Fig 1.
Type I and Type III EBV+ cells express different components of the nuclear lamina.
A) Western blot for key EBV proteins (EBNA2, LMP1) and nuclear lamina components (lamin B1 and A/C) in EBV-negative cell lines (B cells, Akata) and EBV-positive cell lines that express the type I (Mutu I) or the type III (Mutu III, Mutu-LCL, B95.8-LCK, GM12878) latency program. EBV actin was used as a loading control. Images are representative of at least three independent experiments. B) Top: Confocal microscopy analysis of EBV+ Mutu I and LCL cells stained with anti-Lamin B1 and anti-Lamin A/C antibodies and DAPI. The images show that lamin A/C is differentially expressed, depending on the presence of EBV and its latency state. Bottom: Higher magnifications of the boxed areas showing colocalization of the lamin proteins at the nuclear periphery. C) Representative quality analysis of the fluorescence intensity in LCL cell nuclei immunostained as described above. The fluorescence intensity was measured along the dotted white line (x-axis) using a Leica software analysis.
Fig 2.
EBV infection of primary B cells induces the expression of lamin A/C.
A) Immunofluorescence confocal microscopy analysis of B cells from a healthy donor immunostained with lamin B1 (red) and lamin A/C (green) antibodies and DAPI (blu). Top: control (uninfected) cells; bottom: 2 days post-infection with EBV viral particles. B) RNA-seq of primary B cells from 3 different healthy donors that were infected with EBV and collected at the indicated time points after viral infection. The plot shows the normalized reads of lamin A/C mRNA. The t test p values for each time point are indicated. C) Whole cell proteomic analysis of lamin A/C expression at the indicated time points following infection of primary human B cells as described in B. The plot shows the relative abundance of lamin A/C from 3 biological replicates, representing 12 human donors. The t test p values for each time point are indicated. N = 3, Mean ± SD.
Fig 3.
Activation of B cells by CD40 treatment induces lamin A/C expression.
A) Confocal analysis of resting (top) and activated (bottom) primary B cells from a healthy donor, immunostained with lamin A/C (green) and lamin B1 (red) antibodies and DAPI (blue). For activation, cells were treated for 24 hours with a stimulatory cocktail containing 20 ng/mL interleukin 4 (IL-4), 5uM CD40 ligand (CD40L), and 25 ng/mL CpG oligodeoxynucleotides. The right panel shows higher magnification of the boxed areas in the left panel to show colocalization of the lamin proteins at the nuclear periphery. B) RNA-seq of primary B cells from 2 donors. The plot shows the normalized reads for lamin A/C mRNA after a 24-hour stimulation with the indicated ligands (control = untreated cells). The values of two different experiments performed in parallel is shown. C) Proteomic analysis of lamin A/C expression in B cells treated as described above with the indicated stimulatory compounds (control = untreated cells). Data are presented as Mean ± SD (N = 3). The t test p values for each time point are indicated.
Fig 4.
The EBV genome interacts with the nuclear lamins.
A) ChIP-Seq analysis of lamin B1 and lamin A/C binding in EBV+ B cells adopting Type I (Mutu I) or Type III (LCLs) latency. Blue track profile: lamin B1 binding in type I cells; green track profile: lamin B1 binding in type III cells; orange track profile: lamin A/C binding in type III cells. The peaks identified through peak calling method are indicated above each track. Tracks are aligned with the annotated EBV genome shown at the bottom.
Fig 5.
Patterns of lamin B1 and lamin A/C binding on the EBV genome in different types of latency.
A) Upset plot of EBV peaks identified in three ChIP-seq datasets. Each bar in the upper plot corresponds to the number of peaks identified in the datasets indicated by black dots in the lower plot. Connected dots indicate an intersection of peaks between ChIP-Seq datasets. The number of EBV regions in each condition is indicated above the corresponding columns. B), C) and D) Lamin B1 and lamin A/C ChIP-Seq tracks for unique and overlapping peaks across the viral genome identified in A: LADs with lamin B1 binding in Mutu I and LCL cells and lamin A/C binding in LCL cells (B); LADs with lamin B1 binding in Mutu I cells and lamin A/C binding in LCL cells (C); LADs with lamin B1 binding only in Mutu I cells and lamin A/C binding only in LCL cells (D).
Fig 6.
Lamin A/C knockout alters interaction of the EBV genome with the nuclear lamina and changes viral chromatin composition.
A) Western blot analysis for lamin A/C, lamin B1 and actin protein expression in Ctr and LMNA KO GM12878 LCL cells. CRISPR/Cas9 gene editing was used to knock out lamin A/C expression in GM12878 cell lines using two different single-guide RNAs (sgRNAs) that target distinct regions of the LMNA gene. We generated two lamin A/C KO stable bulk populations. B) Immunofluorescence confocal microscopy analysis of Ctr and KO cells immunostained with lamin A/C (green) and lamin B1 (red) antibodies and DAPI (blue). C) Quantitative chromatin immunoprecipitation (ChIP-qPCR) analysis of EBV chromatin extracted from Ctr and LMNA KO GM12878 cells. EBV chromatin was analyzed for the binding of lamin B1 and lamin A/C and the deposition of the repressive histone marks H3K9me2 and H3K27me3 at the indicated regions. Data are presented as %input. N = 3, Mean ± SD. The t test p values for the KO/ctr comparison are indicated.
Fig 7.
Lamin A/C knockout deregulates EBV gene expression in Type III B cells.
A) Principal Component Analysis (PCA) of RNA-Seq analysis of Ctr and KO LCL cells (3 samples and 2 samples, respectively). The samples are shown as a function of principal component 1, or PC1 (x-axis), and principal component 2, or PC2 (y-axis). The percentage of variance explained by PC1 and PC2 is indicated. B) Heat map of RNA-seq data from Ctr and LMNA KO LCL cells showing genes whose expression was significantly altered (p<0.05) by lamin A/C depletion, which include EBNAs and LMP1 family members; differences were calculated using p-values.