Sensitive in vitro analysis of HIV-1 Rev multimerization.
Oligomerization of the Rev protein of human immuno-deficiency virus type 1 on its cognate response element is essential for export of the late viral mRNAs from the nucleus. Two regions of the protein, flanking the RNA binding site, have been defined as oligomerization sites after mutants (M4 and M7) had been reported to bind specifically to the response element but not to oligomerize in vivo or in vitro. These mutants are often used as paradigms for studies of Rev multimerization. We have re-examined the in vitro binding of these mutants to model Rev response elements, using improved gel mobility assays. We find that both mutants will form oligomers on the Rev response element, but have somewhat lower affinities for RNA than the wild-type protein. M7 has lower specific affinity, but shows little deficiency in oligomerization once binding starts. In contrast, M4 is multimerization deficient, as previously reported. Therefore, whilethe sites are correctly defined, it is inappropriate to employ the original M7 deletion mutant to study Rev oligomerization.
Brice
P C
PC
MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK. pjgb@mrc-lmb.cam.ac.uk
Kelley
A C
AC
Butler
P J
PJ
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D015694:Gene Products, rev / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015497:HIV-1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics; D054321:rev Gene Products, Human Immunodeficiency Virus
D004279:DNA, Viral; D015694:Gene Products, rev; D012367:RNA, Viral; D054321:rev Gene Products, Human Immunodeficiency Virus