Mapping the RNA binding sites for human immunodeficiency virus type-1 gag and NC proteins within the complete HIV-1 and -2 untranslated leader regions.
Encapsidation of HIV-1 genomic RNA is mediated by specific interactions between the RNA packaging signal and the Gag protein. During maturation of the virion, the Gag protein is processed into smaller fragments, including the nucleocapsid (NC) domain which remains associated with the viral genomic RNA. We have investigated the binding of glutathione- S -transferase (GST) Gag and NC fusion proteins from HIV-1, to the entire HIV-1 and -2 leader RNAencompassing the packaging signal. We have mapped the binding sites at conditions where only about two complexes are formed and find that GST-Gag and GST-NC fusion proteins bind specifically to discrete sites within the leader. Analysis of the HIV-1 leader indicated that GST-Gag strongly associates with the PSI stem-loop and to a lesser extent with regions near the primer binding site. GST-NC binds the same regions but with reversed preferences. The HIV-1 proteins also interact specifically with the 5'-leader of HIV-2 and the major site of interaction mapped to a stem-loop, with homology to the HIV-1 PSI stem-loop structure. The different specificities of Gag and NC may reflect functionally distinct roles in the viral replication, and suggest that the RNA binding specificity of NC is modulated by its structural context.
Damgaard
C K
CK
Department of Molecular and Structural Biology, University of Aarhus, C.F. Mollers Allé, Building 130,DK-8000 Aarhus C, Denmark.
Dyhr-Mikkelsen
H
H
Kjems
J
J
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D015683:Gene Products, gag / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000235:genetics* / Q000378:metabolism* / Q000502:physiology; D015498:HIV-2 / Q000235:genetics* / Q000378:metabolism* / Q000502:physiology; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D019590:Nucleocapsid Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010449:Peptide Mapping; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication
D017931:DNA Primers; D015683:Gene Products, gag; D019590:Nucleocapsid Proteins; D012367:RNA, Viral; D011993:Recombinant Fusion Proteins