Specific binding of human immunodeficiency virus type 1 gag polyprotein and nucleocapsid protein to viral RNAs detected by RNA mobility shift assays.
Packaging of retroviral genomic RNA during virion assembly is thought to be mediated by specific interactions between the gag polyprotein and RNA sequences (often termed the psi or E region) near the 5' end of the genome. For many retroviruses, including human immunodeficiency virus type 1 (HIV-1), the portions of the gag protein and the RNA that are required for this interaction remain poorly defined. We have used an RNA gel mobility shift assay to measure the in vitro binding of purified glutathione S-transferase-HIV-1 gag fusion proteins to RNA riboprobes. Both the complete gag polyprotein and the nucleocapsid (NC) protein alone were found to bind specifically to an HIV-1 riboprobe. Either Cys-His box of NC could be removed without eliminating specific binding to the psi riboprobe, but portions of gag containing only the MA and CA proteins without NC did not bind to RNA. There were at least two binding sites in HIV-1 genomic RNA that bound to the gag polyprotein: one entirely 5' to gag and one entirely within gag. The HIV-1 NC protein bound to riboprobes containing other retroviral psi sequences almost as well as to the HIV-1 psi riboprobe.
Berkowitz
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RD
Department of Biochemistry and Molecular Biophysics, College of Physicians and Surgeons, Columbia University, New York, New York 10032.
Luban
J
J
Goff
S P
SP
D000595:Amino Acid Sequence; D001665:Binding Sites; D015153:Blotting, Western; D002213:Capsid / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D015683:Gene Products, gag / Q000235:genetics / Q000378:metabolism*; D005982:Glutathione Transferase / Q000235:genetics; D015497:HIV-1 / Q000378:metabolism*; D008969:Molecular Sequence Data; D009068:Movement; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D015347:RNA Probes; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014758:Viral Core Proteins / Q000378:metabolism*
D015683:Gene Products, gag; D011498:Protein Precursors; D015347:RNA Probes; D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; D014758:Viral Core Proteins; D005982:Glutathione Transferase