NMR structure of the HIV-1 nucleocapsid protein bound to stem-loop SL2 of the Psi-RNA packaging signal. Implications for genome recognition

The RNA genome of the human immunodeficiency virus type-1 (HIV-1) contains a similar to 120 nucleotide Psi-packaging signal that is recognized by the nucleocapsid (NC) domain of the Gag polyprotein during virus assembly. The Psi-site contains four stem-loops (SL1-SL4) that possess overlapping and po ssibly redundant functions. The present studies demonstrate that the 19 res idue SL2 stem-loop binds NC with affinity (K-d=110(+/-50) nM) similar to th at observed for NC binding to SL3 (K-d = 170(+/-65) nM) and tighter than ex pected on the basis of earlier work, suggesting that NC-SL2 interactions pr obably play a direct role in the specific recognition and packaging of the full-length, unspliced genome. The structure of the NC-SL2 complex was dete rmined by heteronuclear NMR methods using N-15,C-13-isotopically labeled NC protein and SL2 RNA. The N and C-terminal "zinc knuckles" (Cys-X-2-Cys-X-4 -His-X-4-Cys; X = variable amino acid) of HIV-1 NC bind to exposed guanosin e bases G9 and G11, respectively, of the G8-G9-U10-G11 tetraloop, and resid ues Lys3-Lys11 of the N-terminal tail forms a 3(10) helix that packs agains t the proximal zinc knuckle and interacts with the RNA stem. These structur al features are similar to those observed previously in the NMR structure o f NC bound to SL3. Other features of the complex are substantially differen t. In particular, the N-terminal zinc knuckle interacts with an A-U-A base triple platform in the minor groove of the SL2 RNA stem, but binds to the m ajor groove of SL3. Ln addition, the relative orientations of the N and C-t erminal zinc knuckles differ in the NC-SL2 and NC-SL3 complexes, and the si de-chain of Phe6 makes minor groove hydrophobic contacts with G11 in the NC -SL2 complex but does not interact with RNA in the NC-SL3 complex. Finally, the N-terminal helix of NC interacts with the phosphodiester backbone of t he SL2 XNA stem mainly via electrostatic interactions, but does not bind in the major groove or make specific H-bonding contacts as observed in the NC -SL3 structure. These findings demonstrate that NC binds in an adaptive man ner to SL2 and SL3 via different subsets of inter and intra-molecular inter actions, and support a genome recognition/packaging mechanism that involves interactions of two or more NC domains of assembling HIV-1 Gag molecules w ith multiple Psi-site stem-loop packaging elements during the early stages of retrovirus assembly. (C) 2000 Academic Press.