Barrier to autointegration factor (BAF) inhibits vaccinia virus intermediate transcription in the absence of the viral B1 kinase

Ibrahim, Nouhou; Wicklund, April; Jamin, Augusta; Wiebe, Matthew S.

doi:10.1016/j.virol.2013.07.002

Cited by 27 publications

(50 citation statements)

References 74 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…CV-1 cells (1.2 ϫ 10 6 ) were freshly collected, pelleted, and resuspended in 300 l of SDS sample buffer supplemented with 10 units of benzonase as described previously (25). Lysate volumes equivalent to 10 5 cells were resolved by 18% SDS-PAGE followed by protein transfer to polyvinylidene difluoride (PVDF) membranes overnight.…”

Section: Methodsmentioning

confidence: 99%

“…Chromatin immunoprecipitation was performed as described previously (25) with the following slight modification. After overnight incubation of lysates and antibodies and 2 h of incubation of Dynabeads, beads were washed 3 times with final wash buffer (20 mM Tris-HCl [pH 8.0], 500 mM NaCl, 2 mM EDTA [pH 8], 1% Triton X-100, and 0.1% SDS), followed by elution of bound complexes.…”

Section: Methodsmentioning

confidence: 99%

“…CV1 cells expressing empty-vector control, WT, or BAF mutants were transfected with 150 ng pG8-Luc plasmid for 24 h and subsequently fixed with paraformaldehyde. This pG8-Luc plasmid contains the promoter of the vaccinia virus G8R gene driving the expression of a luciferase gene (23,25). Our recent study utilizing this plasmid demonstrated that pG8-Luc coprecipitates with BAF in ChIP analysis (25), validating this plasmid as a source of foreign DNA that can be bound by BAF.…”

Section: Stable Expression Of Baf Phosphorylation Mutants In Cv1 Cellsmentioning

confidence: 99%

“…Both in vitro and in vivo data support that B1 can directly phosphorylate BAF at Thr2, Thr3, and Ser4 at BAF's N terminus (27,28). If not phosphorylated by B1, BAF localizes to the sites of viral DNA and interferes with genome replication (27,29) and transcription (25), thereby leading to a sharp reduction in the number of viral progeny produced (29).…”

mentioning

confidence: 94%

“…Such autonomy is attributed to the large poxviral genome, which encodes essential replication and transcription proteins (18,19). One of these essential proteins is the B1 Ser/Thr kinase, which is expressed early in the viral life cycle and performs functions in viral DNA replication and intermediate gene expression (20)(21)(22)(23)(24)(25)(26). The temperature-sensitive mutant viruses Cts2 and Cts25 harbor point mutations within the B1 locus and serve as critical tools for gaining insights about B1's functions.…”

mentioning

confidence: 99%

See 4 more Smart Citations

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

Jamin

Wicklund

Wiebe

2014

J Virol

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Stable Expression Of Baf Phosphorylation Mutants In Cv1 Cellsmentioning

confidence: 99%

mentioning

confidence: 94%

mentioning

confidence: 99%

See 3 more Smart Citations

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

Jamin

Wicklund

Wiebe

2014

J Virol

Self Cite

View full text Add to dashboard Cite

show abstract

The TFAMoplex—Conversion of the Mitochondrial Transcription Factor A into a DNA Transfection Agent

2022

View full text Add to dashboard Cite

Non‐viral gene delivery agents, such as cationic lipids, polymers, and peptides, mainly rely on charge‐based and hydrophobic interactions for the condensation of DNA molecules into nanoparticles. The human protein mitochondrial transcription factor A (TFAM), on the other hand, has evolved to form nanoparticles with DNA through highly specific protein‐protein and protein‐DNA interactions. Here, the properties of TFAM are repurposed to create a DNA transfection agent by means of protein engineering. TFAM is covalently fused to Listeria monocytogenes phospholipase C (PLC), an enzyme that lyses lipid membranes under acidic conditions, to enable endosomal escape and human vaccinia‐related kinase 1 (VRK1), which is intended to protect the DNA from cytoplasmic defense mechanisms. The TFAM/DNA complexes (TFAMoplexes) are stabilized by cysteine point mutations introduced rationally in the TFAM homodimerization site, resulting in particles, which show maximal activity when formed in 80% serum and transfect HeLa cells in vitro after 30 min of incubation under challenging cell culture conditions. The herein developed TFAM‐based DNA scaffolds combine interesting characteristics in an easy‐to‐use system and can be readily expanded with further protein factors. This makes the TFAMoplex a promising tool in protein‐based gene delivery.

show abstract

Proteomic analysis of BmN cell lipid rafts reveals roles in Bombyx mori nucleopolyhedrovirus infection

Zhu

Liang

et al. 2017

Mol Genet Genomics

View full text Add to dashboard Cite

The mechanism of how Bombyx mori nucleopolyhedrovirus (BmNPV) enters cells is unknown. The primary components of membrane lipid rafts are proteins and cholesterol, and membrane lipid rafts are thought to be an active region for host-viral interactions. However, whether they contribute to the entry of BmNPV into silkworm cells remains unclear. In this study, we explored the membrane protein components of lipid rafts from BmN cells with mass spectrometry (MS). Proteins and cholesterol were investigated after establishing infection with BmNPV in BmN cells. In total, 222 proteins were identified in the lipid rafts, and Gene Ontology (GO) annotation analysis showed that more than 10% of these proteins had binding and catalytic functions. We then identified proteins that potentially interact between lipid rafts and BmNPV virions using the Virus Overlay Protein Blot Assay (VOPBA). A total of 65 proteins were analyzed with MS, and 7 were predicted to be binding proteins involved in BmNPV cellular invasion, including actin, kinesin light chain-like isoform X2, annexin B13, heat-shock protein 90, barrier-to-autointegration factor B-like and serine/arginine-rich splicing factor 1 A-like. When the cholesterol of the lipid rafts from the membrane was depleted by methyl-β-cyclodextrin (MβCD), BmNPV entry into BmN cells was blocked. However, supplying cholesterol into the medium rescued the BmNPV infection ability. These results show that membrane lipid rafts may be the active regions for the entry of BmNPV into cells, and the components of membrane lipid rafts may be candidate targets for improving the resistance of the silkworm to BmNPV.

show abstract

Barrier to autointegration factor (BAF) inhibits vaccinia virus intermediate transcription in the absence of the viral B1 kinase

Cited by 27 publications

References 74 publications

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

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

The TFAMoplex—Conversion of the Mitochondrial Transcription Factor A into a DNA Transfection Agent

Proteomic analysis of BmN cell lipid rafts reveals roles in Bombyx mori nucleopolyhedrovirus infection

Contact Info

Product

Resources

About