Osuya, I. O.; Kamalaldinezabadi, S. S.; Sengupta, A.; Seo, G.; Chapman, M. S.; Stagg, S. M.

The AAV2 capsid assembles from three viral proteins, VP1, VP2, and VP3, expressed from a single cap gene. In the assembled T=1 60-subunit icosahedral capsid, they have a canonical stoichiometry of 1:1:10. Relative to VP3, VP1 and VP2 have N-terminal extensions that are required for endosomal escape and nuclear trafficking; however, these regions remain poorly resolved due to low stoichiometry, symmetry mismatches, and conformational heterogeneity of their N-termini. Here, we generated empty AAV2 capsids using a baculovirus expression system with time-course harvests, yielding approximately 2:3:5 and 1:2:7. VP1:VP2:VP3 ratios on days 3 and 5 post-infection, respectively. Cryo-EM single-particle and subparticle reconstructions revealed distinct N-terminal configurations associated with these different VP stoichiometries. Day 3 capsids contained an internal density protruding from the twofold axis and lacked the ordered {beta}A preceding residue 237, whereas day 5 capsids lacked the twofold protrusion and instead showed density associated with the fivefold axis, with {beta}A ordered to approximately residues 219-221. Fivefold subparticle reconstruction of day 5 capsids further resolved a density plug occluding the pore and an underlying density ring. These findings link VP stoichiometry to distinct conformations of the VP1/VP2 N-termini and provide structural evidence for alternative organization of these regions within the AAV2 capsid, offering new insights into the conformational landscape of AAV capsid heterogeneity.