2015 dissection of amino-terminal functional domains of murine coronavirus nonstructural protein 3
TRANSCRIPT
JVI Accepted Manuscript Posted Online 25 March 2015J. Virol. doi:10.1128/JVI.00197-15Copyright © 2015, American Society for Microbiology. All Rights Reserved.
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per se
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in vitro
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In vitro
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cis
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mfold
mfold
in vitro
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cis
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cis
Thosea asigna
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in vitro
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in vitro in vivo
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in vitro
ts
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in vivo
in vitro
in
vivo
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cis
cis
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cis
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in vitro in vivo
Thosea asigna
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i.e.
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ts
ts
ts
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In
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cis
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Ubl1
Ac PLP1 ADRP
Ubl2 PLP2 NAB
G2M TM1 and 2
Y
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in vitro
In vitro
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in vitro
in vitro
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MHV
nsp3
3’ 5’ A n 1 2 3 4 5 6 7-10 11
12 13 14 15 16rep 1a rep 1b
S E M N
hydrophobic region(cleavage site )(cleavage sites )
N- -C
1161 252233 484 622 827775 1085 12321113 1273 1390 1633 2005
PLP2PLP1
Ubl2
[SUD]Ac ADRPUbl1 NAB G2M Y
TM1 TM2
A
B
C
U
G
C
A
A
U
C G
GC
GUGU
G
A
C
U
C
U
G
G
UC
AG
GA U
U
CGCG
GC
CC
U
G
A
UU
G
AG
A
A AA G
G CG C
CG
AA AGU
AU
A
GU
A
G CU
CGC
GUA
AU
GC
CU
AC
UAA
C
GG C
CC
C
GCGU
U
UUU
U G
C
A
GU
SL4
SL5
SL6
SL7
SL8
C
GUGC G
C
CUA
AU
GCAU
UA
UC
UG
U
A
U
U
CG
G
A UU
CG
CGUG
C
C
U
GCU
GA
UU
CGAG
A UAG
G
U
C
UG
C
GC
GAUA
A
A
GU
SLC
G C
CUGG
C
U AC
U
CCG
UUUAA
AAC
U
C GG
UUG
CUAGU
C
G
G
U
GA UA
C
U
GGC
U
A
CUAUC GUG
AC
A
GU
C GG C
UA UC
C
G
GCG
GU
U
CA
GCUG
G
A
C
U
GC G
U
U
UG
U
UC
AC
U
U
U
CC
C G
C G
CGC
G
SLB
GC
GCG U
G CU
A G
U
CG
G
U AU
GC
UAGC
G
C
U
UG
CG
GUA
SLA
GC
C
GGA
C
CU
C
AG
ACU
U
A
G
GG
A
GA A
A
CC G
GU
CC GU
G
A
UA
A
GU
GCUGCC
C
GGCAAGGUA5’81 131 172 226 234 316 318 336 377 447 454 491 492 519 521 580 594 2707
A
AG
G C
C
U
U
U
CC G
GG UAC GG
U
C
U G
CCA
GU G
U
GU A
C
CCA
GG C
AC
C
GG A
A
UA
CC
AC
AC
UA
A
GUA
GU
A
nsp1startcodon
5’*
*
* *** ** *
*
*
123
4
5
6
7
8A
BC
LRAUG- nsp3
wild type
N3S mutant
nsp1 nsp2 nsp3
5’1
23
4
5
6
7
8A
BC
LR
B
C
[nsp3]nsp1-[nsp2]
pA-WT/BstZ17IpA-N3S/SpeI
pA-N3S 1/SpeIpA-N3S 2/SpeIm
ock
2025
100150
806050
40
30
kDa
AN3S mutant
pA-WT RNA
pA-N3S RNA
pA-N3S 1 RNA
pA-N3S
5’ nsp140.2 kDa
BstZ17I210 nt
35.9 kDa
5’ * * ** **** * * * nsp3
SpeI
5’ nsp3
5’ ** * nsp3
591 nt
249 nt
25 nt
A
B nsp3 E851G D1791V
D1791G rev1, rev3
I813T rev2V1449F rev6
A1437S rev12E146(EKEM) rev12
D142G rev4
A38V rev6A60T rev7, rev8
D98Y rev8
L480I rev9
D1757E rev2, rev7N1705S rev4
D1791A rev5, rev8-rev12
N- -C
1 252233 484 827775 1085 1390 1633 2005
PLP2PLP1
Ubl2
Ac ADRPUbl1 NAB G2M YTM1 TM2
wild type
N3S mutant N3S rev2 39oC
N3S rev12 39oC37oC
37oC
39oC
39oC
33oC
33oC
A
B C
wild type
SSN3
SSN3x lethal
BstZ17I
5’ nsp1 nsp2 nsp340.2 kDa
His7 --- FMDV 2A stop-start: (49 aa) - G D V E S N P G P SSN3 mutant
His7 --- FMDV 2A stop-start: (49 aa) - G D V E S N L A P SSN3x mutantx
5’ [nsp1] nsp357.4 kDa
x
SpeI
5’ [nsp1] nsp321.5 kDa 35.9 kDa
wild type
SSN3 mutant
37oC
37oC
39oC
39oC
33oC
33oC
[nsp1]-2A-[nsp3]
[nsp1]-2A
[nsp3]nsp1-[nsp2]
pA-WT/BstZ17IpA-SSN3/SpeI
pA-SSN3x/SpeIpA-N3S 1/SpeIm
ock
2025
100150
806050
40
30
kDa
B
A
SSN3HASSN3 SSN3HAmutP SSN3HA P3wild type
37oC
SSN3HA
SSN3HAmutP
SSN3HA P1 lethal
SSN3HA P2 lethal
SSN3HA P3
234 484HA
5’ [nsp1] nsp3
274 460HA
5’ [nsp1] nsp3
234 651HA
5’ [nsp1] nsp3
5’ [nsp1] nsp3HA
5’ [nsp1] nsp3
C289AHA
827116
PLP2PLP1
Ubl2
Ac ADRPUbl1
1 233 484 622 775 1085252
B
A
IP: -nsp3 (VU164)WB: -HA
IP: -nsp3 (D3)WB: -HA
moc
k
SSN
3HA
SSN
3HA
SSN
3HA m
utP
SSN
3
190135
245kDa
190135
245kDa
C
D
WB: -nsp1
WB: -nsp8m
ock
wild
type
SSN
3HA
SSN
3HA
SSN
3HA m
utP
SSN
3
2217
25
32kDa
2217
25
3246
kDa
B
A
h postinfection
log
PFU
/ml
2
3
4
5
6
7
8
168 24 32 40 48
wild typeSSN3HA
SSN3HA P3
gRNA
sgRNA4
sgRNA7
sgRNA6
sgRNA5
sgRNA3
sgRNA2
wild
type
moc
k
SSN
3HA
SSN
3HA
P3
wild
type
moc
k
SSN
3HA
SSN
3HA
P3