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. 2013 Sep;444(1-2):363-73.
doi: 10.1016/j.virol.2013.07.002. Epub 2013 Jul 24.

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

Nouhou Ibrahim  1 , April Wicklund, Augusta Jamin, Matthew S Wiebe
Affiliations

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

Nouhou Ibrahim et al. Virology. 2013 Sep.

Abstract

Barrier to autointegration factor (BAF/BANF1) is a cellular DNA-binding protein found in the nucleus and cytoplasm. Cytoplasmic BAF binds to foreign DNA and can act as a defense against vaccinia DNA replication. To evade BAF, vaccinia expresses the B1 kinase, which phosphorylates BAF and blocks its ability to bind DNA. Interestingly, B1 is also needed for viral intermediate gene expression via an unknown mechanism. Therefore, we evaluated the impact of B1-BAF signaling on vaccinia transcription. Strikingly, the decrease in vaccinia transcription caused by loss of B1 can be rescued by depletion of BAF. The repressive action of BAF is greatest on a viral promoter, and is more modest when non-vaccinia promoters are employed, which suggests BAF acts in a gene specific manner. These studies expand our understanding of the role of the B1 kinase during infection and provide the first evidence that BAF is a defense against viral gene expression.

Keywords: BAF; BANF1; Barrier to autointegration; Transcription; Vaccinia; Virus host interaction.

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Figures

Figure 1
Figure 1. The ts2 defect in intermediate transcription is observed on multiple promoters and is rescued by B1 expression in L929 cells
(A-B) L929 cells were transfected with the reporter construct shown for 7hr, then infected at 37°C with WT, ts2, or ts2/B1 as indicated (MOI=3) in the presence of 50 μM AraC. Lysates were prepared at 16h after infection and assayed for luciferase activity. RLU shown is normalized to total protein measured by BCA assay. Data were obtained from three independent experiments performed in triplicate wells. Data from a representative experiment is shown. Error bars represent the standard deviation. (C) L929 cells were infected with WT, ts2, or ts2/B1 at MOI= 0.1 or MOI=5 for 24h at 37°C. After lysates were collected virus yield was determined by a plaque titration on monolayers of BSC40 cells at 32°C. Data were obtained from triplicate experiments, and standard deviation is presented by the error bars. (*** indicates a p-value <0.05)
Figure 2
Figure 2. The defect in intermediate transcription is specific to ts2 and is rescued by depletion of BAF
(A). DNA replication assays. Total DNA was isolated at 30 minutes and 24 hours after infection (MOI=3) with WT, ts2, ts42, or ts24 at 37°C, then quantified by qPCR. Data was obtained from two independent experiments, PCR amplified in duplicate. Data is shown as a fold difference compared to the WT sample at 0.5 hpi. Error bars represent standard deviation. (B). Immunoblot analysis of BAF expression in L929 cells stably depleted of BAF using specific shRNA. Lysates from equivalent numbers of cells were collected and analyzed using antibody against BAF. The total amounts of BAF in each lane were quantified using a Bio-Rad Chemidoc XRS instrument. GAPDH level was used as loading control. (C) L929 cells stably expressing shBAF or shControl were transfected with pG8-Luciferase for 7hr, then infected at 37°C with WT, ts2, ts42 or ts24 at MOI=3 in the presence of 50 μM AraC. Lysates were prepared at 16h after infection and assayed for luciferase activity. RLU shown is normalized to total protein measured by BCA assay. Data were obtained from three independent experiments performed in triplicate wells. Data from a representative experiment is shown. Error bars represent standard deviation. D) Data from the luciferase expression shown in (C) was replotted as a fold difference between the shControl and shBAF cell lines for each virus. (*** indicates a p-value <0.05)
Figure 3
Figure 3. Depletion of B1 impairs intermediate transcription following WT virus infection, and can be rescued by BAF depletion
(A) L-929 cells were transfected with 100 nM of siRNA siControl, siB1R-1, or siB1R-2. At 24 hours post transfection, cells were infected with WT virus at a MOI of 3 and total RNA harvested at 4hpi. Following reverse transcription, cDNA was quantified by qPCR. (B) L929 stably expressing shBAF or shControl were transfected with 100nM siRNA specific to B1 kinase (siB1-1) or siControl for 12h, and then transfected with pG8-Luciferase for 7hr, then infected at 37°C with WT virus at MOI=3 in the presence of 50 uM AraC. An infection of L929 with ts2 at MOI=3 in the presence of AraC at 37°C was also performed. Lysates were prepared at 12h after infection and assayed for luciferase activity, and RLU normalized to protein level. Data were obtained from three independent experiments performed in triplicate wells. Data from a representative experiment is shown. Error bars represent standard deviation. (*** indicates a p-value <0.05)
Figure 4
Figure 4. BAF immunoprecipitates pG8-Luc DNA in L929 cells and depletion of BAF rescues viral intermediate gene expression at the transcriptional level
(A) ChIP assays were performed to determine the binding of BAF to DNA. L929 cells stably expressing MCS plasmid (control) or 3xFlag-BAF were transfected with 150 ng pG8-luciferase plasmid for 24h. Following fixation and sonication, BAF-DNA complex was immunoprecipitated using anti-FLAG antibody and analyzed by quantitative PCR using primers flanking the G8 promoter (A) or Neomycin resistance gene (B) of pG8-Luc DNA or the beta-actin cellular gene (C). Data shown is a representative from three independent experiments, each performed in duplicate. Error bars represent standard deviation. Data from the Raw Ct values (shown in Supp Fig. 2) was plotted as fold enrichment relative to MCS. (D) L929 cells stably expressing shBAF or shControl were transfected with 10, 100, and 500ng of pG8-luciferase for 7h, then infected with 37°C with WT or ts2 at MOI=3 in the presence of AraC. Lysates were collected for RNA extraction and luciferase transcripts quantified by RT-qPCR. (*** indicates a p-value <0.05)
Figure 5
Figure 5. The role of B1 kinase and BAF in viral intermediate gene expression is promoter dependent
(A) L929 stably expressing shBAF or shControl were transfected with siRNA specific to B1 kinase (siB1-1) or siControl for 12h, and then transfected with 1 ng of T7-Luciferase for 7hr, then infected with 37°C with Wt-VTF7.3 at MOI=3 in the presence of AraC. Lysates were prepared at 12h after infection and assayed for luciferase activity, with RLU shown normalized to protein level. (B-C) L929 stably expressing shBAF or shControl were transfected with the indicated amounts of minP-Luc (B) or CMV-Luc (C). Lysates were prepared at 24h after transfection and assayed for luciferase activity, with RLU shown normalized to protein level. For all graphs, data were obtained from three independent experiments performed in triplicate wells. Data from a representative experiment is shown. Error bars represent standard deviation. (*** indicates a p-value <0.05)
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