HIV-1 Gag binds specifically to RNA stem-loops in the 5' leader sequence.
GST-Gag(p55) binds specifically to HIV-1 RNA sequences 1-406, in vitro, with a Kd of about 50 nM. This RNA transcript contains a number of stem loop (SL) structures. The binding is due to the Gag moiety of the fusion protein, not GST. There is a high affinity binding site for Gag in an RNA containing nucleotides 325-362. SL4 is predicted by both biochemical studies and computer folding to be located between nucleotides 335 and 358. An RNA transcript ending at nucleotide 335 does not bind Gag. The deletion of nucleotides 334-358 from HIV-1 RNAs does not affect Gag binding. Digestions with RNase V1 and T1 show that nucleotides 297-300 in SL2, 310, 312, 313, 315, 317, 318, 325 in SL3, and 342 and 343 in SL4 are protected in the presence of Gag. The cleavage of nucleotides 348-351 in SL4 by RNAse V1 is enhanced by Gag binding. At least two Gag binding sites are therefore located in the leader RNA. Those located 5' of nucleotide 335 require the presence of additional 3' sequences.
Maddison
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Department of Microbiology and Immunology, University of Leicester, Medical School, University Road, Leicester LE1 9HN, UK.
Marya
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Heaphy
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D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D015683:Gene Products, gag / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005982:Glutathione Transferase / Q000235:genetics / Q000302:isolation & purification; D015497:HIV-1 / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides; D011498:Protein Precursors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D012367:RNA, Viral / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012260:Ribonucleases / Q000378:metabolism; D013329:Structure-Activity Relationship
D015683:Gene Products, gag; D009838:Oligodeoxyribonucleotides; D011498:Protein Precursors; D012333:RNA, Messenger; D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; C059767:p55 gag precursor protein, Human immunodeficiency virus 1; D005982:Glutathione Transferase; D012260:Ribonucleases