Rescue of dnapol-null Autographa californica multiple nucleopolyhedrovirus with DNA polymerase (DNApol) of Spodoptera litura nucleopolyhedrovirus (SpltNPV) and identification of a nuclear localization signal in SpltNPV DNApol.

Abstract

DNA polymerase (DNApol) is highly conserved in all baculoviruses and plays an essential role in viral DNA replication. It determines the fidelity of baculovirus DNA replication by inserting the correct nucleotides into the primer terminus and proofreading any mispaired nucleotides. DNApols of groups I and II of the genus Alphabaculovirus in the family Baculoviridae share many common structural features. However, it is not clear whether a group I Autographa californica multiple nucleopolyhedrovirus (AcMNPV) DNApol can be substituted by a group II NPV DNApol. Here we report the successful generation of AcMNPV dnapol-null virus being rescued by a group II Spodoptera litura NPV (SpltNPV) dnapol (Bac-AcΔPol : Slpol). Viral growth curves and quantitative real-time PCR showed that the dnapol replacement reduced the level of viral production and DNA replication of Bac-AcΔPol : SlPol compared with WTrep, a native dnapol insertion in an AcMNPV dnapol-null virus. Light microscopy showed that production of occlusion bodies for Bac-AcΔPol : Slpol was reduced. We also identified a nuclear localization signal (NLS) for the SpltNPV DNApol C terminus at residues 827-838 by mutational analysis and confocal microscopy. Multiple point substitution of SpltNPV DNApol NLS abrogated virus production and viral DNA replication. Overall, these data suggested that the NLS plays an important role in SpltNPV DNApol nuclear localization and that SpltNPV DNApol cannot efficiently substitute the AcMNPV DNApol in AcMNPV.