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 embe dded within a crystalline occlusion body or spheroid. Spheroids are co mposed 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 r eplaced with those of a heterologous reporter gene encoding chloramphe nicol 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 th at flanks the spheroidin gene. The flanking sequences provide the tran scriptional control signals and also guide homologous recombination so that the spheroidin gene coding region can be replaced with that of t he foreign gene. The transfer vector was designed so that the translat ional start codon of the introduced foreign gene would be utilized. A recombinant virus, AmEPV.CAT, was produced by transfecting AmEPV-infec ted cells with the transfer vector encoding the CAT gene. The recombin ant virus was isolated from wild-type virus by identifying plaques wit h a spheroidin-negative phenotype. Light microscopy and SDS-PAGE analy sis demonstrated that no spheroids or spheroidin protein were produced in the recombinant virus-infected cells. The recombinant virus was ab le to replicate to high titres (10(7) p.f.u./ml) in insect cells indic ating 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 fol lowed the pattern of spheroidin production suggesting that the spheroi din gene promoter was functioning under normal regulatory control in t he genetically modified virus.