Fig. 5: K25A in vitro capsid assembly is rescued by compensating mutations.

A Assembly of 200 µM K25A (left) or K25T (right) CA variants in 2.5 mM NaCl, measuring absorbance over time at 350 nm. B Negative stain EM images of assembly reactions from (A). A, B WT data are reproduced in each pair of graphs to allow easier comparison with mutants. C Assembly of 200 µM K25A (left) or K25T (right) variants in 1.25 mM IP6. Where indicated by the asterisk, assembly reactions were performed in 400 µM CA and 10 mM IP6. D Negative stain EM images of assembly reactions from (C). Scalebar: 200 nm. E–G Representations of packing interactions involving capsid pentamers (8CKW) in an HIV-1 capsid assembled in the presence of IP6⁸⁷. E N21-proximal residues at the interface between neighbouring monomers in the pentamer, including the key pawl and ratchet residue M39⁴⁶. Dotted lines from N21 highlight the relative distance and positioning of nearby residues and do not necessarily indicate non-covalent interactions. F As (E), but with S21 modelled in place of N21 and T25 instead of K25. G Interaction networks that promote packing at multiple pentamer (8CKW) interfaces. Two monomers of the same pentamer are shown in orange and green and parts of monomers from adjacent capsomers in pink and blue. A proposed allosteric network connects a linear ratchet motif (V24, K25 & M49) to a ‘TVGG’ motif and gate residue M66 that modulate hexamer or pentamer formation⁴⁶. The TVGG motif sits behind a three-fold interface where T216I is located. Yellow arrows indicate that interactions at each interface determine how closely monomers pack within and between pentamers.