Supplementary Information The latent origin of replication of Epstein-Barr virus directs viral genomes to active regions of the nucleus Manuel J. Deutsch, Elisabeth Ott, Peer Papior, and Aloys Schepers* Department of Gene Vectors HelmholtzZentrum muenchen Marchioninistrasse Munich Germany *Corresponding Author: Aloys Schepers Phone: FAX:

Figure S1 HEK293 EBV + AB CD Supplementary Figure S1 The interaction domain between the dense chromatin (CD) and the interchromatin compartment (IC) defines the perichromatin (PC). A) DAPI DNA counterstain of HEK293 EBV + cells after incubation in isotonic buffer. B) DAPI DNA counterstain of hypertonically treated HEK293 EBV + displaying characteristic condensation of the chromatin. C) enlarged single cell from B) D) enlarged section of C) indicating the condensed chromatin or chromatic domain (CD), the enlarged nuclear travelling channels of the interchromatin domain (IC) and the interaction zone of the CD and IC, the perichromatic domain (PC) depicted in a red line. Enlargements are indicated as white-lined squares. Scale bar = 2 µm.

Figure S2 A B C hypertone Raji hypertone LCL2908 wt-oriP hypertone HEK293 EBV ,424,837,25 58,9 distance in µm greyscale value ,887,7611,6 68,7 distance in µm greyscale value ,272,533,8 59,2 distance in µm greyscale value Supplementary Figure S2 Signal-intensity scans of EBV (red) and DNA-counterstain (blue) along the indicated line in HEK293-EBV + (A), Raji (B) and LCL2908 cells (C). Localization of EBV is not observed in the peaks of the DNA-counterstain but next to it, indicating perichromatic localisation. Scale bar = 2µm. (D) Peak classification. (top) Signals that show a complete overlap were classified as colocalizing. (middle) Signals that show overlaping shoulders but no but no complete overlap were classified as associated. (bottom) Signals that show no overlapping intensities were classified as non-associated. Distance in µm greyscale value peaks overlap = colocalization Distance in µm greyscale value shoulders overlap = association Distance in µm greyscale value peaks are separated = not associated D

Figure S3 - Part 1 H3K9me3 H3K9ac H3K27me3 3D-reconstruction turned 172° along x-axis C A B D Raji H3K4me3 Raji 3D-reconstruction

Figure S3 - Part ,194,376,55 71,3 distance in µm greyscale value ,164,316,47 73,3 distance in µm greyscale value ,14,26,3 73,3 distance in µm greyscale value ,517,0210,5 73,3 distance in µm greyscale value H3K4me3 H3K9ac H3K9me3 H3K27me3 F E H G Distance in µm greyscale value peaks overlap = colocalization Distance in µm greyscale value shoulders overlap = association Distance in µm greyscale value peaks are separated = not associated I Raji

Supplementary Figure S3 Raji-cells after combined immunofluorescence and fluorescence in situ hybridization. EBV-genomes were visualized by FISH using an EBV-specific probe (red). Colocalization with histone3 trimethylated at lysine 4 (H3K4me3; first panel) (A), histone3 trimethylated at lysine 9 (H3K9me3; second panel) (B), histone3 trimethylated at lysine 27 (H3K27me3; third panel) (C), and histone3 acetylated at lysine 9 (H3K9ac; fourth panel) (D) are shown in green. The cells outlined with white-lined squares were 3D-reconstructed for localization analysis. Reconstruction image of H3K9me3 has been rotated 172° along its x-axis for better understanding. (E-H) Signal-intensity scans of the respective histone modifications (green; channel 1), EBV (red; channel 0) and DNA-counterstain (blue; channel 2) along the indicated line in Raji-cells. EBV-peaks colocalize with peaks for H3K4me3 (E) and H3K9ac (H). Colocalization occurs not with the peaks of H3K9me3 (F) and only sporadically with H3K27me3 (H). Scale bar = 2 µm. (I) Peak classification. (top) Signals that show a complete overlap were classified as colocalizing. (middle) Signals that show overlaping shoulders but no but no complete overlap were classified as associated. (bottom) Signals that show no overlapping intensities were classified as non-associated.

Figure S4 Supplementary Figure S4 The correctness of the different mini-EBV-mutants the LCLs resulting from the infection experiments were analyzed by Southern blot hybridization and PCR. A) Genomic DNA of LCL subclones was digested with the indicated restriction enzyme. The hybridization probe detected oriP-fragments. The expected sizes of the fragments at the oriP and ectopic integration site are given below. B) The DS-fragment translocated to the ectopic site is not easily detected by Southern blotting. Therefore, we confirmed the integrity of the translocated DS-fragment by PCR using a primer pair that encompasses the integration site. Successful integration was indicated by a shift from 160 bp to 280 bp. A BamHI XhoI 9,25,85,610,0 and 5,410,7 and 4,5.II.4.XI.XII.4.II.XII  DS 2912eFR2913eDS.4.I translocated size (kbp) oriP enzyme B 2908H2OH2O 280 bp M 2913eDS: PCR Analysis.4.II.XII 160 bp clone ID

Figure S5 - Part1 LCL2908 wt-oriP HEK eFR LCL2913eDS hypertone LCL2910-  DS hypertone3D-reconstruction C A B D

Figure S5 - Part ,595,187,76 70,4 distance in µm greyscale value ,444,897,33 66,4 distance in µm greyscale value ,585,127,68 57,5 distance in µm greyscale value ,136,279,4 58,9 distance in µm greyscale value LCL2908 wt-oriP HEK eFR LCL2913eDS hypertone LCL2910-  DS hypertone G E F H

Supplementary Figure S5 Lymphoblast-cells after combined immunofluorescence and fluorescence in situ hybridization. Cells were infected with a mini-EBV-genome lacking the lytic genes of EBV; EBV genomic DNA (red), EBNA1 (green) and DNA (blue); EBV and EBNA1 are colocalizing in the chromatin-poorer regions of the nucleus. A) Perichromatic localization is revealed by the hypertonic treatment of cells carrying a mini-EBV-genome with wild-type oriP (2908 wt-oriP). B) Deletion of the dyad symmetry element (DS) of oriP (2910  DS) does not lead to a deviation of the EBV:EBNA1-colocalisation in perichromatin. The alteration of the spatial integrity of oriP by translocating either the family of repeats (FR) (2912eFR; C) or, the dyad symmetry element (DS) (2913eDS; D) does neither affect the EBV:EBNA1-colocalisation nor the perichromatic localization of the EBV-genomes. (E-H) Signal-intensity scans for EBNA1 (green; channel 1), Mini-EBV-genomes (red; channel 0) and DNA-counterstain (blue; channel 2) along the indicated line in the respective LCLs. EBV-peaks colocalize with peaks for EBNA1. Colocalization does not occurr in the peaks of the DNA-counterstain, but next to it, indicating perichromatic localization. Scale bar = 2 µm.

Figure S6 - Part ,6815,423 73,3 distance in µm greyscale value ,753,515,26 73,3 distance in µm greyscale value ,322,653,97 73,3 distance in µm greyscale value H3K4me3 H3K9me3 H3K27me3 LCL2908 wt-oriP C A B

Figure S6 - Part ,583,164,74 58,1 distance in µm greyscale value ,272,533,8 73,3 distance in µm greyscale value ,092,193,28 73,3 distance in µm greyscale value LCL2910-  DS H3K4me3 H3K9me3 H3K27me3 F D E Supplementary Figure S6 Lymphoblast-cells LCL2908 wt-oriP A)-C) and LCL2910 ∆DS D)-F) after combined immunofluorescence and fluorescence in situ hybridization. EBV-genomes were visualized by FISH using an EBV-specific probe (red). Colocalization with histone3 trimethylated at lysine 4 (H3K4me3) (A+D), histone3 trimethylated at lysine 9 (H3K9me3) (B+E) and histone3 trimethylated at lysine 27 (H3K27me3) (C+F) are shown in green. Signal-intensity scans of the respective histone modifications (green), EBV (red) and DNA-counterstain (blue) along the indicated line in the respective cells. EBV-peaks colocalize with peaks for H3K4me3. Colocalization occurs rarely with the peaks of H3K9me3. Colocalization with H3K27me3 is increased in LCL2910∆DS. Scalebar = 2 µm.