Investigating the cellular distribution and interactions of HIV-1 nucleocapsid protein by quantitative fluorescence microscopy.
The nucleocapsid protein (NCp7) of the Human immunodeficiency virus type 1 (HIV-1) is a small basic protein containing two zinc fingers. About 2000 NCp7 molecules coat the genomic RNA in the HIV-1 virion. After infection of a target cell, the viral core enters into the cytoplasm, where NCp7 chaperones the reverse transcription of the genomic RNA into the proviral DNA. As a consequence of their much lower affinity for double-stranded DNA as compared to single-stranded RNAs, NCp7 molecules are thought to be released in the cytoplasm and the nucleus of infected cells in the late steps of reverse transcription. Yet, little is known on the cellular distribution of the released NCp7 molecules and on their possible interactions with cell components. Hence, the aim of this study was to identify potential cellular partners of NCp7 and to monitor its intracellular distribution and dynamics by means of confocal fluorescence microscopy, fluorescence lifetime imaging microscopy, fluorescence recovery after photobleaching, fluorescence correlation and cross-correlation spectroscopy, and raster imaging correlation spectroscopy. HeLa cells transfected with eGFP-labeled NCp7 were used as a model system. We found that NCp7-eGFP localizes mainly in the cytoplasm and the nucleoli, where it binds to cellular RNAs, and notably to ribosomal RNAs which are the most abundant. The binding of NCp7 to ribosomes was further substantiated by the intracellular co-diffusion of NCp7 with the ribosomal protein 26, a component of the large ribosomal subunit. Finally, gradient centrifugation experiments demonstrate a direct association of NCp7 with purified 80S ribosomes. Thus, our data suggest that NCp7 molecules released in newly infected cells may primarily bind to ribosomes, where they may exert a new potential role in HIV-1 infection.
Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Université de Strasbourg, Faculté de Pharmacie, Illkirch, France.
Halina
H
Anton
Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Université de Strasbourg, Faculté de Pharmacie, Illkirch, France.
Nedal
N
Taha
Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Université de Strasbourg, Faculté de Pharmacie, Illkirch, France.
Emmanuel
E
Boutant
Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Université de Strasbourg, Faculté de Pharmacie, Illkirch, France.
Ludovic
L
Richert
Institut de Génétique et de Biologie Moléculaire et Cellulaire, UMR 7104 CNRS, U964 Inserm, Université de Strasbourg, Illkirch, France.
Heena
H
Khatter
Institut de Génétique et de Biologie Moléculaire et Cellulaire, UMR 7104 CNRS, U964 Inserm, Université de Strasbourg, Illkirch, France.
Bruno
B
Klaholz
Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Université de Strasbourg, Faculté de Pharmacie, Illkirch, France.
Philippe
P
Rondé
Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Université de Strasbourg, Faculté de Pharmacie, Illkirch, France.
Eléonore
E
Réal
Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Université de Strasbourg, Faculté de Pharmacie, Illkirch, France.
Hugues
H
de Rocquigny
Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Université de Strasbourg, Faculté de Pharmacie, Illkirch, France.
Yves
Y
Mély
D002352:Carrier Proteins / Q000378:metabolism; D002460:Cell Line; D015870:Gene Expression; D017930:Genes, Reporter; D015658:HIV Infections / Q000378:metabolism* / Q000821:virology*; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D019590:Nucleocapsid Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D002352:Carrier Proteins; D019590:Nucleocapsid Proteins; D011993:Recombinant Fusion Proteins