Densovirus of Aedes aegypti as an expression vector in mosquito cells.
We have constructed an infectious DNA clone containing the genome of Aedes aegypti densovirus (AeDNV) in a bacterial plasmid. When this clone was transfected into Aedes albopictus C6/36 mosquito cells, the AeDNV genome rescued from the plasmid and replicated as the wild-type virus. To investigate the cloned virus as an expression vector, the reporter gene encoding beta-galactosidase (beta-gal) was inserted into four large open reading frames (ORF) observed in the AeDNV genome. When these recombinant constructs were transfected into Aedes albopictus C6/36 cells, the beta-gal was expressed efficiently from the right ORF (encoding capsid proteins, Vps) and the mid ORF (encoding putative nonstructural protein 2). A low level of expression was found from the left ORF (encoding nonstructural protein 1, NS1), and no expression was detected from the ORF observed on the minus strand of the AeDNV genome. The expression from the right, mid, and left ORFs can be trans-activated with NS1. A putative nuclear targeting sequence observed in the N-terminus of the AeDNV Vps is presumed to be responsible for transport of the chimeric beta-gal into nucleus. The recombinant AeDNV genomes (carrying the beta-gal gene) supplied with the AeDNV capsid proteins can be packaged into infectious transducing particles. Our results indicate that the genome of AeDNV can serve as a vector for delivery and expression of foreign genes in mosquito cells with subsequent targeting of the product to the desired cell compartment.
Engelhardt Institute of Molecular Biology, Moscow, Russia.
B N
BN
Afanasiev
Y V
YV
Kozlov
J O
JO
Carlson
B J
BJ
Beaty
D000330:Aedes / Q000382:microbiology*; D000595:Amino Acid Sequence; D000818:Animals; D015139:Blotting, Southern; D002460:Cell Line; D003001:Cloning, Molecular; D004261:DNA Replication; D004274:DNA, Recombinant; D004279:DNA, Viral / Q000032:analysis; D017990:Densovirus / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005260:Female; D015967:Gene Expression Regulation, Viral; D005798:Genes, Bacterial; D005822:Genetic Vectors; D016679:Genome, Viral; D008297:Male; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010957:Plasmids; D011401:Promoter Regions, Genetic; D014161:Transduction, Genetic; D014162:Transfection; D014764:Viral Proteins / Q000737:chemistry; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics
D004274:DNA, Recombinant; D004279:DNA, Viral; D014764:Viral Proteins; D001616:beta-Galactosidase