Affinities of the nucleocapsid protein for variants of SL3 RNA in HIV-1.
Efficient packaging of genomic RNA into new HIV-1 virus particles requires that nucleocapsid domains of precursor proteins bind the SL3 tetraloop (G317-G-A-G320) from the 5'-untranslated region. This paper presents the affinities of 35 RNA variants of SL3 for the mature 55mer NC protein, as measured by fluorescence quenching of tryptophan-37 in the protein by nucleobases. The 1:1 complexes that form in 0.2 M NaCl have dissociation constants ranging from 8 nM (GGUG) to 20 microM (GAUA). The highly conserved (GGAG) sequence for the wild type is not the most stable (K(d) = 28 nM), suggesting that other selective pressures beyond the stability of the complex must be satisfied. The leading requirement for strong interaction is for G320, followed closely by G318. Replacing either with U, A, or C reduces affinity by a factor of 15-120. NC-domains from multiple proteins combine to recognize unpaired G(2)-loci, where two guanines are in close proximity. We have previously measured affinities of the NC protein for the important stem-loops of the major packaging domain [Shubsda, M. F., Paoletti, A. C., Hudson, B. S., and Borer, P. N. (2002) Biochemistry 41, 5276-82]. Comparison with the present work shows that the nature of the stem also modulates NC-RNA interactions. Placing the G(2)-loci from the apical SL2 or SL1 loops on the SL3 stem increases affinity by a factor of 2-3, while placing the SL4 loop on the SL3 stem reduces affinity 50-fold. These results are interesting in the context of RNA-protein interaction, as well as for the discovery of antiNC agents for AIDS therapy.
Paoletti
Andrew C
AC
Department of Chemistry, Graduate Program in Structural Biology, Biochemistry, and Biophysics, Syracuse University, Syracuse, New York 13244-4100, USA.
Shubsda
Michael F
MF
Hudson
Bruce S
BS
Borer
Philip N
PN
D000595:Amino Acid Sequence; D020137:Base Pair Mismatch; D002213:Capsid / Q000737:chemistry*; D036022:Capsid Proteins; D004279:DNA, Viral / Q000737:chemistry; D015683:Gene Products, gag / Q000737:chemistry* / Q000235:genetics; D014644:Genetic Variation; D015497:HIV-1 / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D008969:Molecular Sequence Data; D019590:Nucleocapsid Proteins / Q000737:chemistry* / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D017433:Protein Structure, Secondary / Q000235:genetics; D020038:RNA, Spliced Leader / Q000737:chemistry* / Q000235:genetics; D012367:RNA, Viral / Q000737:chemistry*; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D014764:Viral Proteins; D019065:Virus Assembly / Q000235:genetics; D054301:gag Gene Products, Human Immunodeficiency Virus
D036022:Capsid Proteins; D004279:DNA, Viral; D015683:Gene Products, gag; C071884:NCP7 protein, Human immunodeficiency virus 1; D019590:Nucleocapsid Proteins; D020038:RNA, Spliced Leader; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D014764:Viral Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus