A dynamic in vivo view of the HIV-I Rev-RRE interaction.
The export of pre-mRNAs coding for the structural genes of the human immunodeficiency virus type I depends on the interaction of the Rev protein with a highly structured viral RNA sequence, the Rev-responsive element (RRE). To gain information about the structure of the RRE and the determinants of the in vivo RRE-Rev interaction, we have analyzed the structure of the 351 nt RRE RNA within living yeast (Saccharomyces cerevisiae) by dimethyl sulfate probing with or without Rev. The in vivo structure in the absence of Rev is generally similar to the previously established solution structure. In addition, we observe a single hypermethylated guanine residue (G128), located within the Rev high-affinity binding site, in vitro as well as in vivo. The important homopurine interaction between residues 129 and 106 is required for the hyperreactivity, confirming its biological relevance. Expression of wild-type Rev leads to a protection of this region and to modifications of the RRE structure: the high-affinity site becomes further structured, and Stem IIA is destabilized. High-level expression of the oligomerization-defective mutant M4 protein leads to the same protections without destabilization of Stem IIA. Taken together with other observations, the data suggest that Rev captures the unusual conformation of the high-affinity site, followed by additional changes in the structure of the RRE.
Charpentier
B
B
Department of Biology, Brandeis University, Waltham, MA 02254, USA.
Stutz
F
F
Rosbash
M
M
D001483:Base Sequence; D001665:Binding Sites; D015694:Gene Products, rev / Q000378:metabolism*; D015752:Genes, env; D006147:Guanine / Q000378:metabolism; D015497:HIV-1 / Q000235:genetics*; D008957:Models, Genetic; D015335:Molecular Probes; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D012313:RNA / Q000378:metabolism; D012367:RNA, Viral / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013463:Sulfuric Acid Esters; D054321:rev Gene Products, Human Immunodeficiency Virus
D015694:Gene Products, rev; D015335:Molecular Probes; D012367:RNA, Viral; C039919:RNA, recombinant; D016601:RNA-Binding Proteins; D013463:Sulfuric Acid Esters; D054321:rev Gene Products, Human Immunodeficiency Virus; D006147:Guanine; D012313:RNA; C007482:dimethyl sulfate