The Trichoplusia ni granulovirus helicase is unable to support replication of Autographa californica multicapsid nucleopolyhedrovirus in cells and larvae of T-ni

Baculovirus DNA helicases are essential for replication and are determinants of host range. Helicases of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) and Trichoplusia ni granulovirus (TnGV) differ markedly, although both viruses replicate efficiently in the cabbage looper, T. ni. To determine whether the TnGV helicase (P137) could support replication of AcMNPV in T. ni cells or larvae, the native AcMNPV helicase gene (p143) was disrupted and substituted with p137. P137 did not support replication when synthesized by the P143-deficient AcMNPV. Moreover, P137 did not inhibit AcMNPV replication when co-synthesized in the presence of the AcMNPV P143. These results suggest that although TnGV and AcMNPV replicate efficiently in T. ni, specific protein-protein or protein-DNA interactions between baculoviral helicases and viral-specific factors which form the replicase complex are required for virus replication. A novel and rapid method for disrupting AcMNPV genes in E. coli using the commercial Bac-to-Bac AcMNPV baculovirus expression vector is described.