was supported by a Rio Hortega contract. J. Chojnacki is supported by European Union's Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant . The funding bodies had no role in the design of the study and collection, analysis, and interpretation of data.We aimed to determine whether 3D imaging reconstruction allows identifying molecular:clinical associations in myotonic dystrophy type 1 (DM1). We obtained myoblasts from 6 patients with DM1 and 6 controls. We measured cytosine-thymine-guanine (CTG) expansion and detected RNA foci and muscleblind like 1 (MBNL1) through 3D reconstruction. We studied dystrophia myotonica protein kinase (DMPK) expression and splicing alterations of MBNL1, insulin receptor, and sarcoplasmic reticulum Ca(2+)-ATPase 1. Three-dimensional analysis showed that RNA foci (nuclear and/or cytoplasmic) were present in 45%-100% of DM1-derived myoblasts we studied (range: 0-6 foci per cell). RNA foci represented <0.6% of the total myoblast nuclear volume. CTG expansion size was associated with the number of RNA foci per myoblast (r = 0.876 [95% confidence interval 0.222-0.986]) as well as with the number of cytoplasmic RNA foci (r = 0.943 [0.559-0.994]). Although MBNL1 colocalized with RNA foci in all DM1 myoblast cell lines, colocalization only accounted for 1% of total MBNL1 expression, with the absence of DM1 alternative splicing patterns. The number of RNA foci was associated with DMPK expression (r = 0.967 [0.079-0.999]). On the other hand, the number of cytoplasmic RNA foci was correlated with the age at disease onset (r = −0.818 [−0.979 to 0.019]). CTG expansion size modulates RNA foci number in myoblasts derived from patients with DM1. MBNL1 sequestration plays only a minor role in the pathobiology of the disease in these cells. Higher number of cytoplasmic RNA foci is related to an early onset of the disease, a finding that should be corroborated in future studies

Dissemination of <i>Mycobacterium tuberculosis</i> is associated to a <i>SIGLEC1</i> null variant that limits antigen exchange via trafficking extracellular vesicles

The identification of individuals with null alleles enables studying how the loss of gene function affects infection. We previously described a non‐functional variant in SIGLEC1, which encodes the myeloid‐cell receptor Siglec‐1/CD169 implicated in HIV‐1 cell‐to‐cell transmission. Here we report a significant association between the SIGLEC1 null variant and extrapulmonary dissemination of Mycobacterium tuberculosis (Mtb) in two clinical cohorts comprising 6,256 individuals. Local spread of bacteria within the lung is apparent in Mtb‐infected Siglec‐1 knockout mice which, despite having similar bacterial load, developed more extensive lesions compared to wild type mice. We find that Siglec‐1 is necessary to induce antigen presentation through extracellular vesicle uptake. We postulate that lack of Siglec‐1 delays the onset of protective immunity against Mtb by limiting antigen exchange via extracellular vesicles, allowing for an early local spread of mycobacteria that increases the risk for extrapulmonary dissemination

Siglec-1 on dendritic cells mediates SARS-CoV-2 trans-infection of target cells while on macrophages triggers proinflammatory responses

COVID-19 pandemic is not yet under control by vaccination, and effective antivirals are critical for preparedness. Here we report that macrophages and dendritic cells, key antigen presenting myeloid cells (APCs), are largely resistant to SARS-CoV-2 infection. APCs effectively captured viruses within cellular compartments that lead to antigen degradation. Macrophages sense SARS-CoV-2 and released higher levels of cytokines, including those related to cytokine storm in severe COVID-19. The sialic acid-binding Ig-like lectin 1 (Siglec-1/CD169) present on APCs, which interacts with sialylated gangliosides on membranes of retroviruses or filoviruses, also binds SARS-CoV-2 via GM1. Blockage of Siglec-1 receptors by monoclonal antibodies reduces SARS-CoV-2 uptake and transfer to susceptible target cells. APCs expressing Siglec-1 and carrying SARS-CoV-2 are found in pulmonary tissues of non-human primates. Single cell analysis reveals the in vivo induction of cytokines in those macrophages. Targeting Siglec-1 could offer cross-protection against SARS-CoV-2 and other enveloped viruses that exploit APCs for viral dissemination, including those yet to come in future outbreaks.The research of CBIG consortium (constituted by IRTA-CReSA, BSC, & IrsiCaixa) is supported by Grifols pharmaceutical. The authors also acknowledge the crowdfunding initiative #Yomecorono (https://www.yomecorono.com). J.M-P. is supported by grant PID2019-109870RB-I00 from the Spanish Ministry of Science and Innovation and in part also by Grifols. CR lab is funded by RTI2018-094445-B100 (MCIU/AEI/FEDER, UE). The authors also acknowledge the crowdfunding initiative #Yomecorono (https://www.yomecorono.com). The NHP study was primarily supported by YNPRC Coronavirus Pilot Research Project Program grant to M.Pa. under award P51 OD11132, Emergent Venture Fast grant program to MPa under awards #2206 and #2144, and William and Lula Pitts Foundation (to MPa).N