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.