Evidence for a direct interaction of Rev protein with nuclear envelop mRNA-translocation system.
The interaction of the Rev protein from human immunodeficiency virus type 1 (HIV-1) with the nucleocytoplasmic mRNA-transport system was investigated. In gel-shift assay, the recombinant Rev protein used in this study selectively bound to the Rev-responsive element (RRE) region of HIV-1 env-specific RNA. Nitrocellulose-filter-binding studies and Northern/Western-blotting experiments revealed an association constant of approximately 1 x 10(10) M-1. The Rev protein also strongly bound to isolated nuclear envelopes from H9 cells, containing the poly(A)-binding site (= mRNA carrier) and the nucleoside triphosphatase (= NTPase), which are thought to be involved in nuclear export of poly(A)-rich mRNA. Binding of 125I-Rev to a 110-kDa nuclear-envelope protein, the putative mRNA carrier, could be demonstrated in in vitro experiments. Both efflux of cellular poly(A)-rich RNA, such as actin RNA [but not efflux of poly(A)-free RNA] from isolated nuclei and the nuclear-envelope NTPase activity were strongly inhibited by Rev protein. On the other hand, transport of viral env RNA, containing the Rev-responsive element, was increased in the presence of Rev. Studying the release of RNA from closed nuclear-envelope vesicles containing entrapped RNA, the action of Rev was found to occur at the level of translocation of RNA through the nuclear pore. Evidence is presented that Rev down-regulates the NTPase-driven transport of mRNA lacking the RRE, most likely via binding to the mRNA carrier within the envelope. In contrast to the efflux of RRE-free RNA, ATP-dependent efflux of RRE-containing RNA from resealed nuclear-envelope vesicles was found to be increased, if the RNA was entrapped in the vesicles together with Rev protein. In addition, it was found that phosphorylated Rev, which is transported together with RRE-containing RNA out of the vesicles, becomes dephosphorylated during transport. In the vesicle experiments it is demonstrated for the first time that a protein selectively channels a specific mRNA across the nuclear-envelope pore complex.
Pfeifer
K
K
Institut für Physiologische Chemie, Universität Mainz, Federal Republic of Germany.
Weiler
B E
BE
Ugarkovic
D
D
Bachmann
M
M
Schröder
H C
HC
Müller
W E
WE
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001692:Biological Transport; D015152:Blotting, Northern; D015153:Blotting, Western; D002460:Cell Line; D015694:Gene Products, rev / Q000096:biosynthesis / Q000378:metabolism*; D015497:HIV-1 / Q000378:metabolism*; D009685:Nuclear Envelope / Q000201:enzymology / Q000378:metabolism*; D043583:Nucleoside-Triphosphatase; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D010766:Phosphorylation; D011061:Poly A / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D012367:RNA, Viral / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D054321:rev Gene Products, Human Immunodeficiency Virus
D015694:Gene Products, rev; D012333:RNA, Messenger; D012367:RNA, Viral; D011994:Recombinant Proteins; D054321:rev Gene Products, Human Immunodeficiency Virus; D011061:Poly A; D000255:Adenosine Triphosphate; D010744:Phosphoric Monoester Hydrolases; D043583:Nucleoside-Triphosphatase