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 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) of Aedes densonucleosis 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 and Aedes aegypti larvae, 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.
Allen-Miura
T M
TM
Department of Microbiology, Colorado State University, Fort Collins, Colorado, 80523, USA.
Afanasiev
B N
BN
Olson
K E
KE
Beaty
B J
BJ
Carlson
J O
JO
D000330:Aedes / Q000821:virology*; D000818:Animals; D002478:Cells, Cultured; D017990:Densovirus / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D015967:Gene Expression Regulation, Viral; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics; D011993:Recombinant Fusion Proteins; D012845:Sindbis Virus / Q000378:metabolism*; D014162:Transfection / Q000379:methods; D015678:Viral Structural Proteins / Q000096:biosynthesis / Q000235:genetics*
D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D015678:Viral Structural Proteins; D049452:Green Fluorescent Proteins