The deficiency in nuclear localization signal of Neodiprion lecontei nucleopolyhedrovirus DNA polymerase prevents rescue of viral DNA replication and virus production in dnapol-null Autographa californica multiple nucleopolyhedrovirus

Abstract

DNA polymerase (DNApol) is highly conserved in baculovirus and is required for viral DNA replication. However, little is known about gammabaculovirus DNApol. Here DNApol of the gammabaculovirus Neodiprion lecontei nucleopolyhedrovirus (NeleNPV) was cloned into a dnapol-null alphabaculovirus AcMNPV bacmid, creating Bac-GFP-AcΔPol-NlPol. The resulting recombinant bacmid did not spread to neighboring cells, virus growth curve and real-time PCR revealed that NeleNPV dnapol substitution did not rescue AcMNPV DNA replication and virus production. Immunofluorescence microscopy revealed that NeleNPV DNApol was expressed but could not localize to the nucleus. Subsequently NeleNPV DNApol was fused to SpltNPV DNApol nuclear localization signal (NLS) and the fused DNApol could import into nucleus. The NLS-fusing NeleNPV DNApol was further transposed into the dnapol-null AcMNPV bacmid, creating Bac-GFP-AcΔPol-HA:NlPolNLS. The recombinant virus could replicate and produce infectious virus in Sf9 cells, albeit at reduced levels compared to wild type AcMNPV. Taken together, our results suggested that the NLS deficiency of NeleNPV DNApol blocked viral DNA replication and production of infectious virus in dnapol-null AcMNPV bacmid.