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 VectorsHelmholtzZentrum muenchenMarchioninistrasse 2581377 MunichGermany      

 *Corresponding Author:Aloys SchepersPhone: 0049 89 7099509FAX: 0049 89 7099225e-mail: schepers@helmholtz-muenchen.de

Figure S1

HEK293 EBV+

A B

C D

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+

177

118

0 2,42 4,83 7,25

58,9

distance in µm

grey

scal

e va

lue

206

137

0 3,88 7,76 11,6

68,7

distance in µm

grey

scal

e va

lue

178

118

0 1,27 2,53 3,8

59,2

distance in µm

grey

scal

e va

lue

Supplementary Figure S2Signal-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

grey

scal

e va

lue

peaks overlap

= colocalization

Distance in µm

grey

scal

e va

lue

shoulders overlap

= association

Distance in µm

grey

scal

e va

lue

peaks are separated

= not associated

D

Figure S3 - Part 1

H3K9me3

H3K9ac

H3K27me3

3D-reconstruction

3D-reconstruction

3D-reconstruction

turned 172° along x-axis

C

A

B

D

Raji

Raji

Raji

H3K4me3

Raji

3D-reconstruction

Figure S3 - Part 2

214

143

0 2,19 4,37 6,55

71,3

distance in µmgr

eysc

ale

valu

e

220

147

0 2,16 4,31 6,47

73,3

distance in µm

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scal

e va

lue

220

147

0 2,1 4,2 6,3

73,3

distance in µm

grey

scal

e va

lue

220

147

0 3,51 7,02 10,5

73,3

distance in µm

grey

scal

e va

lue

H3K4me3

H3K9ac

H3K9me3

H3K27me3

F

E

H

G

Distance in µm

grey

scal

e va

lue

peaks overlap

= colocalization

Distance in µm

grey

scal

e va

lue

shoulders overlap

= association

Distance in µm

grey

scal

e va

lue

peaks are separated

= not associated

IRaji

Raji

Raji

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

9,2 5,8 5,6 10,0 and 5,4 10,7 and 4,5

.II .4 .XI .XII .4 .II .XII.3

2800 2908 2910 DS 2912eFR 2913eDS

.4 .I

translocated

size (kbp)

oriP

enzyme

B2908 H2O

280 bp

M

2913eDS: PCR Analysis.4 .II .XII

160 bp

clone ID

clone ID

Figure S5 - Part1

LCL2908 wt-oriP

HEK293-2912eFR

LCL2913eDS

hypertone

hypertone

hypertone

LCL2910-DS

hypertone 3D-reconstruction

3D-reconstruction

3D-reconstruction

3D-reconstruction

C

A

B

D

Figure S5 - Part2

211

145

0 2,59 5,18 7,76

70,4

distance in µm

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lue

198

132

0 2,44 4,89 7,33

66,4

distance in µm

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scal

e va

lue

172

135

0 2,58 5,12 7,68

57,5

distance in µm

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scal

e va

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177

138

0 3,13 6,27 9,4

58,9

distance in µm

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scal

e va

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LCL2908 wt-oriP

HEK293-2912eFR

LCL2913eDS

hypertone

hypertone

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 (2910DS) 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 - Part1

220

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0 7,68 15,4 23

73,3

distance in µm

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220

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0 1,75 3,51 5,26

73,3

distance in µm

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220

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0 1,32 2,65 3,97

73,3

distance in µm

grey

scal

e va

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H3K4me3

H3K9me3

H3K27me3

LCL2908 wt-oriP

C

A

B

Figure S6 - Part2

174

116

0 1,58 3,16 4,74

58,1

distance in µm

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lue

220

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0 1,27 2,53 3,8

73,3

distance in µm

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scal

e va

lue

220

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0 1,09 2,19 3,28

73,3

distance in µm

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scal

e va

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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.