Molecular Characterization of the Host Defense Activity of the Barrier to Autointegration Factor against Vaccinia Virus

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

doi:10.1128/jvi.00641-11

Cited by 38 publications

(68 citation statements)

References 45 publications

(82 reference statements)

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“…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%

“…The stable depletion of BAF was performed using a BAF shRNA or scrambled shRNA (control) as previously described in (29). Cells were then passaged in medium containing 10 g/ml of puromycin to select for stable lentiviral integration.…”

Section: Methodsmentioning

confidence: 99%

“…Each lentiviral vector carrying BAF and BAF mutants was used to generate lentivirus as previously described (29). The stable overexpression of BAF in CV1 cells was then performed by transducing cells using these lentivirus preparations expressing 1ϫ FLAG-BAF or BAF mutants.…”

Section: Methodsmentioning

confidence: 99%

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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

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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.

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confidence: 94%

Section: Methodsmentioning

confidence: 99%

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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

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“…In the cytoplasm, the amount of DNA decreased within a few hours without completely disappearing, suggesting a rapid degradation of a major fraction and the persistence of a minor fraction of the molecules (13). Within a few hours after introducing DNA into the cytoplasm, tubular membranes and Emerin, a protein synthesized in the endoplasmic reticulum (ER) and subsequently predominantly present in the inner nuclear membrane, appear in the cytoplasm (10,11). However, the functional relevance of these observations is not clear.…”

mentioning

confidence: 99%

“…In animal cells, DNA sensors mediate the early detection of exogenous DNA, such as DNA of invading pathogens and artificially introduced DNA (9)(10)(11). Both in leukocytes and nonprofessional immune cells, these can trigger innate immune responses, such as cytokine production, autophagy, and apoptosis (9).…”

mentioning

confidence: 99%

Asymmetric partitioning of transfected DNA during mammalian cell division

Wang

Denoth-Lippuner

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Foreign DNA molecules and chromosomal fragments are generally eliminated from proliferating cells, but we know little about how mammalian cells prevent their propagation. Here, we show that dividing human and canine cells partition transfected plasmid DNA asymmetrically, preferentially into the daughter cell harboring the young centrosome. Independently of how they entered the cell, most plasmids clustered in the cytoplasm. Unlike polystyrene beads of similar size, these clusters remained relatively immobile and physically associated to endoplasmic reticulum-derived membranes, as revealed by live cell and electron microscopy imaging. At entry of mitosis, most clusters localized near the centrosomes. As the two centrosomes split to assemble the bipolar spindle, predominantly the old centrosome migrated away, biasing the partition of the plasmid cluster toward the young centrosome. Down-regulation of the centrosomal proteins Ninein and adenomatous polyposis coli abolished this bias. Thus, we suggest that DNA clustering, cluster immobilization through association to the endoplasmic reticulum membrane, initial proximity between the cluster and centrosomes, and subsequent differential behavior of the two centrosomes together bias the partition of plasmid DNA during mitosis. This process leads to their progressive elimination from the proliferating population and might apply to any kind of foreign DNA molecule in mammalian cells. Furthermore, the functional difference of the centrosomes might also promote the asymmetric partitioning of other cellular components in other mammalian and possibly stem cells.foreign DNA | asymmetric cell division | centrosome | endoplasmic reticulum | Ninein G enerally, noncentromeric DNA molecules are mitotically instable in eukaryotes. This results in their apparent disappearance from an ever-increasing proportion of the progeny of an affected cell (e.g., 1-3). Endogenous sources of such DNA are recombination byproducts [double minutes, extrachromosomal ribosomal (r)DNA circles (ERCs) and other DNA circles (3-6)] or mitotic defects generating noncentromeric chromosomal fragments and cytoplasmic micronuclei (1, 7). Exogenous sources are DNA of pathogens or DNA, typically plasmids, artificially introduced into cells. For the latter, decades of work established that plasmid-born protein expression is transient, persisting only for a few cell cycles (8). This finding is consistent with plasmid DNA being somehow eliminated through divisions. Thus, some mechanisms seem to prevent the propagation of foreign DNA and extrachromosomal DNA in proliferating eukaryotic cells. However, how this is achieved is unclear.In animal cells, DNA sensors mediate the early detection of exogenous DNA, such as DNA of invading pathogens and artificially introduced DNA (9-11). Both in leukocytes and nonprofessional immune cells, these can trigger innate immune responses, such as cytokine production, autophagy, and apoptosis (9). However, what happens to the DNA molecules themselves over time is unclear. When microi...

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

2022

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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.

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Molecular Characterization of the Host Defense Activity of the Barrier to Autointegration Factor against Vaccinia Virus

Cited by 38 publications

References 45 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

Asymmetric partitioning of transfected DNA during mammalian cell division

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

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