Cell- and Virus-Mediated Regulation of the Barrier-to-Autointegration Factor's Phosphorylation State Controls Its DNA Binding, Dimerization, Subcellular Localization, and Antipoxviral Activity

Abstract

Barrier-to-autointegration factor (BAF) is a DNA binding protein with multiple cellular functions, including the ability to act as a potent defense against vaccinia virus infection. This antiviral function involves BAF's ability to condense double-stranded DNA and subsequently prevent viral DNA replication. In recent years, it has become increasingly evident that dynamic phosphorylation involving the vaccinia virus B1 kinase and cellular enzymes is likely a key regulator of multiple BAF functions; however, the precise mechanisms are poorly understood. Here we analyzed how phosphorylation impacts BAF's DNA binding, subcellular localization, dimerization, and antipoxviral activity through the characterization of BAF phosphomimetic and unphosphorylatable mutants. Our studies demonstrate that increased phosphorylation enhances BAF's mobilization from the nucleus to the cytosol, while dephosphorylation restricts BAF to the nucleus. Phosphorylation also impairs both BAF's dimerization and its DNA binding activity. Furthermore, our studies of BAF's antiviral activity revealed that hyperphosphorylated BAF is unable to suppress viral DNA replication or virus production. Interestingly, the unphosphorylatable BAF mutant, which is capable of binding DNA but localizes predominantly to the nucleus, was also incapable of suppressing viral replication. Thus, both DNA binding and localization are important determinants of BAF's antiviral function. Finally, our examination of how phosphatases are involved in regulating BAF revealed that PP2A dephosphorylates BAF during vaccinia infection, thus counterbalancing the activity of the B1 kinase. Altogether, these data demonstrate that phosphoregulation of BAF by viral and cellular enzymes modulates this protein at multiple molecular levels, thus determining its effectiveness as an antiviral factor and likely other functions as well. IMPORTANCEThe barrier-to-autointegration factor (BAF) contributes to cellular genomic integrity in multiple ways, the best characterized of which are as a host defense against cytoplasmic DNA and as a regulator of mitotic nuclear reassembly. Although dynamic phosphorylation involving both viral and cellular enzymes is likely a key regulator of multiple BAF functions, the precise mechanisms involved are poorly understood. Here we demonstrate that phosphorylation coordinately regulates BAF's DNA binding, subcellular localization, dimerization, and antipoxviral activity. Overall, our findings provide new insights into how phosphoregulation of BAF modulates this protein at multiple levels and governs its effectiveness as an antiviral factor against foreign DNA.

Deletion of the Vaccinia Virus B1 Kinase Reveals Essential Functions of This Enzyme Complemented Partly by the Homologous Cellular Kinase VRK2

“…Previous studies of the B1 kinase determined that this kinase is needed to directly regulate the phosphorylation and thus the antiviral activity of BAF, a cellular DNA binding protein. Specifically, hyperphosphorylated BAF has reduced affinity for dsDNA and reduced homodimerization activity, both of which are required to facilitate dsDNA condensation and inhibition of vaccinia virus DNA replication and intermediate transcription (13,(27)(28)(29). Despite our growing understanding of this B1-BAF signaling axis, it is unknown if the B1 kinase activity mediating BAF phosphorylation in cells is enhanced by other viral factors.…”

“…In addition to these cellular functions, BAF is also capable of strongly inhibiting vaccinia virus DNA replication (25,26) and intermediate transcription (27). The host defense activity of BAF against vaccinia virus is dependent on its DNA-binding property, which can be blocked through phosphorylation mediated by B1 (25,28), thus highlighting the importance of this kinase during poxvirus DNA replication.…”

Vaccinia Virus B1 Kinase Is Required for Postreplicative Stages of the Viral Life Cycle in a BAF-Independent Manner in U2OS Cells

“…The activity of the B1 kinase is required during DNA replication in many cell lines in order to inactivate the cellular DNA-binding protein BAF (24). As such, the inhibition of Cts2 viral DNA accumulation can be exacerbated in cells that overexpress BAF (24,29,32). To test the impact of BAF overexpression in U2OS cells, cell lines that stably express a FLAG epitope-tagged form of BAF were generated using a lentiviral transduction system as previously described (29,32).…”

“…Lentiviral expression vectors for 1ϫ FLAG-tagged BAF (FLAG-BAF) (32) were used to generate lentivirus as previously described (29). The stable overexpression of BAF was accomplished by transducing cells with these lentivirus preparations as described previously (32). Transduced cells were selected with 200 g/ml of hygromycin prior to further analysis.…”

“…Quantifications of the chemiluminescence signal were performed by using Bio-Rad ImageLab software. The primary antibodies used were as follows: antibody against GAPDH (glyceraldehyde-3-phosphate dehydrogenase; 1:500; catalog number sc-25778; Santa Cruz Biotechnology), antibody against BAF (custom) (32), and custom antibodies against vaccinia virus proteins I3, A11, F17, and L4.…”