Identification of the nucleolar localization signal in Autographa californica multiple nucleopolyhedrovirus multifunctional protein Ac16

<i>Autographa californica</i> multiple nucleopolyhedrovirus (AcMNPV) Ac16 (BV/ODV-E26) is a multifunctional protein found exclusively in group I NPV genomes. Here, we report a novel role for Ac16 in nucleolar localization. Using protein truncation and subcellular localization analysis, we characterized that the residues 78-113 of Ac16 contain nuclear (NLS) and nucleolar (NoLS) localization signals. Further multiple point mutation analysis within this region demonstrated that two basic-amino-acid-rich clusters, ⁷⁸HKKKLRH⁸⁴ and ¹⁰⁸KKTTHR¹¹³, function as NLSs, and together, they constitute a functional NoLS. However, Ac16 itself was not observed to localize to nucleoli, while it displayed an overlapping distribution with IE1 during AcMNPV infection. Co-expression assay revealed that IE1, an Ac16-interacting protein, alters the subcellular localization of Ac16, and this effect is independent of the Ac16 NoLS. By yeast two-hybrid library screening, vacuolar (H⁺)-ATPase subunit D (SfVhaD) was identified as a candidate interaction partner of Ac16, which was further confirmed by co-immunoprecipitation. Ac16 affected the localization of SfVhaD; both proteins predominantly colocalized in nucleoli in transient co-expression assays, while they primarily colocalized within the virogenic stroma during AcMNPV infection. Together, these data suggest that Ac16 contains a functional NoLS and may facilitate the transport of its interaction partners from nucleoli to viral replication centres during AcMNPV infection.