In vitro interaction between human immunodeficiency virus type 1 Rev protein and splicing factor ASF/SF2-associated protein, p32.
Continuous replication of human immunodeficiency virus type 1 requires the expression of the regulatory protein Rev, which binds to the Rev response element (RRE) and up-regulates the cytoplasmic appearance of singly spliced and unspliced mRNA species. It has been demonstrated that the murine protein YL2 interacts with Rev in vivo and modulates the activity of Rev (Luo, Y., Yu, H., and Peterlin, B. M. (1994) J. Virol. 68, 3850-3856). Here we show that the YL2 human homologue, the p32 protein, which co-purifies with alternative splicing factor ASF/SF2, interacts directly with the basic domain of Rev in vitro and that the Rev-p32 complex is resistant to high concentrations of salt or nonionic detergent. Protein footprinting data suggest that Rev interacts specifically with amino acids within the 196-208 region of p32. An analysis of the ternary complex, formed among p32, Rev, and RRE RNA, shows that Rev can bridge the association of p32 and RRE. Furthermore, we demonstrate that exogenously added p32 specifically relieves the inhibition of splicing in vitro exerted by the basic domain of Rev. Our data are consistent with a model in which p32 functions as a link between Rev and the cellular splicing apparatus.
Tange
T O
TO
Department of Molecular Biology, University of Aarhus, C. F. Møllers Allé, Building 130, DK-8000 Aarhus C, Denmark.
Jensen
T H
TH
Kjems
J
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins; D017931:DNA Primers / Q000737:chemistry; D015967:Gene Expression Regulation, Viral; D015694:Gene Products, rev / Q000378:metabolism*; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D018960:Hyaluronan Receptors; D024101:Mitochondrial Proteins; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000378:metabolism*; D012326:RNA Splicing; D012333:RNA, Messenger / Q000378:metabolism; D012367:RNA, Viral / Q000378:metabolism; D016601:RNA-Binding Proteins; D000068103:Serine-Arginine Splicing Factors; D054321:rev Gene Products, Human Immunodeficiency Virus
C087299:C1QBP protein, human; D002352:Carrier Proteins; D017931:DNA Primers; D015694:Gene Products, rev; D018960:Hyaluronan Receptors; D024101:Mitochondrial Proteins; D009687:Nuclear Proteins; D011506:Proteins; D012333:RNA, Messenger; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D054321:rev Gene Products, Human Immunodeficiency Virus; D000068103:Serine-Arginine Splicing Factors