TY - JOUR
T1 - First snapshots of the HIV-1 RNA structure in infected cells and in virions
AU - Paillart, Jean Christophe
AU - Dettenhofer, Markus
AU - Yu, Xiao Fang
AU - Ehresmann, Chantal
AU - Ehresmann, Bernard
AU - Marquet, Roland
PY - 2004/11/12
Y1 - 2004/11/12
N2 - 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 tRNA3Lys 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.
AB - 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 tRNA3Lys 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.
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U2 - 10.1074/jbc.M408294200
DO - 10.1074/jbc.M408294200
M3 - Article
C2 - 15355993
AN - SCOPUS:9144247039
SN - 0021-9258
VL - 279
SP - 48397
EP - 48403
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 46
ER -