Membrane Domains and Viral Assembly

  • 5 nov 2021: Congrats to Rayane et Coline whom successfully animated two different workshops during  MiFoBio 2021!


    Coline and Sebastien Lyonnais (CEMIPAI) co-lead an atomic force coupled to fluorescence microscopy workshop to visualise and characterise Sars-CoV2 particles at the nanoscale, in partnership with JPK/Bruker. 





    Rayane et Delphine on their side, designed a 2D/3D STED super-resolution to visualise HIV-1 particles on actin protrusion, in partnership with Abberior.


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  • 29 oct 2021: Congrats to Manon & Coline ont received  a Montpellier University grant to respectively attend the 2022 CROI congress  and 2021 MiFoBio.
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  • 27-30 sept 2021: Joint Congress of the Biophysical French Society and the Membrane Research Consortium.
    Our work on plasma membrane lipid sorting induced by HIV assembly has been selected for an oral communication.

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  • 03 juin 2021: Congrats to Delphine, organising comittee member of the 23rd Exo-Endo Congress. An outstanding membrane trafficking congress.

    The whole team will be attending and Manon will briefly present her work. Delphine will chair the "Physics and membrane dynamics" session w/. L. Johannes from Curie Inst.





  • 01 mai 2021 : Welcome to ISEV 2021!




    If you are interested in I-BAR EVs, don't hesitate to listen to the oral presentation on demand OD09.04 at ISEV2021 annual meeting! It's accessible until June 14th!



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  • 16 mar 2021 : Our webinar "Imaging Viruses: From Single Molecule to Diagnosis" has been a real success!
    World-renowned speakers. More than 100 registrants all over Europe and even the world! Warmful thanks to GdR ImaBio & Abbelight for their support during the organisation.

  • 04 oct 2020 -  Delphine as a scientific advisor for this interesting animation of Influenza viral entry.

Watch it here.


 

 




Our team is interested in the role of matrix and capsid proteins as well as lipids and lipid domains in the assembly of viruses in their host cells.
We study these phenomena quantitatively at the single-molecule level in living cells and in model systems.




 

Our research focuses on 5 main topics (click on each to access details).

 

HIV-1 assembly molecular mechanism occuring at CD4 Tcells plasma membrane (Mariani et al., 2014)




1. Lipid sorting during self-assembly of HIV-1 Gag and influenza A M1 proteins

(Funding: ANR Fluobuds, CNRS, Univ Montp, GdR IMABIO, ERC Erasmus Mundus)


This project aims at deciphering the role of pre-existing or generated lipid nanodomains in the plasma membrane of the host cell during viral assembly.
In previous studies, we have shown that the interaction between the Matrix domain of the Gag protein and specific plasma membrane lipids (PS/PIP2) induces retrovirus assembly (Hamard-Péron et al., 2010 ; Hamard-Péron & Muriaux, 2011). We proposed that acidic lipid-enriched microdomains (ALEMs) are created by oligomerization of the HIV-1 Gag protein in the host cell plasma membrane during viral assembly (Kerviel et al., 2013, Yandapalli et al., 2014, Mariani et al., 2014 & Saad & Muriaux, Editorial 2015).

To show that, we first have characterized the nature and the effects of the MA domain interaction of retroviral Gag proteins (HIV-1 and MLV) with lipid membranes in silico  (Kerviel et al., 2013, Charlier et al., 2014).

In vitro
, we investigated the impact of HIV-1 Gag protein self-assembly on lipid plasma membrane using model membranes with controlled molecular composition mimicking this membrane. We were able to show that, during its self-assembly, the viral Gag protein sorts membrane lipids and generates lipid nano-domains enriched in PIP2 and Cholesterol, while excluding Sphingomyelin (Yandrapalli et al., 2016).


We finally quantified this phenomenon in viral host cells (HIV-1-infected or Gag-transfected CD4 T cells) by measuring the diffusional changes of lipids (PIP2, Cholesterol, SM and PE) into and out of the viral bud on the T cell surface by sSTED-FCS (Scanning STED FCS), in collaboration with C. Eggeling (U. Oxford/ Univ Jena, UK-Germany) and J Chojnacki (U. Oxford/ IRSI Caixa, Barcelona, SP) (Favard et al., 2019)
This work was highlighted by the Company of Biologists, by the CNRS-INSB in its monthly news as well as in its  2019 highlights and also by ANRS.

Back to research topics




2. Single molecule microscopy of HIV-1 assembly in living CD4 T cells

(Funding : ANR Fluobuds, CNRS, Univ Montp, GdR IMABIO, Erasmus Mundus)

In parallel, we have studied the dynamics
of capsid protein assembly in the cell using super-resolution optical microscopy methods (Live PALM, spt-PALM), in collaboration with the teams of JB Sibarita (IINS, Bordeaux) and M. Dahan (Institut Curie, Paris).
We followed the Gag self-assembly, molecule by molecule, at a resolution of a tenths of nanometers, generating billions of individual trajectories. (Mariani et al., 2016, Journal Cover)



We rely on methods from Big Data, for the automatic analysis of these trajectories using Bayesian inferences. In collaboration with J.B. Masson (Institut Pasteur), M. El Beheiry and M. Dahan (I. Curie), we were able to follow, during 25 minutes, molecules after molecules, the self-assembly of Gag during the formation of a VLP of HIV-1 in a T lymphocyte. This was done by measuring both the changes in the diffusion of these molecules (left part of the movie) but also in the attraction energy (right part of the movie) that is sensed by each Gag in the viccinity of the assembly site. (Mariani et al., 2018)

Our work on the study of HIV-1 assembly using super-resolution microscopies of the living has resulted in 2 general interest reviews (Inamdar et al., 2019 et Arone et al., 2021).

 
Back to research topics




3. Role of membrane co-factors and cortical actin during viral assembly .

(Funding : ANRS, Sidaction, FRM)

This project aims to undesrtand the role of filamentous cortical actin (F-actin) and newly identified cellular co-factors from HIV-1 host cells (T-CD4+ lymphocytes) in the modulation of the dynamics and organization of membrane domains as well as in the membrane curvature generation during HIV-1 assembly as well as filamentous cortical actin (F-actin).



In our previous research, we have shown that CD81 tetraspanin is a host cell transmembrane protein involved in HIV-1 assembly in CD4 (Grigorov et al., 2009) and that the clathrin-dynamin endocytosis pathway was involved in the transmission of HIV-1 from CD4 T cells to CD4 T cells (Bosch et al., 2008). We are now interested in studying the role of these tetraspanins in the assembly of another human retrovirus HTLV-1 chronically infecting T cells in collaboration with Hélène Dutartre (CIRI,ENS Lyon)
In parallel, we also studied the role of a major regulator of the cortical actin network regulated by the Rac1/IRSp53/Wave2/arp2/3 activation pathway in the assembly of the HIV-1 Gag protein and the release of the viral particle in CD4 T cells, the main hosts of HIV-1. (Thomas et al., 2015).

We recently showed that the membrane bending factor IRSp53 was indeed involved in the assembly and budding of the HIV-1 Gag viral particle (Inamdar et al., 2021). During the reduction of IRSp53 expression by interfering RNA in HIV-1 Gag particle-producing cells, we observe a blockage of virus assembly at midstream (Figure A). IRSp53 is found at the site of virus assembly, as observed by PALM (Gag)/STORM (IRSp53) microscopy (Figure B) and quantified by correlating experimental single molecule coordinate based colocalization (CBC) measurements with those obtained by numerical simulations on pre-defined models (Figure C). The cellular protein IRSp53 assists in membrane bending during HIV-1 particle formation to finalize HIV-1 assembly and budding.


From Inamdar et al., 2021.

Back to research topics




4. Role of M-matrix proteins and cellular cofactors in the assembly of influenza virus and SARS-CoV-2.

We are currently studying other enveloped RNA viruses. The influenza virus, A (H1N1) pdm09, still circulating, which caused the first pandemic of the 21st century. We are interested in the role of the matrix protein M1 (and M2) in the membrane assembly of this virus. M1 is the most abundant viral protein and is essential for influenza virus assembly. We have recently shown that the arginine triplet (R76/77/78) located on helix 5 of M1, a highly conserved motif among influenza A subtypes, is essential for its localization to the cell plasma membrane, for the membrane attachment of M1, for its incorporation into viral particles and for its infectivity (in collaboration with O.Moncorgé, IRIM CNRS Montpellier and P. Roingeard, University of Tours). Its mutation completely abolishes the production of infectious virus. This study also allowed us to develop a minimal system for the production of non-infectious influenza A VLP-M (Kerviel, Dash et al., 2016).



We are now interested in the role of cortical actin and its dynamic regulators in the assembly and budding of influenza A virus and SARS-CoV-2 (Bracquemond & Muriaux, 2021), that we explore using RNA interference, infection, and cell biology techniques coupled with advanced photonic microscopies.


5. HIV-1, IAV and SARS-CoV-2 assembly visualized by Atomic force microscopy (AFM) on single virus particles.

(Funding : ANRS 2015, ANRS 2019, MUSE)

We have shown using atomic force microscopy at the level of single virus particles that genomic RNA is a polydispersity factor in the morphology of HIV-1 (Faivre-Moskalenko et al.,2014). In collaboration with the physicists C. Moskalenko and M. Castelnovo at ENS Lyon, atomic force microscopy is used to quantitatively study the variation of the size of viral particles and cores produced by host cells, as a function of various microenvironments (Bernaud et al., 2015). This polydispersity is an estimator of the self-assembly efficiency of the HIV-1 Gag protein.
We have recently imaged the infectious and the inactivated SARS-CoV2 virus by AFM in collaboration with Sébastien Lyon and the Cemipai team (Lyonnais et al., 2021).


We continue to analyze all types of VLPs derived from HIV-1, IAV and SARS-CoV-2 viruses by these fluorescence techniques, which represent wonderful tools to study the assembly or the entry of viruses into their host cell (outside a BSL3).

Back to research topics






 

 




*Co-last and/or corresponding, # Co first, In Bold, team members


Publication Year: 2022

Publication Year: 2021


Publication Year: 2020


Publication Year: 2019


Publication Year: 2018

 


Publication Year: 2016

 


Publication Year: 2015


Publication Year: 2014

Publication Year: 2013


Publication Year: 2012

 



Released previous to the team began.

Publication Year: 2011
Publication Year: 2010 Publication Year: 2009
  • Gasecka A, Han TJ, Favard C, Cho BR, Brasselet S. Quantitative imaging of molecular order in lipid membranes using two-photon fluorescence polarimetry. Biophysical Journal ; 97(10):2854-62.
  • Grigorov B, Attuil-Audenis V, Perugi F, Nedelec M, Watson S, Pique C, Darlix JL, Conjeaud H and D.Muriaux A role for CD81 on the late steps of HIV-1 replication in a chronically infected T cell line. Retrovirology ; 6:28.
  • F.Perugi, D.Muriaux, B.C.Ramirez, S.Chabani, E.Decroly, JL.Darlix, V.Blot & C.Pique Human Discs Large is a new negative regulator of HIV-1 infectivity. Molelcular & Cellular Biology ; 20:498-508.
  • Taulet N, Comunale F, Favard C, Charrasse S, Bodin S, Gauthier-Rouvière C. N-cadherin/p120 catenin association at cell-cell contacts occurs in cholesterol-rich membrane domains and is required for RhoA activation and myogenesis.Journal of Biological Chemistry ; 284(34):23137-45.
  • Portet T, Camps i Febrer F, Escoffre JM, Favard C, Rols MP, Dean DS. Visualization of membrane loss during the shrinkage of giant vesicles under electropulsation. Biophysical Journal ; 96(10):4109-21.
Publication Year: 2008
  • Macia E, Partisani M, Favard C, Mortier E, Zimmermann P, Carlier MF, Gounon P, Luton F, Franco M. The pleckstrin homology domain of the Arf6-specific exchange factor EFA6 localizes to the plasma membrane by interacting with phosphatidylinositol 4,5-bisphosphate and F-actin. Journal of Biological Chemistry 283(28), 19836-44.
  • Fritz JV, Didier P, Clamme JP, Schaub E, Muriaux D, Cabanne C, Morellet N, Bouaziz S, Darlix JL, Mély Y, de Rocquigny H. Direct Vpr-Vpr interaction in cells monitored by two photon fluorescence correlation spectroscopy and fluorescence lifetime imaging Retrovirology ; 22, 87.
  • Bosch, B., Grigorov, B., JL. Darlix, D. Muriaux & J. Esté. A clathrin-dynamin-dependent endocytic pathway for the uptake of HIV-1 by direct T cell-T cell transmission. Antiviral Research ; 80:185-93.
  • L. Houzet, Z. Morichaud, L. Didier, D. Muriaux, JL. Darlix & M.Mougel. Deletion in nucleocapsid turn HIV-1 into a DNA-containing virus. Nucleic Acids Research ; 36, 2311-9.
  • Corbin, B. Grigorov, P. Roingeard, J.-L. Darlix et D. Muriaux. Une nouvelle vision de l’assemblage du HIV-1 / Revisiting HIV-1 assembly. Médecine & Sciences ; 24:49-55.

Current Team Members (2020-2021)




  • Jittendriya Swain - Post Doc Membrane Biophysics and Virology
  • Rayane Dibsy - Ph.D. Student CBS2 Montpellier.
  • Manon Gourdelier - Ph.D. Student  CBS2 Montpellier.
  • Anita Mouttou - Ph.D. Student CBS2 Montpellier.
  • Coline Arone Ph.D. Student CBS2 Montpellier.
  • Erwan Bremaud - Chemical Engineer.

 


Alumni

2020
  • Aurore de Poret  CIFRE-CILOA. Now working for Bayer.
  • Kaushik Inamdar, ANRS, starting his post-doc at MPI for Biophysical Chemistry (S. Jakobs group) in Goettingen, Germany. 
  • David Bracquemond, Chemical Engineer for CEMIPAI.
  • Aurélie Favarin (w/L. Picas) - Infectious disease master student , Univ. Montp. Now doing a Ph.D. in Paris
2019 
2018
2017

  • Shantoshini Dash - Ph.D. CBS2 Montpellier, Now Post-Doc Pennsylvania University
  • Elise Boilley - Master EPHE Paris Sorbonne. Consultant for Efor Lyon
  • Salma Guerfallah - Master Infectious Disease, Univ. Montpellier
  • Aida Houhou - Master Biopysics Univ. Montpellier
  • Sarah Hadouch - B. Sc. Chemistry
  • Mélanie Amigues - Biotechnology Technician, CNRS
2016
  • Charlotte Mariani, Ph.D. "Quantifying the nano-dynamic of HIV-1 Gag protein on the plasma membrane of T-Cells during viral assembly." Now Bioprocess Validation Specialist @ Merck Inc.
  • Naresh Yandrapalli, Ph.D., "Role of HIV-1 Gag protein multimerization in the generation of nanodomains in lipid membranes", Now Post-Doc at Max Planck Institute Postdam.
  • Julien Noero, Master Biophysics NUM
  • Lisa Morichon, Biotechnology Technician Student
2015 
  • Nathalie Gros, Lab Manager, Now CNRS Permanent position at CNRS at Cemipai.
  • Alexis Chesseron, Master Physics of the living systems, NUM
  • Jordan Tremoulet, Biotechnology Technician Student
2014
  • Adeline Kerviel - Ph.D. Now Post Doc at NIH Bethesda.
  • Jan Prchal - Visiting Scientist - Sanofi Price - Institute of Chemical Technology - Prague (CZ)
  • Emmanuel Aurouet - Master EPHE Now @ Beckman Coulter
  • Rony Midahuen - Master Biotechnology.
2013
  • Audrey Thomas - Ph.D. Student.
  • Sophie Guillaume - B Sc Chemistry
  • Alexandre Pasquiou – Master Biological Imaging (Nice, Zeiss Fellowship) - Now @ Amplitech
  • Taoufik Lahdidioui - Master Software Engineering (Montpellier)
2012
  •  Charlotte Berliat –Engineer Chemistry/Physics/Electronics Lyon - Now @ Laboratoire Aguettant
  • Fatimata Diagne – Master EPHE.
  • Stéphane Pelletier - BSc Cell Biology
  • Elise Faye - BSc Chemistry

 




Past and present in pictures


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ACADEMICS COLLABORATORS

INTERNATIONAL

NATIONAL

LOCAL/IN HOUSE

INDUSTRIAL COLLABORATORS

 

Team Leader

Delphine Muriaux

Research Director CNRS,
Head of CEMIPAI BSL3 facilities,
Virologist

Cyril Favard

Research Engineer CNRS,
Biophysicist

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At a Glance

We aim at characterizing the respective roles of viral proteins and host-cell lipids or sub-plasma membrane constituents during the assembly of enveloped viruses affecting human beings (such as HIV-1 and influenza viruses).

We are biologists and biophysicists using multi-disciplinary approaches.

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IRIM
Institut de Recherche en Infectiologie de Montpellier
UMR 9004 - CNRS / UM
1919 route de Mende - 34293 Montpellier cedex 5
FRANCE

 

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