Figure 1.
HSV-1 infection induces dephosphorylation of BAF.
(A) Western blot analysis of cellular BAF and viral proteins upon infection of L929 cells. Cells were infected with HSV-1 virus at MOI = 1 for the indicated times prior to harvest. Following cell harvest, protein samples were resolved by SDS-PAGE and transfer to membrane. Membranes were incubated with antibodies specific to the proteins indicated at left. Data were obtained from three independent experiments and representative blots are shown.
Figure 2.
Stable expression of BAF proteins in L929 cells.
(A) Point mutations were introduced to BAF N-terminal residues Thr2, Thr3, or Ser4 as indicated in the amino acid alignment. In each case the residue were mutated to A (alanine). (B–C) Representative western blot analysis of whole cell lysates from cells stably-expressing BAF-MTTSQ and BAF-MAAAQ. B) Anti-BAF antibody recognizes both endogenous BAF protein (arrowheads) and FLAG-tagged proteins (bracketed). C) Lysates from the same blot probed either withαGAPDH (top) or αFLAG (bottom).
Figure 3.
Subcellular distribution and DNA-binding activity of BAF mutants.
(A) Immunofluorescence analyses of FLAG-BAF-MTTSQ and FLAG-BAF-MAAAQ with an anti-FLAG antibody (AlexaFluor488-conjugated secondary antibody). (B) Subcellular fractionation analyses of cells indicated in (A). Cell lysates were fractionated to saponin-soluble cytosolic fraction (grey bars) and insoluble nuclear fraction (black bars). Each fraction was analyzed by western blot with an anti-FLAG antibody and quantified with ImageLab software (BioRad) (n = 3). % fraction was calculated from the amount of protein on each fraction relative to the total protein level. Error bars represent standard deviations. (C) ChIP analyses of FLAG-BAF-MTTSQ and BAF-MAAAQ. Cells were transfected with 150 ng of pUC-Neo plasmid DNA for 24 h followed by fixation, immunoprecipitation with anti-FLAG antibody, and reverse crosslinking of protein-DNA complexes. Purified DNA was analyzed by qPCR using primers specific for the pUC-Neo plasmid (Plasmid) or the β-actin locus of chromatin DNA (Genomic). Fold enrichment was obtained relative to empty vector control and normalized to input DNA. Error bars represent standard deviations.
Figure 4.
HSV-1 infection triggers relocalization of BAF to the nucleus where unphosphorylatable BAF can suppress HSV-1 viral yield.
(A) Immunofluorescence analyses of FLAG-BAF-MTTSQ or MAAAQ cells infected with HSV-1 virus at MOI = 1 for 6 h. Following fixation, cells were then stained with an anti-FLAG antibody (AlexaFluor488-conjugated). (B) Viral yield analyses of HSV-1 and VACV Cts2-infected cells. Indicated cell lines were infected with HSV-1 or VACV Cts2 virus at MOI = 0.01 for 48 h at 37°C. Viral yield was calculated relative to vector control for each infection (n = 3). Error bars represent standard deviations (*indicates a p-value<0.05 from t-test analysis).
Figure 5.
Unphosphorylatable BAF mutant suppresses HSV-1 viral DNA replication and exhibits increased relative binding to HSV-1 viral DNA.
(A) Relative viral DNA accumulation of HSV-1-infected FLAG-BAF-MTTSQ and MAAAQ cells. Cells were infected with HSV-1 virus at MOI = 0.1 for 3 h and 24 h. Following harvest, cells were analyzed for viral DNA accumulation via qPCR by using HSV-1 DNA-specific primers. DNA accumulation was calculated relative to vector control at 3 hpi (n = 3). Error bars represent standard deviations. (* indicates a p-value<0.05 from t-test analysis) (B) ChIP analyses of viral DNA bound to FLAG-BAF-MTTSQ and MAAAQ HSV-infected cells. Cells were infected with HSV-1 virus at MOI = 1 for 6 h followed by fixation, immunoprecipitation with anti-FLAG antibody, and reverse crosslinking of protein-DNA complexes. Purified DNA was analyzed by qPCR using primers specific for the ICP0 promoter region. Fold enrichment was obtained relative to empty vector control and normalized to input DNA. Data were obtained from three independent experiments performed in triplicate wells and data from a representative experiment is shown. Error bars represent standard deviations.
Figure 6.
Decreased HSV-1 viral protein expression can be observed in unphosphorylatable BAF mutant cells.
Western blot analysis of HSV-1 viral proteins upon infection of FLAG-BAF-MTTSQ and FLAG-BAF-MAAAQ cells. Cells were infected with HSV-1 virus at MOI = 1 for 20 h. Following cell harvest, protein samples were resolved by SDS-PAGE and transfer to membrane. Membranes were incubated with antibodies against HSV-1 proteins: ICP0, ICP4, ICP27, ICP8, TK, gD, and VP16. Anti-tubulin antibody was used as a loading control. Data were obtained from three independent experiments and a representative blot is shown. Values represent average relative signal intensity from three experiments as quantified by ImageLab software (BioRad).