Genetic modification of an entomopoxvirus: deletion of the spheroidin gene does not affect virus replication in vitro.
In the late stages of an entomopoxvirus infection, virions become embedded within a crystalline occlusion body or spheroid. Spheroids are composed primarily of a single polypeptide, spheroidin. We describe the construction of a genetically modified Amsacta moorei entomopoxvirus (AmEPV) in which the spheroidin gene coding sequences are deleted and replaced with those of a heterologous reporter gene encoding chloramphenicol acetyltransferase (CAT). A transfer vector, pAmCP1, was prepared containing a unique BamHI site in lieu of the spheroidin gene coding region, together with 1 kbp of upstream and downstream DNA sequence that flanks the spheroidin gene. The flanking sequences provide the transcriptional control signals and also guide homologous recombination so that the spheroidin gene coding region can be replaced with that of the foreign gene. The transfer vector was designed so that the translational start codon of the introduced foreign gene would be utilized. A recombinant virus, AmEPV.CAT, was produced by transfecting AmEPV-infected cells with the transfer vector encoding the CAT gene. The recombinant virus was isolated from wild-type virus by identifying plaques with a spheroidin-negative phenotype. Light microscopy and SDS-PAGE analysis demonstrated that no spheroids or spheroidin protein were produced in the recombinant virus-infected cells. The recombinant virus was able to replicate to high titres (10(7) p.f.u./ml) in insect cells indicating that the spheroidin gene is non-essential for AmEPV replication in vitro. Moderate levels of CAT were synthesized in recombinant virus-infected cells and temporal analyses indicated that CAT synthesis followed the pattern of spheroidin production suggesting that the spheroidin gene promoter was functioning under normal regulatory control in the genetically modified virus.
School of Biological Science, Oxford Brookes University, UK.
C P
CP
Palmer
D P
DP
Miller
S A
SA
Marlow
L E
LE
Wilson
A M
AM
Lawrie
L A
LA
King
D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis / Q000235:genetics; D017996:Entomopoxvirinae / Q000235:genetics*; D017353:Gene Deletion; D005814:Genes, Viral; D008969:Molecular Sequence Data; D014764:Viral Proteins / Q000235:genetics* / Q000502:physiology; D015678:Viral Structural Proteins; D014779:Virus Replication
D014764:Viral Proteins; D015678:Viral Structural Proteins; C063744:spheroidin protein, Entomopoxvirus; D015500:Chloramphenicol O-Acetyltransferase