Expression of heterologous genes in the Amsacta moorei entomopoxvirus.
Spheroidin (SPH) is the most abundant late protein in cells infected with the Amsacta moorei entomopoxvirus (AMEV). This locus can be used for expression of exogenous genes because it is not essential for virus replication. The sph promoter contains a conserved TAAATG motif, which serves as the site of initiation for both transcription and translation. Additional sequences downstream of the conserved motif have been shown to be involved in high-level expression of the sph gene. As a first step towards developing a protein expression vector based on the sph locus, four recombinant AMEV viruses expressing either gfp or lacZ were constructed. Both reporter genes were expressed under the control of the sph promoter containing the TAAATG motif. An additional 6 bp or 21 bp of sph coding region was included in three of the recombinants, to be expressed as an N-terminal fusion protein of GFP or LacZ. GFP and beta-galactosidase expression was observed at 2 days post-infection and continued throughout the observation period. The highest level of reporter gene expression was observed in the recombinant containing 21 bp from the sph coding region. These results indicate that sph locus of AMEV can be used successfully to express exogenous genes.
Perera
Srini C
SC
Laboratory for Molecular Virology, Great Lakes Forestry Centre, 1219 Queen St E, Sault Ste Marie, ON P6A 2E5, Canada. Anjali.Perera@NRCan-RNCan.gc.ca
Wong
Philip
P
Krell
Peter J
PJ
Arif
Basil M
BM
D000818:Animals; D002460:Cell Line; D017996:Entomopoxvirinae / Q000235:genetics*; D015870:Gene Expression; D017930:Genes, Reporter; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D007915:Lepidoptera / Q000821:virology; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis*; D015678:Viral Structural Proteins / Q000235:genetics; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics
D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D015678:Viral Structural Proteins; C063744:spheroidin protein, Entomopoxvirus; D049452:Green Fluorescent Proteins; D001616:beta-Galactosidase