First snapshots of the HIV-1 RNA structure in infected cells and in virions.
With the increasing interest of RNAs in regulating a range of cell biological processes, very little is known about the structure of RNAs in tissue culture cells. We focused on the 5'-untranslated region of the human immunodeficiency virus type 1 RNA genome, a highly conserved RNA region, which contains structural domains that regulate key steps in the viral replication cycle. Up until now, structural information only came from in vitro studies. Here, we developed chemical modification assays to test nucleotide accessibility directly in infected cells and viral particles, thus circumventing possible biases and artifacts linked to in vitro assays. The secondary structure of the 5'-untranslated region in infected cells points to the existence of the various stem-loop motifs associated to distinct functions, proposed from in vitro probing, mutagenesis, and phylogeny. However, compared with in vitro data, subtle differences were observed in the dimerization initiation site hairpin, and none of the proposed long range interactions were observed between the functional domains. Moreover, no global RNA rearrangement was observed; structural differences between infected cells and viral particles were limited to the primer binding site, which became protected against chemical modification upon tRNA(3) (Lys) annealing in virions and to the main packaging signal. In addition, our data suggested that the genomic RNA could already dimerize in the cytoplasm of infected cells. Taken together, our results provided the first analysis of the dynamic of RNA structure of the human immunodeficiency virus type 1 RNA genome during virus assembly ex vivo.
Paillart
Jean-Christophe
JC
Unité Propre de Recherche 9002 du CNRS conventionnée à l'Université Louis Pasteur, Institut de Biologie Moléculaire et Cellulaire, 15 rue René Descartes, 67084 Strasbourg cedex, France. jc.paillart@ibmc.u-strasbg.fr
Dettenhofer
Markus
M
Yu
Xiao-Fang
XF
Ehresmann
Chantal
C
Ehresmann
Bernard
B
Marquet
Roland
R
D020121:5' Untranslated Regions; D001483:Base Sequence; D002460:Cell Line; D019281:Dimerization; D016679:Genome, Viral; D015497:HIV-1 / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012367:RNA, Viral / Q000737:chemistry*; D014771:Virion / Q000235:genetics* / Q000378:metabolism; D014779:Virus Replication
D020121:5' Untranslated Regions; D012367:RNA, Viral