Characterization of the interactions of baculovirus transcription and replication regulators by bimolecular fluorescence complementation
Breznik, Jessica Ann
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Baculovirus gene expression is temporally divided into early and late stages. Viral late expression factors (LEFs) are essential for the transition between the early and late transcriptional stages, as well as for late gene expression. A virus-encoded RNA polymerase (RNApol) transcribes late and very late genes. Four LEFs comprise the core viral RNApol sufficient for in vitro transcription: LEF-4, LEF-8, LEF-9, and P47. LEF-4 has triphosphatase, guanylyltransferase, and ATPase activities. LEF-8 and LEF-9 contain catalytic motifs that are conserved in the active sites of other multisubunit RNApols. These motifs are required for in vitro baculovirus RNApol transcription. P47 has no demonstrated enzymatic activity. How these four subunits associate in vivo within the viral RNApol complex and initiate transcription is undetermined, as is whether its transcription mechanism requires the host TATA binding protein (TBP). Viral DNA replication coincides with the transition between early and late transcription and it is closely associated with the regulation of late gene expression. Two LEFs essential for viral DNA synthesis, LEF-3 and P143, have also been implicated in the regulation of baculovirus RNApol transcription. Bimolecular fluorescence complementation (BiFC) assays were adapted to characterize the in vivo interactions of the viral RNApol subunits LEF-4, LEF-8, LEF-9, and P47, in the context of viral infection, as well as to investigate their potential interactions with viral DNA replication proteins LEF-3 and P143, and host Spodoptera frugiperda TBP. The proteins of interest were modified by N-terminal fusions of the Venus1 (aa1-158) or Venus2 (aa159-239) fragments of the Venus fluorescent protein. Recombinant plasmids and baculoviruses were constructed. Nuclear fluorescence was detected from all BiFC assays investigating the self-association and inter-subunit interactions of the viral RNApol. BiFC assays also suggested that the viral RNApol proteins interact with LEF-3, P143, and TBP. The potential effects of endogenous viral proteins and viral DNA on the detected protein-protein interactions are discussed. The results highlight extensive interactions between the core baculovirus RNApol subunits, possible roles of viral DNA replication proteins in the nuclear localization and transcription mechanism of the viral RNApol, as well as the involvement of host TBP in in vivo late stage transcription.