FIG. 6.

HvAV-miR-1 specifically targets HvAV DNA polymerase I. (a) A recombinant baculovirus was constructed expressing a fragment of the HvAV DNA polymerase I gene containing the target sequence for HvAV-miR-1. Sf9 cells were initially transfected with pIZ empty vector, pIZ-MCP, pIZ-MCP1 (lacking HvAV-miR-1), and pIZ-MCP2 (containing HvAV-miR-1). Twenty-four hours following transfection, cells were infected with the recombinant baculovirus. At 72 hpi, cells were collected and analyzed by Western blotting in which an anti-His monoclonal antibody was used as a probe (1:5,000). Expression of a 69-kDa protein corresponding to the HvAV DNA polymerase fusion protein was significantly reduced in cells transfected with vectors containing the HvAV-miR-1 miRNA (pIZ-MCP and pIZ-MCP2). (b) The blot in panel “a” was reprobed with a monoclonal antibody to the Autographa californica multiple nucleopolyhedrovirus gp64 protein (1:5,000) to show that the miRNA did not affect replication of the virus but specifically affected expression of the recombinant HvAV DNA polymerase.