Heterologous recombination between Autographa californica nuclear polyhedrosis virus DNA and foreign DNA in non-polyhedrin segments of the viral genome

Abstract

We used the expression vector system of Autographa californica nuclear polyhedrosis virus (AcNPV) and Spodoptera frugiperda insect cells to study mechanisms of recombination in insect cells. We concentrated on the isolation and analysis of heterologous recombinants. The E1 region of human adenovirus type 2 (Ad2) was inserted into regions of the AcNPV genome which lacked apparent homologies to the polyhedrin region. Out of a total of 122 recombinant AcNPV plaques, which hybridized to Ad2 DNA in plaque annealing experiments, 13 recombinants proved heterologous, and 5 of these recombinants could be grown to titers that facilitated virus replication and further investigations of the recombinant DNA. Restriction and Southern blot analyses for all of the recombinants and nucleotide sequence determinations for one of them permitted the mapping of the sites of foreign DNA integration into the AcNPV genome for the heterologous recombinants. These sites were located in the EcoRI-C (map units 42.5–52.4), the EcoRI-L (map units 69.5–72.5), the EcoRI-O (map units 32.6–34.5), and the EcoRI-Q (map units 88.2–89.7) segments of the plaque isolate E AcNPV genome. Two of the heterologous recombinants carried the insert in the EcoRI-L fragment. The nucleotide sequence determinations across the sites of junction between the AcNPV DNA and the foreign (Ad2) DNA in one of the heterologous recombinants, AcNPV-Ad2E1-D, revealed no sequence similarities at or close to the sites of junctions. A short sequence of six nucleotides was deleted from the original EcoRI-O sequence of AcNPV at the site of insertion. The inserted Ad2E1 DNA fragment comprised nucleotides 183–2763; thus nucleotides at the termini had been deleted. In the usual polyhedrin gene-located recombinants, the foreign Ad2 DNA segment was fused to the polyhedrin promoter and recombined presumably via polyhedrin sequence segments in the vector into the polyhedrin gene of AcNPV. In one of the control recombinants, AcNPV-Ad2E1-192, the Ad2E1 DNA segment between nucleotides 1 and 3117 (out of 3322 original nucleotides) was inserted in an inverted orientation between nucleotides − 115 and + 735 of the polyhedrin gene of AcNPV. This particular polyhedrin sequence was deleted in the process. It was uncertain how this recombinant had been generated. The infectivities of the polyhedrin-located recombinant AcNPV-Ad2E1-192 and of the five heterologous recombinants were compared by single-cycle growth curves to the infectivity of non-recombinant AcNPV. Within a factor of about 1.5, these values were found to be identical at 72 h postinfection (p.i.), although there were differences in the timing of virus production. However, some of the heterologous recombinants proved unstable. There was no evidence that the Ad2E1 region inserted in the heterologous recombinants was transcribed in Spodoptera frugiperda cells. The data presented document that regions other than the polyhedrin gene in the AcNPV genome are capable of accepting foreign DNA.