Functional characterization of Tat protein from human immunodeficiency virus. Evidence that Tat links viral RNAs to nuclear matrix.
The processes of transcription and posttranscription are assumed to proceed in close association with the nuclear matrix. In this study we demonstrated that Tat, the trans-activating protein from human immunodeficiency virus type 1 (HIV-1), binds both to the TAR region of the nascent HIV mRNAs and the nuclear matrix with high affinity. Both North/Western blotting experiments and nitrocellulose binding studies revealed that Tat binds with an association constant (K alpha) of approximately 1 x 10(9) M-1 to the TAR segment of HIV RNA; binding of Tat to this sequence which is present between position 32 and 82 downstream from the TATA box was also confirmed by gel retardation assays. Binding of Tat to TAR only occurs if the loop segment in the proposed stem-loop secondary structure of HIV leader mRNA is present. Likewise, Tat binds to the nuclear matrix with a K alpha of 7.5 x 10(7) M-1. The number of binding sites has been estimated to be 2 x 10(8)/micrograms of matrix protein, corresponding to 4 x 10(3) sites/nucleus. Tat displays its bimodal function only in the presence of Zn2+ ions. In vitro transcription experiments, using HIV-1 infected nuclei, demonstrate that beyond the TAR-region HIV RNA synthesis occurs only in the presence of Tat. Present studies indicate that Tat may function as a linker by binding of nascent HIV RNAs to the nuclear matrix.
Müller
W E
WE
Institut für Physiologische Chemie, Universität, Mainz, West Germany.
Okamoto
T
T
Reuter
P
P
Ugarkovic
D
D
Schröder
H C
HC
D002460:Cell Line; D003001:Cloning, Molecular; D015683:Gene Products, gag / Q000235:genetics; D015696:Gene Products, tat / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D015530:Nuclear Matrix / Q000378:metabolism*; D012367:RNA, Viral / Q000378:metabolism*; D015534:Trans-Activators / Q000378:metabolism*; D014158:Transcription, Genetic; D054322:tat Gene Products, Human Immunodeficiency Virus
D015683:Gene Products, gag; D015696:Gene Products, tat; D012367:RNA, Viral; D015534:Trans-Activators; D054322:tat Gene Products, Human Immunodeficiency Virus