2016 Analyses of Coronavirus Assembly Interactions with Interspecies Membrane and Nucleocapsid Protein Chimeras

JVI Accepted Manuscript Posted Online 17 February 2016J. Virol. doi:10.1128/JVI.03212-15Copyright © 2016, American Society for Microbiology. All Rights Reserved.

JVI03212-15 Version 2 page 2 of 30 02.04.16






g












in

vitro






In






Tm NTD (N1b)

SR CTD (N2b)

B N3

mock



mock

AM

M ecto M endoTm1 Tm2 Tm3

M monomer-monomercontacts

M dimer-dimer contactsM-N, M-S contacts

NNTD (N1b) CTD (N2b) N3SR B

N-nsp3interaction

N-RNA binding N-RNA bindingN-N interaction

M-N contacts

C

N3

CTD (N2b) N3spacer BN MHV ...ELQYSGAVRFDSTLPGFETIMKVLNENLNAYQKDGGADVVSPKPQRKGRRQAQEKKDEVDNVSVAKPKS 417N SCoV ...WLTYHGAIKLDDKDPQFKDNVILLNKHIDAYKTFPPTEPKKDKKKKTDEAQPLPQRQKKQPTVTLLPAA 399

N MHV SVQRNVSRELTPEDRSLLAQILDDGVVPDGLEDDSNV 454N SCoV DMDDF-SRQLQNSMSGASADSTQA 422

B M ecto Tm1 Tm2

Tm3 M endo

M endo

M MHV MSSTTQAPEPVYQWTADEAVQFLKEWNFSLGIILLFITIILQFGYTSRSMFIYVVKMIILWLMWPLTIVLCIFNCVY 77M SCoV MADNGTITVEELKQLLEQWNLVIGFLFLAWIMLLQFAYSNRNRFLYIIKLVFLWLLWPVTLACFVLAAVY 70

M MHV ALNNVYLGFSIVFTIVSIVIWIMYFVNSIRLFIRTGSWWSFNPETNNLMCIDMKGTVYVRPIIEDYHTLTATIIRGH 154M SCoV RINWVTGGIAIAMACIVGLMWLSYFVASFRLFARTRSMWSFNPETNILLNVPLRGTIVTRPLMESELVIGAVIIRGH 147

M MHV LYMQGVKLGTGFSLSDLPAYVTVAKVSHLCTYKRAFLDKVDGVSGFAVYVKSKVGNYRLPS-NKPSGADTALLRT 228M SCoV LRMAGHPLGRCD-IKDLPKEITVATSRTLSYYKLGASQRVGTDSGFAAYNRYRIGNYKLNTDHAGSNDNIALLVQ 221

C

B

kDa

25

32

46

5880

135

moc

k

MN

3

wild

type

-N (MHV)

kDa

2522

17

32

moc

k

moc

k (V

)

MN

3

wild

type

SCoV

(V)

-M (SCoV)

kDa

252217

32m

ock

moc

k (V

)

MN

3

wild

type

SCoV

(V)

-M (MHV)

MN3 wild type

37oC

39oC

33oC

ANTD CTD N3ecto endoTm1,2,3

M N

MHV SARS-CoV

MHV wt

MN2 lethal

MN1 lethal

MN3

A

E MHV VLSPSIYLYDRSKQLYKYYNEEMRLPLLEVDDI 83E SCoV LVKPTVYVYSRVKNL....NSSEGVPDLLV 76

I rev1 K rev2K rev4, rev6

E MHV MFNLFLTDTVWYVGQIIFIFAVCLMVTIIVVAFLASIKLCIQLCGLCNTL 50E SCoV MYSFVSEETGTLIVNSVLLFLAFVVFLLVTLAILTAHRLCAYCCNIVNVS 50

A rev5V rev7S rev3

B ME N

MHV SARS-CoV

MHV wt

MN6

MN3

MN7F20S

C MN3 MN3rev3 MN6 MN7wild type

39oC

37oC

39oC

37oC

1.2 x 108 1.1 x 106 4.8 x 107 4.1 x 105

5.5 x 1056.1 x 107

3.5 x 107

3.3 x 1071.7 x 1061.2 x 108

A

B

ME N

MHV SARS-CoV

MHV wt

MN9

MN8

MN10F20S

F20S

MN8 MN9 MN10wild type

37oC

C

MN4B

MN4A

MN4C

SGFA

YK

GNY

MN5A

MN5B GNY

SGFA

1.1 x 108 6.0 x 106 1.1 x 106 3.7 x 106

A

4321 8765 1211109 151413 < bottomtop >

dens

ity (g

/ml)

Infe

ctiv

ity (l

og P

FU/m

l)9

8

7

6

1.150

1.130

1.110

1.090

1.070

MN8wild type

Dwildtype RNA

1.00

0.50

0.25

CMN8

kDa

2522

324658 N

M

F

EMN8 RNA

1.00

0.50

0.25

Bwildtype

kDa

2522

32

4658

N

M

N protein M protein gRNA PFU

1.00 1.00 1.00

1.05 0.85 1.58

wild type 3.56 x 1010

MN8 1.23 x 109

C

virion

MN

8

wild

type

SC

221711

3258

kDa

B

intracellular

MN

8

wild

type

moc

k

S2

S0

kDa245190

58

80

100135

A

N

M

S2

S0

MN8wild type7 8 9 10 4 5 56 7kDa

2522

17

32

465880100135

virion

Eecto endoTm

M

ES

N

MHV SARS-CoV

MN11F20S

D

virion

MN

8

MN

3

MN

3rev

3

wild

type

S2

S0

kDa

190

58

80

100

135

245

Documents

2016 Analyses of Coronavirus Assembly Interactions with Interspecies Membrane and Nucleocapsid Protein Chimeras