Stem-loop SL4 of the HIV-1 Ψ RNA packaging signal exhibits weak affinity for the nucleocapsid protein. Structural studies and implications for genome recognition

Gaya K. Amarasinghe, Jing Zhou, Matthew Miskimon, Kalola J. Chancellor, Jasmine A. McDonald, Akida G. Matthews, Robyn R. Miller, Michael D. Rouse, Michael F. Summers

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77 Scopus citations

Abstract

Encapsidation of the genome of the human immunodeficiency virus type-1 (HIV-1) during retrovirus assembly is mediated by interactions between the nucleocapsid (NC) domains of assembling Gag polyproteins and a ∼110 nucleotide segment of the genome known as the Ψ-site. The HIV-1 Ψ-site contains four stem-loops (SL1 through SL4), all of which are important for genome packaging. Recent isothermal titration calorimetry (ITC) studies have demonstrated that SL2 and SL3 are capable of binding NC with high affinity (Kd∼ 140 nM), consistent with proposals for protein-interactive functions during packaging. To determine if SL4 may have a similar function, NC-interactive studies were conducted by NMR and gel-shift methods. In contrast to previous reports, we find that SL4 binds weakly to NC (Kd= (±14 μM), suggesting an alternative function. NMR studies indicate that the GAGA tetraloop of SL4 adopts a classical GNRA-type fold (R = purine, N = G, C, A or U), a motif that stabilizes RNA tertiary structures in other systems. In combination with previously reported gel mobility studies of Ψ-site deletion mutants, these findings suggest that SL4 functions in genome recognition not by binding to Gag, but by stabilizing the structure of the Ψ-site. Differences in the affinities of NC for SL2, SL3 and SL4 stem-loops can now be rationalized in terms of the different structural properties of stem loops that contain GGNG (SL2 and SL3) and GNRA (SL4) sequences.

Original languageEnglish
Pages (from-to)961-970
Number of pages10
JournalJournal of Molecular Biology
Volume314
Issue number5
DOIs
StatePublished - Dec 14 2001

Keywords

  • Human immunodeficiency virus
  • NMR
  • Nucleocapsid protein
  • Psi-site
  • RNA

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