2016 Analyses of Coronavirus Assembly Interactions with Interspecies Membrane and Nucleocapsid...
Transcript of 2016 Analyses of Coronavirus Assembly Interactions with Interspecies Membrane and Nucleocapsid...
JVI Accepted Manuscript Posted Online 17 February 2016J. Virol. doi:10.1128/JVI.03212-15Copyright © 2016, American Society for Microbiology. All Rights Reserved.
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g
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in
vitro
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In
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Tm NTD (N1b)
SR CTD (N2b)
B N3
mock
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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