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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 Marchioninistras
se 25 81377 Munich Germany               Corresp
onding Author Aloys Schepers Phone 0049 89
7099509 FAX [phone] e-mail
schepers_at_helmholtz-muenchen.de
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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 S1
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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.
Figure S2
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Figure S3 - Part 1
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Raji
Raji
Raji
Raji
Figure S3 - Part 2
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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.
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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.
Figure S4
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Figure S5 - Part1
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Figure S5 - Part2
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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 (2910DDS) does not lead to a
deviation of the EBVEBNA1-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 EBVEBNA1-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.
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Figure S6 - Part1
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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) (AD), histone3 trimethylated
at lysine 9 (H3K9me3) (BE) and histone3
trimethylated at lysine 27 (H3K27me3) (CF) 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.
Figure S6 - Part2