Assembly of adeno-associated virus type 2 capsids in vitro.
Capsid proteins VP1, VP2 and VP3 of adeno-associated virus type 2 (AAV-2) were separately expressed by recombinant baculoviruses, purified under denaturing conditions and renatured in the presence of 0.5 M arginine, followed by dialysis against buffers of physiological ionic strength. At a protein concentration of 0.05 mg/ml, the three capsid proteins predominantly formed monomers and, to a lesser extent, oligomers, as determined by sedimentation analysis. Oligomerization increased at higher protein concentrations. The capsid protein oligomers consisted of globular, non-capsid-like structures, as detected by electron microscopy. Addition of a HeLa cell extract significantly stimulated oligomerization of the capsid proteins, probably due to interactions with HeLa cell proteins. Characterization of structures sedimenting around 60S by immunoprecipitation and electron microscopy showed that, in addition to other aggregates, empty capsid-like structures were formed in vitro. The identity of these structures as empty AAV capsids was verified by immunoelectron microscopy. Analysis of capsid formation in HeLa cells by transfection of VP expression constructs allowing separate expression of VP1, VP2 and VP3 showed that they were able to form capsids, although with a reduced efficiency as compared to VP proteins expressed from the wt cap gene. This finding suggests that the mutations introduced to allow separate capsid protein expression reduced the efficiency of capsid assembly in vivo and might also explain the reduced recovery of empty capsids employing the in vitro assembly procedure.
Steinbach
S
S
Deutsches Krebsforschungszentrum, Forschungsschwerpunkt Angewandte Tumorvirologie, Heidelberg, Germany.
Wistuba
A
A
Bock
T
T
Kleinschmidt
J A
JA
D000818:Animals; D002213:Capsid / Q000737:chemistry / Q000502:physiology*; D002478:Cells, Cultured; D000229:Dependovirus / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D011994:Recombinant Proteins; D018411:Spodoptera; D019065:Virus Assembly
D011994:Recombinant Proteins