2015 dissection of amino-terminal functional domains of murine coronavirus nonstructural protein 3

JVI00197-15 Version 2 page 3 of 39 03.16.15


per se


in vitro


In vitro


cis


mfold

mfold

in vitro


cis


cis

Thosea asigna


in vitro


in vitro in vivo


in vitro

ts


in vivo

in vitro

in

vivo


cis

cis


cis


in vitro in vivo

Thosea asigna


i.e.


ts

ts

ts


In


cis


Ubl1

Ac PLP1 ADRP

Ubl2 PLP2 NAB

G2M TM1 and 2

Y


in vitro

In vitro


in vitro

in vitro

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

2015 dissection of amino-terminal functional domains of murine coronavirus nonstructural protein 3

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