Fig 6. Favourable interaction energetics between high-order Pr55^Gag oligomers and Adenosine-containing RNA persists with authentic HIV A-containing sequences.

(A) On the left is a top-down view of the published immature Pr55^Gag capsid hexamer cryo-EM structure (PDB ID:4usn) [32]. Intra-hexameric subunits are coloured in alternating green-cyan and dark green. Helix6 mutations (TTSTLQ 239–44 AASALA) disrupting intra-hexameric interactions of the NTD-capsid are shown as spheres and coloured in magenta. Helix10 mutation (D329A) and MHR mutation (K290A) disrupting intra-hexameric interactions of the CTD-capsid are shown as spheres and coloured in orange and blue-white, respectively. On the right is the side view of immature Pr55^Gag capsid. The WM mutation (WM316-7AA) disrupting inter-hexameric dimerization is highlighted in blue. Pr55^Gag (CA All 4) contains 4 sets of mutations that disrupt CA oligomerization (Helix6, Helix10, WM and MHR) [28]. ITC binding curves indicating heat released per mole of oligonucleotide titrated when 40 μM of 4x 5’-GAUGG-3’ (GU-containing) and 4x 5’-GAGAA-3’ (A-containing) RNA were injected in 1.5 ul aliquots into 8 μM of Pr55^Gag (CA Helix 6) (Fig 6A left panel, dark pink and light pink), Pr55^Gag (CA Helix 10) (Fig 6A middle panel, dark yellow and light yellow), and Pr55^Gag (CA All 4) (Fig 6A right panel, red-brown and purple). (n = 3) (B) Representative ITC binding curves indicating heat released per mole of oligonucleotide titrated when 40 μM of 4x 5’-GAUGG-3’ (GU-containing) and 4x 5’-GAGAA-3’ (A-containing) RNA were injected in 1.5 ul aliquots into 8 μM of Pr55^Gag-Tev (Fig 6B left panel, dark blue and light blue), Pr50^{Gag Δp6-TEV} (Fig 6B middle panel, dark orange and light orange), and Pr55^{Gag WM-TEV} (Fig 6B right panel, green and dark yellow). (n = 3) (C) Representative ITC binding curves indicating heat released per mole of oligonucleotide titrated when 40 μM of 5’- CTTAGAAATAGGGCAGCATAGAACAAAAATAGAGGAA-3’ (HIV_NL4.3RNA_2671-2710) and 5’- CTATCTTTTAGGGCAGCAATCTTCAAAAATAGTCCTT-3’ (HIV_NL4.3RNA_2671-2710 with AGAAA mutation) RNA were injected in 1.5 ul aliquots into 8 μM of Pr55^Gag-Tev (Fig 6C left panel, dark blue and light blue), Pr50^{Gag Δp6-TEV} (Fig 6C middle panel, dark orange and light orange), and Pr55^{Gag WM-TEV} (Fig 6C right panel, green and dark yellow). (n = 3)