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- PDB-7mt0: Structure of the adeno-associated virus 9 capsid at pH 7.4 -

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Basic information

Entry
Database: PDB / ID: 7mt0
TitleStructure of the adeno-associated virus 9 capsid at pH 7.4
ComponentsCapsid protein VP1
KeywordsVIRUS LIKE PARTICLE / adeno-associated virus / pH / phospholipase-A2 / capsid / gene therapy / AAV9 / dependoparvovirus / intracellular trafficking / endosome
Function / homologyPhospholipase A2-like domain / Phospholipase A2-like domain / Parvovirus coat protein VP2 / Parvovirus coat protein VP1/VP2 / Parvovirus coat protein VP2 / Capsid/spike protein, ssDNA virus / T=1 icosahedral viral capsid / structural molecule activity / Capsid protein VP1
Function and homology information
Biological speciesAdeno-associated virus 9
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.82 Å
AuthorsPenzes, J.J. / Chipman, P. / Bhattacharya, N. / Zeher, A. / Huang, R. / McKenna, R. / Agbandje-McKenna, M.
Funding support United States, 5items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM109524 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM082946 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)S10 OD018142 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)S10 RR025080 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)U24 GM116788 United States
CitationJournal: J Virol / Year: 2021
Title: Adeno-associated Virus 9 Structural Rearrangements Induced by Endosomal Trafficking pH and Glycan Attachment.
Authors: Judit J Penzes / Paul Chipman / Nilakshee Bhattacharya / Allison Zeher / Rick Huang / Robert McKenna / Mavis Agbandje-McKenna /
Abstract: Adeno-associated viruses (AAVs) are small nonenveloped single-stranded DNA (ssDNA) viruses that are currently being developed as gene therapy biologics. After cell entry, AAVs traffic to the nucleus ...Adeno-associated viruses (AAVs) are small nonenveloped single-stranded DNA (ssDNA) viruses that are currently being developed as gene therapy biologics. After cell entry, AAVs traffic to the nucleus using the endo-lysosomal pathway. The subsequent decrease in pH triggers conformational changes to the capsid that enable the externalization of the capsid protein (VP) N termini, including the unique domain of the minor capsid protein VP1 (VP1u), which permits the phospholipase activity required for the capsid lysosomal egress. Here, we report the AAV9 capsid structure, determined at the endosomal pHs (7.4, 6.0, 5.5, and 4.0), and terminal galactose-bound AAV9 capsids at pHs 7.4 and 5.5 using cryo-electron microscopy and three-dimensional image reconstruction. Taken together, these studies provide insight into AAV9 capsid conformational changes at the 5-fold pore during endosomal trafficking, in both the presence and absence of its cellular glycan receptor. We visualized, for the first time, that acidification induces the externalization of the VP3 and possibly VP2 N termini, presumably in prelude to the externalization of VP1u at pH 4.0, which is essential for lysosomal membrane disruption. In addition, the structural study of AAV9-galactose interactions demonstrates that AAV9 remains attached to its glycan receptor at the late endosome pH 5.5. This interaction significantly alters the conformational stability of the variable region I of the VPs, as well as the dynamics associated with VP N terminus externalization. There are 13 distinct Adeno-associated virus (AAV) serotypes that are structurally homologous and whose capsid proteins (VP1 to -3) are similar in amino acid sequence. However, AAV9 is one of the most commonly studied and is used as a gene therapy vector. This is partly because AAV9 is capable of crossing the blood-brain barrier and readily transduces a wide array of tissues, including the central nervous system. In this study, we provide AAV9 capsid structural insight during intracellular trafficking. Although the AAV capsid has been shown to externalize the N termini of its VPs, to enzymatically disrupt the lysosome membrane at low pH, there was no structural evidence to confirm this. By utilizing AAV9 as our model, we provide the first structural evidence that the externalization process occurs at the protein interface at the icosahedral 5-fold symmetry axis and can be triggered by lowering the pH.
History
DepositionMay 12, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 2, 2021Provider: repository / Type: Initial release
Revision 1.1Jul 28, 2021Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID
Revision 1.2Sep 22, 2021Group: Database references / Category: citation / citation_author / database_2
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.title / _citation_author.identifier_ORCID / _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Assembly

Deposited unit
A: Capsid protein VP1
B: Capsid protein VP1
C: Capsid protein VP1
D: Capsid protein VP1
E: Capsid protein VP1
F: Capsid protein VP1
G: Capsid protein VP1
H: Capsid protein VP1
I: Capsid protein VP1
J: Capsid protein VP1
K: Capsid protein VP1
L: Capsid protein VP1
M: Capsid protein VP1
N: Capsid protein VP1
O: Capsid protein VP1
P: Capsid protein VP1
Q: Capsid protein VP1
R: Capsid protein VP1
S: Capsid protein VP1
T: Capsid protein VP1
U: Capsid protein VP1
V: Capsid protein VP1
W: Capsid protein VP1
X: Capsid protein VP1
Y: Capsid protein VP1
Z: Capsid protein VP1
1: Capsid protein VP1
2: Capsid protein VP1
3: Capsid protein VP1
4: Capsid protein VP1
5: Capsid protein VP1
6: Capsid protein VP1
a: Capsid protein VP1
b: Capsid protein VP1
c: Capsid protein VP1
d: Capsid protein VP1
e: Capsid protein VP1
f: Capsid protein VP1
g: Capsid protein VP1
h: Capsid protein VP1
i: Capsid protein VP1
j: Capsid protein VP1
k: Capsid protein VP1
l: Capsid protein VP1
m: Capsid protein VP1
n: Capsid protein VP1
o: Capsid protein VP1
p: Capsid protein VP1
q: Capsid protein VP1
r: Capsid protein VP1
s: Capsid protein VP1
t: Capsid protein VP1
u: Capsid protein VP1
v: Capsid protein VP1
w: Capsid protein VP1
x: Capsid protein VP1
y: Capsid protein VP1
z: Capsid protein VP1
7: Capsid protein VP1
8: Capsid protein VP1


Theoretical massNumber of molelcules
Total (without water)3,508,46560
Polymers3,508,46560
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein ...
Capsid protein VP1 /


Mass: 58474.414 Da / Num. of mol.: 60 / Fragment: UNP residues 219-736
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Adeno-associated virus 9 / Gene: cap / Plasmid: pFastBac1-HM / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q6JC40

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Adeno-associated virus 9 / Type: VIRUS / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Adeno-associated virus 9
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm) / Cell: Sf9 / Plasmid: pFastBac1-HM
Details of virusEmpty: YES / Enveloped: NO / Isolate: SEROTYPE / Type: VIRUS-LIKE PARTICLE
Natural hostOrganism: Homo sapiens
Virus shellName: AAV9 virus like particle / Diameter: 240 nm / Triangulation number (T number): 1
Buffer solutionpH: 7.4
Details: 1x Universal buffer of 20 mM Hepes, 20 mM MES, 20 mM sodium acetate, 0.15 M NaCl, 3.7 mM CaCl2
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: OTHER / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: OTHER / Cs: 2.7 mm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.10-2155_2155: / Classification: refinement
EM software
IDNameVersionCategoryDetails
2Auto3DEM4.05.2image acquisition
4Auto3DEM5.04.2CTF correctionAutopp3X subroutine
7UCSF Chimera1.13.1model fitting
12Auto3DEM5.02.43D reconstruction
13Coot0.8.9.3model refinement
CTF correctionType: PHASE FLIPPING ONLY
SymmetryPoint symmetry: I (icosahedral)
3D reconstructionResolution: 2.82 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 150469 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 3UX1
Pdb chain-ID: A
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.01255360
ELECTRON MICROSCOPYf_angle_d0.852348000
ELECTRON MICROSCOPYf_dihedral_angle_d7.669203400
ELECTRON MICROSCOPYf_chiral_restr0.05135580
ELECTRON MICROSCOPYf_plane_restr0.00546440

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