Impact of capsid conformation and Rep-capsid interactions on adeno-associated virus type 2 genome packaging.
Single-stranded genomes of adeno-associated virus (AAV) are packaged into preformed capsids. It has been proposed that packaging is initiated by interaction of genome-bound Rep proteins to the capsid, thereby targeting the genome to the portal of encapsidation. Here we describe a panel of mutants with amino acid exchanges in the pores at the fivefold axes of symmetry on AAV2 capsids with reduced packaging and reduced Rep-capsid interaction. Mutation of two threonines at the rim of the fivefold pore nearly completely abolished Rep-capsid interaction and packaging. This suggests a Rep-binding site at the highly conserved amino acids at or close to the pores formed by the capsid protein pentamers. A different mutant (P. Wu, W. Xiao, T. Conlon, J. Hughes, M. Agbandje-McKenna, T. Ferkol, T. Flotte, and N. Muzyczka, J. Virol. 74:8635-8647, 2000) with an amino acid exchange at the interface of capsid protein pentamers led to a complete block of DNA encapsidation. Analysis of the capsid conformation of this mutant revealed that the pores at the fivefold axes were occupied by VP1/VP2 N termini, thereby preventing DNA introduction into the capsid. Nevertheless, the corresponding capsids had more Rep proteins bound than wild-type AAV, showing that correct Rep interaction with the capsid depends on a defined capsid conformation. Both mutant types together support the conclusion that the pores at the fivefold symmetry axes are involved in genome packaging and that capsid conformation-dependent Rep-capsid interactions play an essential role in the packaging process.
Bleker
Svenja
S
German Cancer Research Centre, Infection and Cancer Research Program, Division of Tumor Virology, Im Neuenheimer Feld 242, 69120 Heidelberg, Germany.
Pawlita
Michael
M
Kleinschmidt
Jürgen A
JA
D000595:Amino Acid Sequence; D002213:Capsid / Q000378:metabolism*; D036022:Capsid Proteins / Q000737:chemistry / Q000235:genetics; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000378:metabolism*; D000229:Dependovirus / Q000235:genetics / Q000502:physiology*; D016679:Genome, Viral; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D016415:Sequence Alignment; D013912:Threonine / Q000235:genetics; D014764:Viral Proteins / Q000378:metabolism*; D019065:Virus Assembly
D036022:Capsid Proteins; D004268:DNA-Binding Proteins; D014764:Viral Proteins; C068734:rep proteins, Adeno-associated virus 2; D013912:Threonine