Extended Data Fig. 4: Negative stain electron micrographs of mutant HIV-1 capsids.

a, Wildtype or mutant CA was recombinantly expressed, purified, and allowed to assemble into cone-shaped CLPs (in the presence of IP6; see Methods). The assemblies were applied to a Superose 6 size exclusion column, where CLPs elute close to the void volume. This can be considered as a first criterion for a faithful CLP assembly. The corresponding fractions were pooled and further analyzed by negative stain electron microscopy. Representative images are shown in this panel and confirm the proper assembly of all mutants for which we describe a transport phenotype. Several other mutants showed defects in capsid assembly (for example, Q7E, M10E/K, V11E, P122E, I124E). These were excluded from the analysis. Experiments were independently replicated three times, yielding consistent outcomes. b, Wildtype and mutant pentamer-only spheres were assembled as analyzed in a, the differences being (1) that the CA protein contained in addition the G60A+G61P double mutation, which is required to assemble pentamer-only spheres⁸³, (2) that the assemblies were smaller than the CLPs (20 nm instead of 100-200 nm), and (3) that these smaller assemblies also eluted later from the Superose 6 column. Scale bars, 200 nm. Experiments were independently replicated two times with consistent outcomes.