Limited proteolytic digestions identify common structural features of HIV-1 Tat proteins expressed during infection from alternatively spliced mRNAs.
Although multiple forms of the HIV-1 Tat protein are synthesized during infection from alternatively spliced mRNAs, only 72 amino acid residues encoded in the first Tat exon are necessary for full transactivation activity. We used limited proteolytic digestions of proteins expressed in vitro to study the structures of three Tat proteins from isolate HXB2: 72R Tat (first Tat exon); 86R Tat (first and second Tat exons), Tev or TNV (first Tat exon plus Env and Rev exons). For the 86R and Tev proteins, either trypsin or chymotrypsin cleaved the majority of carboxyl residues from the first exon. Moreover, when released from carboxyl residues, the first exon of 86R and Tev was relatively resistant to subsequent proteolysis. The entire 72R Tat protein was relatively resistant to proteolysis. The protease-resistant first exon in all Tat proteins was abolished by EDTA treatment, suggesting that divalent cations are required for its conformation. Our results suggest that the first exon in the 86R, 72R, and Tev proteins is folded into a similar structure which, as defined by partial proteolysis, acts as a single biochemical domain.
Rice
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AP
Division of Molecular Virology, Baylor College of Medicine, Houston, TX 77030.
Wilson
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E
Chan
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F
D000595:Amino Acid Sequence; D010450:Endopeptidases / Q000378:metabolism; D015696:Gene Products, tat / Q000235:genetics / Q000378:metabolism*; D015658:HIV Infections / Q000235:genetics* / Q000378:metabolism; D015497:HIV-1; D011487:Protein Conformation; D012326:RNA Splicing; D012333:RNA, Messenger / Q000235:genetics*; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism; D014760:Viral Fusion Proteins / Q000235:genetics / Q000378:metabolism; D054322:tat Gene Products, Human Immunodeficiency Virus
D015696:Gene Products, tat; D012333:RNA, Messenger; D015534:Trans-Activators; D014760:Viral Fusion Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus; C064085:tev protein p28, Human immunodeficiency virus 1; D010450:Endopeptidases