Packaging of AeDNV-GFP transducing virus by expression of densovirus structural proteins from a Sindbis virus expression system

Genetic recombination resulting in the production of wild-type infectious v irus is an obstacle in the current system for producing densovirus transduc ing particles. In order to eliminate this problem, a double subgenomic Sind bis virus (TE/3'2J/VP) was engineered that expresses the structural protein s (VPs) of Aedes densonucleosis virus (AeDNV) from the second subgenomic pr omoter. Expression of AeDNV VPs from TE/3'2J/VP was confirmed by Northern a nalysis 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 wa s used to infect C6/36 cells transfected with p7NS1-GFP, a plasmid expressi ng the nonstructural genes of AeDNV and green fluorescent protein (GFP) as a reporter gene. This infection resulted in the production of AeDNV-GFP tra nsducing virus, which is infectious to C6/36 cells and Aedes aegypti larvae , as determined by GFP expression. The TE/3'2/VP packaging system produced titers of transducing virus comparable to those produced by the standard tw o-plasmid method. The possibility of recombination resulting in wild-type i nfectious virus in transducing densovirus stocks was eliminated by employin g an RNA virus expression system to supply AeDNV structural proteins. (C) 1 999 Academic Press.