Cryo-EM Reveals VP1/VP2 N-Terminal Conformations in Adeno-Associated Virus 2 Capsids.
- 1Department of Biological Science, Florida State University, Tallahassee, Florida, USA., Institute of Molecular Biophysics, Florida State University, Tallahassee
Abstract
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.
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BibTeX
@misc{osuya2026cryoem,
title = {{Cryo-EM Reveals VP1/VP2 N-Terminal Conformations in Adeno-Associated Virus 2 Capsids.}},
author = {I. O. Osuya and S. S. Kamalaldinezabadi and A. Sengupta and G. Seo and M. S. Chapman and S. M. Stagg},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.16.759763},
url = {https://www.biorxiv.org/content/10.64898/2026.10.16.759763v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - Cryo-EM Reveals VP1/VP2 N-Terminal Conformations in Adeno-Associated Virus 2 Capsids.
AU - I. O. Osuya
AU - S. S. Kamalaldinezabadi
AU - A. Sengupta
AU - G. Seo
AU - M. S. Chapman
AU - S. M. Stagg
PY - 2026
DA - 2026/10/16
PB - bioRxiv
DO - 10.64898/2026.10.16.759763
UR - https://www.biorxiv.org/content/10.64898/2026.10.16.759763v1
AB - 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.
ER -