Imaging HIV-1 RNA dimerization in cells by multicolor super-resolution and fluctuation microscopies.
Dimerization is a unique and vital characteristic of retroviral genomes. It is commonly accepted that genomic RNA (gRNA) must be dimeric at the plasma membrane of the infected cells to be packaged during virus assembly. However, where, when and how HIV-1 gRNA find each other and dimerize in the cell are long-standing questions that cannot be answered using conventional approaches. Here, we combine two state-of-the-art, multicolor RNA labeling strategies with two single-molecule microscopy technologies to address these questions. We used 3D-super-resolution structured illumination microscopy to analyze and quantify the spatial gRNA association throughout the cell and monitored the dynamics of RNA-RNA complexes in living-cells by cross-correlation fluctuation analysis. These sensitive and complementary approaches, combined with trans-complementation experiments, reveal for the first time the presence of interacting gRNA in the cytosol, a challenging observation due to the low frequency of these events and their dilution among the bulk of other RNAs, and allow the determination of the subcellular orchestration of the HIV-1 dimerization process.
Centre d'études d'agents pathogènes et biotechnologies pour la santé, CPBS-CNRS, Université de Montpellier, 1919 Route de Mende, 34293 Montpellier, France.
Mireia
M
Ferrer
CNRS UMR5048, Centre de Biochimie Structurale, 29 rue de Navacelles, 34090 Montpellier, France INSERM U1054, 34090 Montpellier, France Université de Montpellier, 34090 Montpellier, France.
Caroline
C
Clerté
Centre d'études d'agents pathogènes et biotechnologies pour la santé, CPBS-CNRS, Université de Montpellier, 1919 Route de Mende, 34293 Montpellier, France.
Célia
C
Chamontin
Institut de Génétique Moléculaire de Montpellier, University of Montpellier, CNRS UMR 5535, 1919 route de Mende, 34293, Montpellier Cedex 5, France.
Eugenia
E
Basyuk
Centre d'études d'agents pathogènes et biotechnologies pour la santé, CPBS-CNRS, Université de Montpellier, 1919 Route de Mende, 34293 Montpellier, France.
Sébastien
S
Lainé
CNRS UMR5048, Centre de Biochimie Structurale, 29 rue de Navacelles, 34090 Montpellier, France INSERM U1054, 34090 Montpellier, France Université de Montpellier, 34090 Montpellier, France.
Jérome
J
Hottin
Institut de Génétique Moléculaire de Montpellier, University of Montpellier, CNRS UMR 5535, 1919 route de Mende, 34293, Montpellier Cedex 5, France.
Edouard
E
Bertrand
CNRS UMR5048, Centre de Biochimie Structurale, 29 rue de Navacelles, 34090 Montpellier, France INSERM U1054, 34090 Montpellier, France Université de Montpellier, 34090 Montpellier, France.
Emmanuel
E
Margeat
Centre d'études d'agents pathogènes et biotechnologies pour la santé, CPBS-CNRS, Université de Montpellier, 1919 Route de Mende, 34293 Montpellier, France marylene.mougel@cpbs.cnrs.fr.
Marylène
M
Mougel
D002462:Cell Membrane / Q000378:metabolism; D002470:Cell Survival; D003116:Color; D003600:Cytosol / Q000378:metabolism; D019281:Dimerization; D016679:Genome, Viral; D015497:HIV-1 / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D021621:Imaging, Three-Dimensional; D017404:In Situ Hybridization, Fluorescence; D008853:Microscopy / Q000379:methods*; D009690:Nucleic Acid Conformation; D012367:RNA, Viral / Q000737:chemistry* / Q000235:genetics; D013194:Staining and Labeling; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000378:metabolism
D012367:RNA, Viral; D054301:gag Gene Products, Human Immunodeficiency Virus