次世代シークエンスを活用した レトロウイルス研究...
TRANSCRIPT
次世代シークエンスを活用したレトロウイルス研究
佐藤 賢文International Research Center for Medical Sciences (IRCMS)
Center for AIDS Research, Kumamoto University, Japan
第15回ウイルス学キャンプ in湯河原 June 6, 2018 Kanagawa, Japan
HTLV-1 (human T-cell leukemia virus type 1 )
Origin, spread and prevalence of HTLV-1
from Verdonck K. et al (2007): Lancet Infectious Diseases 7(4): 266.
endemic retrovirus in tropical area including south-west Japan
most of the carriers are asymptomatic
about 5 % of the carriers develop leukemia of infected cells
(adult T-cell leukemia; ATL)
Some carriers develop chronic inflammatory diseases,
such as HTLV-1 associated myelopathy (HAM/TSP)
HTLV-1 (human T-cell leukemia virus type 1 )
&'(!)*++,!
Life cycle of retrovirus
"#$%!&'((!
Nucleus )*+,(!-./!
)*+,(!-./!
)*+,(!0+#%'*1$!
2./!
)*+,(!-./!
-%./)*!)*++!012132,$,
&-45
6-78"
7$9/,!192./):2%!$,!;*9<!=$>=
-%./)*!)*++!*?10%,$2%
&'(
6'(78"
7$9/,!192./):2%!;*9<!+2@
HIV-1
Apoptosis AIDSCD4+ cells
Latent/Persistent infection
HTLV-1
CD4 T cells
Asymptomatic carrierVirus Latent/Persistent infection
Virus
5%
Leukemia of CD4+ T cells (ATL)
, (1%)
(CTL )
!!
!!
HTLV-1 carrier
-'%+#3*+,(!4'1#5'"65,1!4'1#5'
Provirus transcription should be regulated to evade host immunity
HTLV-1 replicates mainly via clonal expansion of infected cells
Interaction between the host and retroviral genome
Integration of HTLV-1
3’LTR 5’LTR
IFT81
chr: 12
130kb
H3K4me3
H3K36me3
H3K9Ac
(Promoter)
(Elongation)
(Active promoter)
ChIP-seq (epigenetic marks)
HTLV-1 integration site in an ATL clone
A
small genome size (9 ) both plus and minus strand transcription
Structure of HTLV-1
pX region
'0?!888!0):;0329!2B!#C('D!!!!!!!!!!!!!E/+:1+*!2%)2>*%$)!0):;$3<!!6FG!888!E/+:1+*!2%)2>*%$)!0):;$3<!!!!!!!!!!!!!!!*?10%,$2%!2B!$%B*)3*.!)*++,!
Integration of HTLV-1
3’LTR 5’LTR
H3K4me3
H3K36me3
H3K9Ac
H2AZ
IFT81
chr: 12
Example of integration of HTLV-1 into a host gene
130kb
"H
Epigenome pattern of HTLV-1 provirus
(an ATL cell line, ED)
B
C
Primary HTLV-1+ T-cell clone (TBX-4B)
0
5
10
15
20
H3K4me3
IgG
0
1
2
3
4
0
5
10
15
20
H3K4me3
IgG
0
2.5
5
7.5
10
H3K36me3IgG
ATL-derived T-cell line (ED)
0
25
50
75
100
0
0.2
0.4
0.6
0.8H2A.ZIgG
3'LTR
pX2
pX1
env
pol
gag
5'LTR
3'LTR
pX2
pX1
env
pol
gag
5'LTR
3'LTR
pX2
pX1
env
pol
gag
5'LTR0.08
3'LTR
pX2
env
pol
gag
5'LTR
pX1pX1
3'LTR
pX2
env
pol
gag
5'LTR
3'LTR
pX2
pX1
env
pol
gag
5'LTR
3'LTR
pX2
pX1
env
pol
gag
5'LTR
0
25
50
75
100
3'LTR
pX2
pX1
env
pol
gag
5'LTR
0
0.4
0.8
1.2
1.6
pX1
3'LTR
pX2
env
pol
gag
5'LTR
0
0.2
0.4
0.6
0.8
H3K9AcIgG
3'LTR
pX2
pX1
env
pol
gag
5'LTR
H3K36me3IgG
H3K9Ac
IgG
D
E
% o
f Inp
ut%
of I
nput
% o
f DN
A m
ethy
latio
n%
of
DNA
met
hyla
tion
0.06
0.04
0.02
0
H2A.ZIgG
gag pol env pX 5’LTR 3’LTR
3’LTR
A
5’LTR gag pol env pX1 pX2
Figure 1
F
Input DNA
G
HHIV-1-infected cells
(U1 cells)
0
1
2
3
H3K36me3
IgG
3'LTR
tatenv
vifpol
gag
5'LTR
% o
f Inp
ut
position of HIV-1 genome
DNA methylation
H3K4me3
HTLV-1
H3K36me3
5’LTR 3’LTR
H2A.Z
H3K9Ac
Epigenetic Border
H3K9Ac
H2A.Z
H3K36me3
50
25
0
50
25
0
34
17
0
26
13
0
H3K4me3142
71
0
5’LTR 3’LTRgag pol env pX
B
C
Primary HTLV-1+ T-cell clone (TBX-4B)
0
5
10
15
20
H3K4me3
IgG
0
1
2
3
4
0
5
10
15
20
H3K4me3
IgG
0
2.5
5
7.5
10
H3K36me3IgG
ATL-derived T-cell line (ED)
0
25
50
75
100
0
0.2
0.4
0.6
0.8H2A.ZIgG
3'LTR
pX2
pX1
env
pol
gag
5'LTR
3'LTR
pX2
pX1
env
pol
gag
5'LTR
3'LTR
pX2
pX1
env
pol
gag
5'LTR
0.08
3'LTR
pX2
env
pol
gag
5'LTR
pX1pX1
3'LTR
pX2
env
pol
gag
5'LTR
3'LTR
pX2
pX1
env
pol
gag
5'LTR
3'LTR
pX2
pX1
env
pol
gag
5'LTR
0
25
50
75
100
3'LTR
pX2
pX1
env
pol
gag
5'LTR
0
0.4
0.8
1.2
1.6
pX1
3'LTR
pX2
env
pol
gag
5'LTR
0
0.2
0.4
0.6
0.8
H3K9AcIgG
3'LTR
pX2
pX1
env
pol
gag
5'LTR
H3K36me3IgG
H3K9Ac
IgG
D
E
% o
f Inp
ut%
of I
nput
% o
f DN
A m
ethy
latio
n%
of
DNA
met
hyla
tion
0.06
0.04
0.02
0
H2A.ZIgG
gag pol env pX 5’LTR 3’LTR
3’LTR
A
5’LTR gag pol env pX1 pX2
Figure 1
F
Input DNA
G
HHIV-1-infected cells
(U1 cells)
0
1
2
3
H3K36me3
IgG
3'LTR
tatenv
vifpol
gag
5'LTR
% o
f Inp
ut
position of HIV-1 genome
DNA methylation
H3K4me3
HTLV-1
H3K36me3
5’LTR 3’LTR
H2A.Z
H3K9Ac
Epigenetic Border
H3K9Ac
H2A.Z
H3K36me3
50
25
0
50
25
0
34
17
0
26
13
0
H3K4me3142
71
0
5’LTR 3’LTRgag pol env pX
% o
f Inp
ut
Koiwa et al, JV 2002 Takeda et al, Int J Cancer 2004 Taniguchi, Retrovirology, 2005
HTLV-1 HTLV-1
""
Typical pattern of proviral transcription in ATL cells
5’LTR
3’LTR Virus production
Latency Cell proliferation
?
7! 8! 9! :! ;! <! =! >! ?! 7@!
Suppression! Activation"
Virus production
5’LTR
Virus production
Suppression!
Virus production
Latency Cell proliferation
3’LTRVirus production
Latency Cell proliferationCell proliferation
Activation"
7! 8! 9! :! ;! <! =! >! ?! 7@!
(Satou Y et al PNAS 2006)
"I
ChIP assay, ChIP-seq
B
E
C
Primary HTLV-1+
T-cell clone (11.63)
F
PBMCs
(HAM/TSP)
ATL-derived
0
0.1
0.2
0.3
CTCF
IgG
3'LTRpX2pX1envpolgag5'LTR
0
0.1
0.2
0.3
CTCFIgG
pX2pX1envpolgag
0
0.1
0.2
0.3
3'LTRpX2 pX1env pol gag 5'LTR
3'LTR
% o
f Inp
ut
% o
f Inp
ut%
of I
nput
A
Sim
ilarit
y sc
ore
(%)
Nucleotide position of HTLV-1 genome (kb) PBMCs
(ATL)
% o
f Inp
ut
0
0.1
0.2
0.3
0.4
0.5
pX2pX1envpolgag
CTCF
IgG
5'LTR 3'LTR
CTCF
IgG
20
40
60
80
00
0 1 2 3 4 5 6 7 8 9
antisense
sense
5'LTR
T-cell line (ED)
3'LTRta
t
envvifpol
gag
5'LTR pol2 nef
TC1
% o
f Inp
ut
0.5 HIV-1-infected cells (U1 cells)
CTCF
IgG
D
0.4
0.3
0.2
0.1
0
Figure 2
G
Input DNA50
25
0
5’LTR 3’LTR
56
28
0
gag pol env pX
CTCF
CTCF
ChIP-seq analysis of an ATL cell line
H3K4me3
H3K4me3
H3K4me3
ChIP-seq analysis of an ATL cell line
H3K4me3
*1$6(,%#+
H3K4me3H3K4me3
H3K4me3H3K4me3
H3K4me3H3K4me3
H3K4me3H3K4me3
&):;* -%0):;*
CTCF forms DNA looping and regulates intra- and inter-chromosomal interaction.
Chromatin looping
CTCF (CCCTC-binding factor)・A DNA-binding protein, critical organizer of higher-order chromatin structure
"J
B
E
C
Primary HTLV-1+
T-cell clone (11.63)
F
PBMCs
(HAM/TSP)
ATL-derived
0
0.1
0.2
0.3
CTCF
IgG
3'LTRpX2pX1envpolgag5'LTR
0
0.1
0.2
0.3
CTCFIgG
pX2pX1envpolgag
0
0.1
0.2
0.3
3'LTRpX2 pX1env pol gag 5'LTR
3'LTR
% o
f Inp
ut
% o
f Inp
ut%
of I
nput
A
Sim
ilarit
y sc
ore
(%)
Nucleotide position of HTLV-1 genome (kb) PBMCs
(ATL)
% o
f Inp
ut
0
0.1
0.2
0.3
0.4
0.5
pX2pX1envpolgag
CTCF
IgG
5'LTR 3'LTR
CTCF
IgG
20
40
60
80
00
0 1 2 3 4 5 6 7 8 9
antisense
sense
5'LTR
T-cell line (ED)
3'LTRta
t
envvifpol
gag
5'LTR pol2 nef
TC1
% o
f Inp
ut
0.5 HIV-1-infected cells (U1 cells)
CTCF
IgG
D
0.4
0.3
0.2
0.1
0
Figure 2
G
Input DNA50
25
0
5’LTR 3’LTR
56
28
0
gag pol env pX
CTCF
CTCF
ChIP-seq analysis of an ATL cell line
H3K4me3
H3K4me3
H3K4me3
H3K4me3
H3K36me3
H3K9Ac
H2A.Z
Input DNA
B
C
Primary HTLV-1+ T-cell clone (TBX-4B)
0
5
10
15
20
H3K4me3
IgG
0
1
2
3
4
0
5
10
15
20
H3K4me3
IgG
0
2.5
5
7.5
10
H3K36me3IgG
ATL-derived T-cell line (ED)
0
25
50
75
100
0
0.2
0.4
0.6
0.8H2A.ZIgG
3'LTR
pX2
pX1
env
pol
gag
5'LTR
3'LTR
pX2
pX1
env
pol
gag
5'LTR
3'LTR
pX2
pX1
env
pol
gag
5'LTR
0.08
3'LTR
pX2
env
pol
gag
5'LTR
pX1pX1
3'LTR
pX2
env
pol
gag
5'LTR
3'LTR
pX2
pX1
env
pol
gag
5'LTR
3'LTR
pX2
pX1
env
pol
gag
5'LTR
0
25
50
75
100
3'LTR
pX2
pX1
env
pol
gag
5'LTR
0
0.4
0.8
1.2
1.6
pX1
3'LTR
pX2
env
pol
gag
5'LTR
0
0.2
0.4
0.6
0.8
H3K9AcIgG
3'LTR
pX2
pX1
env
pol
gag
5'LTR
H3K36me3IgG
H3K9Ac
IgG
D
E
% o
f Inp
ut%
of I
nput
% o
f DN
A m
ethy
latio
n%
of
DNA
met
hyla
tion
0.06
0.04
0.02
0
H2A.ZIgG
gag pol env pX 5’LTR 3’LTR
3’LTR
A
5’LTR gag pol env pX1 pX2
Figure 1
F
Input DNA
G
HHIV-1-infected cells
(U1 cells)
0
1
2
3
H3K36me3
IgG
3'LTR
tatenv
vifpol
gag
5'LTR
% o
f Inp
ut
position of HIV-1 genome
DNA methylation
H3K4me3
HTLV-1
H3K36me3
5’LTR 3’LTR
H2A.Z
H3K9Ac
Epigenetic Border
H3K9Ac
H2A.Z
H3K36me3
50
25
0
50
25
0
34
17
0
26
13
0
H3K4me3142
71
0
5’LTR 3’LTRgag pol env pX
H3K4me3
H3K4me3
ChIP-seq analysis of an ATL cell line
"J
A
B
vCTCF-BS
CTC
F-C
hIP-
seq
110,540 kb 110,560 kb 110,580 kb
HTLV-1
5’LTR 3’LTR 110,570 kb 110,550 kb Chr 12
cCTCF-BS
C
WT ΔvCTCF-BS0.0
0.2
0.4
0.6
0.8
1.0
1.2
0.0
0.2
0.4
0.6
0.8
1.0
1.2*
0.0
0.2
0.4
0.6
0.8
1.0
1.2*
WT ΔvCTCF-BS WT ΔvCTCF-BS
Rel
ativ
e le
vel o
f exp
ress
ion
TCHP C12orf76GIT2
ns*
Rel
ativ
e le
vel o
f exp
ress
ion
Rel
ativ
e le
vel o
f exp
ress
ion
D
Chr 12
IFT81
110,400 kb 110,500 kb 110,600 kb
HTLV-1
C12orf76ANKRD13AGIT2TCHP
CTCF-BS
0.0
0.2
0.4
0.6
0.8
1.0
1.2
WT ΔvCTCF-BS
a
*
0.0
0.2
0.4
0.6
0.8
1.0
1.2
WT ΔvCTCF-BS
b
ns
0.0
0.2
0.4
0.6
0.8
1.0
1.2
WT ΔvCTCF-BS
c
ns
a b c
Rel
ativ
e in
tera
ctio
n fre
quen
cy
Rel
ativ
e in
tera
ctio
n fre
quen
cy
Rel
ativ
e in
tera
ctio
n fre
quen
cy
CTCF
""
CTCF
• CTCF
!"#$
%&'() *&'()
+,-$
• CTCF
Spread of heterochromatin
DNA methylation
-%0):;* &):;*
CTCF
methylationmethylationmethylationmethylationmethylationmethylationmethylation
Summary (1)
"K
(Satou Y et al PNAS 2016)
total 80M reads in CTCF ChIP-seq
only 180 viral reads (0.0002%)
Human genome: 3.1 billion bp HTLV-1 genome: 9 kb
Genome size difference between virus and the host
Frequency of HTLV-1 in human genome = 0.00015% L#H!ME
!NL#HMEO!
ABCDE$'F
B
E
C
Primary HTLV-1+
T-cell clone (11.63)
F
PBMCs
(HAM/TSP)
ATL-derived
0
0.1
0.2
0.3
CTCF
IgG
3'LTRpX2pX1envpolgag5'LTR
0
0.1
0.2
0.3
CTCFIgG
pX2pX1envpolgag
0
0.1
0.2
0.3
3'LTRpX2 pX1env pol gag 5'LTR
3'LTR
% o
f Inp
ut
% o
f Inp
ut%
of I
nput
A
Sim
ilarit
y sc
ore
(%)
Nucleotide position of HTLV-1 genome (kb) PBMCs
(ATL)
% o
f Inp
ut
0
0.1
0.2
0.3
0.4
0.5
pX2pX1envpolgag
CTCF
IgG
5'LTR 3'LTR
CTCF
IgG
20
40
60
80
00
0 1 2 3 4 5 6 7 8 9
antisense
sense
5'LTR
T-cell line (ED)
3'LT
Rtat
envvifpol
gag
5'LT
R
pol2 nef
TC1
% o
f Inp
ut
0.5 HIV-1-infected cells (U1 cells)
CTCF
IgG
D
0.4
0.3
0.2
0.1
0
Figure 2
G
Input DNA50
25
0
5’LTR 3’LTR
56
28
0
gag pol env pX
CTCF
N"EO
"P
1_37
1_38
2_26
2_25
3_7
3_8
Enrichment of HTLV-1 sequence
Design of biotinylated 148 probes for HTLV-1 genome 5’LTR 3’LTR
!�"#$%&'()*(+',#$-(./(+0!(1&.2',(3-4('51''-738(/8.9!/.&(7:'('-&#;:6'-7(./(<=>?@)(1&.A#&38(+0!!�
BC>=D� EC>=D�
)FG(21�
HG(21�
!IB)E)EJ�
I�
EK(>#2&3&L(,L-7:',#,�
BK(0MN*((O88%6#-3(P#N'Q(HK(!-38L,#,(
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J3K(=3&$'7'4('-&#;:6'-7�
)2K(R:&.637#-('57&3;7#.-�
)3K(+0!('57&3;7#.-�
J2K(0MN*(O88%6#-3(0'57N'Q(BK(!-38L,#,(
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0123454)*678(79!87::;�
<7*%&)8!-./�
=$'>&7*(79!-./�
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!"#$%&'()&*+,-$-.&/-$0#1%2&&3-45+-$*&'()&*+,-$-.&/-$0#1%2&&6+42+%.*$217&8-4,19&&:2-182$5+7+%&,1$79&&;$0%12�
!"#$%&'()*+,&'$-!./�
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123
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126
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!"#9*/:(;<=
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123
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126
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526!(7(8(325@B
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!"#9*/:(;<=�
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123
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=#$
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>
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&� 9� -�
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&�9� -�1:GHI�6:GHI� !=I(&,.'%M%*N("*)%#+/(
(I*)%#+/(O%0E('#O("*'&0%$*(-#$*"&)*�
!�"#$%&'()*(+',#$-(./(+0!(1&.2',(3-4('51''-738(/8.9!/.&(7:'('-&#;:6'-7(./(<=>?@)(1&.A#&38(+0!!�
BC>=D� EC>=D�
)FG(21�
HG(21�
!IB)E)EJ�
I�
EK(>#2&3&L(,L-7:',#,�
BK(0MN*((O88%6#-3(P#N'Q(HK(!-38L,#,(
F2K((R:OS�
F3K((N.-#;37#.-�
J3K(=3&$'7'4('-&#;:6'-7�
)2K(R:&.637#-('57&3;7#.-�
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J2K(0MN*(O88%6#-3(0'57N'Q(BK(!-38L,#,(
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Hybridization & capture
"A
Enrichment
( # )
( # )
( # )
( )
1000
100
20
0
0
0
10
0
2 / 0.5M reads
CTCF-ChIP-seq
Efficiency of the enrichment for the provirus
29 / 5M reads
100
20
0
0
Num
ber o
f rea
ds
180 / 80M reads
4,500 / 0.5M reads
"Q
(Miyazato P et al, Sci Rep 2016)
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Spread of heterochromatin
DNA methylation
-%0):;* &):;*
CTCF
methylationmethylationmethylationmethylationmethylationmethylationmethylation
Summary (1)
IH
1. HTLV-1
2. HTLV-1
3. HTLV-1
, (1%)
(CTL )
!!
!!
HTLV-1 carrier
JSPS JST CREST
AMED
Paola Miyazato, Misaki Matsuo
Michiyo Tokunaga , Asami Fukuda
Hiroo Katsuya, Benjy Tan Jek Yang
Islam Mohammad Saiful, Yuki Inada
Misaki Kakoki, Iwase Saori
Mitsuyoshi Nakao, Ko Ishihara, Shinjiro Hino
Charles RM Bangham
Atae Utsunomiya
Jun-ichi Fujisawa
Takaharu Ueno
Yoshikazu Uchiyama, Hiroyuki Hata
Acknowledgments
