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Structure paper

TitleStructural basis for Rep-mediated adeno-associated virus packaging.
Journal, issue, pagesCell Rep, Vol. 45, Issue 3, Page 117044, Year 2026
Publish dateMar 5, 2026
AuthorsVictoria Xu / Aiysha McInnes / Matthew Wake / Marta Acebrón-García-de-Eulate / Joseph D Barritt / Doryen Bubeck / Sarah L Rouse /
PubMed AbstractAdeno-associated viruses (AAVs) are parvoviruses utilized as gene therapy vectors. However, the AAV packaging mechanism is unresolved at the molecular level, creating a bottleneck for vector ...Adeno-associated viruses (AAVs) are parvoviruses utilized as gene therapy vectors. However, the AAV packaging mechanism is unresolved at the molecular level, creating a bottleneck for vector manufacturing, safety, and efficacy. Here, cryo-EM structures of the Rep helicase packaging motor in complex with the packaging marker DNA (ITR) and the Rep-AAV8 capsid complex are presented. Rep-ITR complexes reveal dynamic oligomeric states on the DNA, elucidating the strand separation mechanism coupled to its ATPase cycle. We observe Rep preferentially bound to empty capsids, with a binding interface likely conserved across the virus family. This complex also unveils a cryptic capsid ATP-binding site which, alongside Rep binding, triggers structural rearrangements priming the capsid for packaging. Collectively, these findings advance the understanding of Rep-mediated packaging, with significant implications for parvovirus virology and viral vector design.
External linksCell Rep / PubMed:41790558
MethodsEM (single particle)
Resolution1.88 - 3.08 Å
Structure data

54050

9rm5

EMDB-54050, PDB-9rm5:
Hexameric AAV2 Rep40-ssDNA (ITR) complex in presence of ATPyS
Method: EM (single particle) / Resolution: 2.44 Å

EMDB-54181: Consensus map of heptameric Rep40-dsDNA (ITR) in presence of ATPyS
Method: EM (single particle) / Resolution: 2.75 Å

EMDB-54182: Focused map of 3 subunits of Rep40 +dsDNA (ITR) in complex with ATPgS
Method: EM (single particle) / Resolution: 2.63 Å

EMDB-54183: Focused map of 4 subunits of heptameric Rep40-dsDNA (ITR) in complex with ATPgS
Method: EM (single particle) / Resolution: 2.96 Å

54184

9rqt

EMDB-54184, PDB-9rqt:
Heptameric AAV2 Rep40-dsDNA (ITR) complex in presence of ATPgS
Method: EM (single particle) / Resolution: 2.88 Å

54195

9rrs

EMDB-54195, PDB-9rrs:
AAV8 capsid in complex with Rep40 and ADP
Method: EM (single particle) / Resolution: 1.88 Å

54328

9rwg

EMDB-54328, PDB-9rwg:
Pentameric AAV2 Rep40 in complex with AAV8
Method: EM (single particle) / Resolution: 2.19 Å

EMDB-54403: Consensus map of Hexameric AAV2 Rep40-dsDNA (ITR) duplex complex in presence of ATPyS
Method: EM (single particle) / Resolution: 3.07 Å

54416

9s0n

EMDB-54416, PDB-9s0n:
Hexameric AAV2 Rep40-dsDNA (ITR) melting complex in presence of ATPyS
Method: EM (single particle) / Resolution: 3.08 Å

54429

9s10

EMDB-54429, PDB-9s10:
Hexameric AAV2 Rep40-dsDNA (ITR) duplex complex in presence of ATPyS
Method: EM (single particle) / Resolution: 3.07 Å

Chemicals

ChemComp-AGS:
PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-gamma-S, energy-carrying molecule analogue*YM

ChemComp-MG:
Unknown entry

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM

ChemComp-HOH:
WATER

Source
  • adeno-associated virus 2
  • dna molecule (others)
  • adeno-associated virus 8
KeywordsDNA BINDING PROTEIN / Helicase / Adeno-associated Virus / AAA+ ATPase / VIRUS / AAV

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