The interaction between NC(p7) 1-55 and p6 may regulate interactions with nucleic acids during assembly through modulation of Gag folding
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We present the solution structures of HIV-1 proteins NC(p7) 1-55 corresponding to the full-length NC(p7) and mature p6. The studies were carried in water and, to mimic the membrane, in micellar DPC (Dodecylphosphocholine) conditions. Our results unravel for the first time the structure adopted by the N-terminal amino acids of the free NC(p7) 1-55 , with the formation of a small helix spanning residues F6 to R10. Our NMR and Fluorescence Anisotropy data disclose an interaction between NC(p7) 1-55 and p6 both in water and DPC, with respective K d of 2.5mM and 370 mM at 23°C. The interaction is thus strengthened in lipidic conditions. Protein p6 stabilizes the N-terminus of NC(p7) 1-55 while increasing at the same time the dynamic of the first zinc finger. Although the entire p6 sequence is involved in the interaction, we show that its C-terminal region is particularly sensitive to the presence of NC(p7) 1-55 , with a propensity of forming a α helix ranging from amino acids S111 to F116. This study brings experimental evidence of a direct protein-protein interaction between p6 and the N-terminal region of NC(p7) 1-55 . We further show that such interaction is readily accommodated within the NC(p15) framework and hypothesize that it may facilitate the selective assembly of assembly of the viral genomic RNA (gRNA) in the cell.
IMPORTANCE
Using NMR and Fluorescence Anisotropy spectroscopies, we bring the first experimental demonstration of the formation of α helix in the N-terminus of NC(p7) 1-55 and of an interaction between the NC(p7) 1-55 and p6 proteins, both derived from the HIV-1 Gag polyprotein. Such interaction stabilizes the preexisting N-terminal helix while increasing the dynamics of the first zinc finger of NC(p7) 1-55 . The C-terminus of p6 also undergoes conformational changes leading to the formation of a helix. The in vitro interaction is strengthened in DPC conditions above the CMC compared to water, suggesting that it could occur in vivo in the vicinity of the membrane. The intramolecular interaction between NC(p7) and p6 is readily accommodated in a 3D model of NC(p15). This suggests that p6 could modulate the recognition of the viral genomic RNA (gRNA) by interacting with NC(p7) 1-55 during assembly and be a key player in this process.
