Packaging of AeDNV–GFP Transducing Virus by Expression of Densovirus Structural Proteins from a Sindbis Virus Expression System

AbstractGenetic recombination resulting in the production of wild-type infectious virus is an obstacle in the current system for producing densovirus transducing particles. In order to eliminate this problem, a double subgenomic Sindbis virus (TE/3′2J/VP) was engineered that expresses the structural proteins (VPs) ofAedesdensonucleosis virus (AeDNV) from the second subgenomic promoter. Expression of AeDNV VPs from TE/3′2J/VP was confirmed by Northern analysis of RNA from infected C6/36 (Aedes albopictus) cells and by indirect immunofluorescence in infected C6/36 cells and BHK-21 cells. TE/3′2J/VP was used to infect C6/36 cells transfected with p7NS1–GFP, a plasmid expressing the nonstructural genes of AeDNV and green fluorescent protein (GFP) as a reporter gene. This infection resulted in the production of AeDNV–GFP transducing virus, which is infectious to C6/36 cells andAedes aegyptilarvae, as determined by GFP expression. The TE/3′2J/VP packaging system produced titers of transducing virus comparable to those produced by the standard two-plasmid method. The possibility of recombination resulting in wild-type infectious virus in transducing densovirus stocks was eliminated by employing an RNA virus expression system to supply AeDNV structural proteins