The 91-205 amino acid region of AcMNPV ORF34 (Ac34), which comprises a potential C3H zinc finger, is required for its nuclear localization and optimal virus multiplication

During baculovirus infection, most viral proteins must be imported to the nucleus to support virus multiplication. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) orf34 (ac34) is an alphabaculovirus unique gene that is required for optimal virus production. Ac34 distributes in both the cytoplasm and the nuclei of virus-infected Sf9 cells, but contains no conventional nuclear localization signal (NLS). In this study, we investigated the nuclear targeting domains in Ac34. Transient expression assays showed that Ac34 localized in both the cytoplasm and the nuclei of Sf9 cells, indicating that no viral protein is required for Ac34 nuclear localization. Subcellular localization analysis of Ac34 truncations and internal deletions fused with green fluorescent protein in plasmid-transfected Sf9 cells identified that the 91-205 amino acid (aa) region is required for Ac34 nuclear localization. Mutations in a potential C3H zinc finger (aa 116-131) in Ac34 resulted in exclusive cytoplasmic distribution of GFP:Ac34, suggesting that the zinc finger is required for Ac34 nuclear localization. To assess the functional importance of Ac34 in the nucleus during virus replication, recombinant AcMNPV bacmids containing a series of Ac34 truncations, internal deletions, or site mutations fused with HA tags were constructed. Subcellular localization analysis showed that Ac34 with internal deletions in aa 91-205 or site mutations in the potential zinc finger was predominantly distributed in the cytoplasm. Viral plaque assays and virus growth curves indicated that disruption of Ac34 nuclear localization significantly impaired virus replication. Taken together, our findings demonstrated that the nuclear localization of Ac34 requires the 91-205 aa region and its nuclear localization is essential for optimal virus replication.