These are all Class I Virus Fusion Proteins

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These are all Class I Virus Fusion Proteins with
coiled-coil motif
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General features of viruses using a coiled-coil
fusion mechanism (myxo-, paramyxo-, filo-,
retro-,corona- and lentiviruses)

• native fusion proteins are metastable trimers.
• contain computer-predicted coiled-coil domains.
• activation by low pH, receptor-binding, both,
• or receptor/co-receptor binding.
• activated by cleavage of fusion protein (corona
  ?)
• fusion peptide at or close to (cleaved)
  N-terminus

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Class II Virus Fusion Proteins
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Common features of class II, non-coiled coil
viral fusion proteins (flaviviruses,
alphaviruses, ?)

• activated by cleavage of a companion subunit
• internal fusion peptide
• not triggered by heat/denaturants
• native dimer converts to highly stable
  homotrimer
• no computer-predicted coiled-coil domains
• TBE and SFV native structures F. Rey, CNRS

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Tick-borne encephalitis virus (TBE) Flavivbirus
plus-strand RNA, small enveloped viruses Two
envelope proteins E (fusion) and prM
(regulatory) Entry via endocytosis and low pH
triggered fusion reaction
prM form immature pH resistant
M form Furin-processed Mature, activated Low
pH-responsive
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TBE E Protein Rey et al.
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Structure of an enveloped alphavirus
Cheng et al., Cell 1995
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The Semliki Forest virus envelope proteins
N
E3
p
6
2

c
l
e
a
v
a
g
e

s
i
t
e
N
P
u
t
a
t
i
v
e

f
u
s
i
o
n

d
o
m
a
i
n

(
a
a

7
9
-
9
7
)
E2
E1
4
1
3
C
4
3
8
3
C
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Cholesterol and sphingolipid are required for
alphavirus fusion with target membranes
fusion
liposome
cholesterol
sphingolipid
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SFV low pH-induced fusion
Acid treatment 1. E2-E1 dimer
dissociation 2. Lipid bilayer binding 3.
E1 epitope exposure E1 homotrimerization 4
. Lag time..... 5. Fusion requires
cholesterol sphingolipid
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Generation of E1 and E2 ectodomains
N
E3
p
6
2

c
l
e
a
v
a
g
e

s
i
t
e
N
P
u
t
a
t
i
v
e

f
u
s
i
o
n

d
o
m
a
i
n

(
a
a

7
9
-
9
7
)
E2
E1
protease cleavage
4
1
3
C
4
3
8
C
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Domain structure of SFV E1 vs. TBE E protein.
Lescar, Roussel, Wien, Navaza, Fuller, Wengler,
Wengler, and Rey.
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Structure of SFV E1 vs. TBE E protein. Lescar,
Roussel, Wien, Navaza, Fuller, Wengler, Wengler,
and Rey.
C
SFV E1
N
Domain II (dimerization)
Domain I (central)
Domain III (C-terminal)
Fusion peptide
C
TBE E
N
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Key questions on fusion of viruses using a
non-coiled-coil mechanism

• How does the companion subunit interact with
• and regulate the fusion protein?
• How does fusion-active trimer form?
• How does the internal fusion peptide act?
• How are lipid co-factors involved?

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Regulation by the companion subunit and its
proteolytic processing.
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Virus Budding
alphavirus
flavivirus
SFV
TBE
p62 cleavage
PM
Furin
prM cleavage ( pH dependent)
TGN
Golgi
RER
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Semliki Forest Virus envelope proteins
N
C
79-97
E1 438 aa
fp
TM
N
C
N
C
E2 422 aa
E3
p62
Furin processing site of SFV
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Preparation of p62 form of Semliki Forest Virus
N
C
79-97
E1 422 aa
fp
TM
N
C
N
C
E2 438 aa
E3
p62
-R-H-R-R
Furin cleavage site
mut L (non-cleavage mutant)
-R-H-R-L-
Wild type p62--grow in furin-deficient
cells Mutant L--mutated processing site
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Fusion assay of wt/p62 and mut L
wt/p62/trypsin
wt/p62
mL/trypsin
mL
Log infectious centers / ml
pH
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Regulation and membrane interaction of the
Semliki Forest virus fusion peptide
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SFV E1 fusion peptide is masked by E2
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The mAb1F epitope is within residues 85-95 of
the fusion peptide
Residues 85-95 mAb1F
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The SFV fusion peptide is accessible in the
native E1 ectodomain
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mAb1f inhibits E1- liposome association and
homotrimerization
E1 Floatation
n3
n3
n2
n3
n2
Control
mAb1f
E1a-1
mAb1n
irr.
Antibodies
n.d.
63.8
11.5
62.7
70.4
HT (n2)
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Gradient assay of detergent-resistant membranes
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The SFV E1 fusion peptide associates with
detergent-resistant membrane domains
3H-chol ( top)
pH 7.0, TX-100
74
pH 5.5, -TX-100
74
pH 5.5, TX-100
49
top
bottom
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SFV E1 fusion peptide
mAb epitope 85-95
82-TGVYPFMWGGAYCFCDSEN-100
E99
C63
C68
C62
C78
C96
c
N100
b
F95
G83
d
D97
V84
C94
Q102
P86
F87
i
Y85
P58
e
S57
A92
M88
P226
j
W89
a
Y93
P232
H230
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Structure and function of the E1 homotrimer
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The Native E1 HT is Remarkably Stable

• Denaturants (up to 5M urea) and detergents (ionic
  and non-ionic).
• Heating alone or in combination with detergents
  or denaturants.

• A wide range of specific and non-specific
  proteases
• trypsin
• a-chymotrypsin
• thermolysin
• proteinase K
• papain
• endoproteinase Glu-C
• bromelain
• elastase

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Trypsin and Elastase Cleavage Sites
fusion peptide loop
80
102
81
97
95
79
230
1 38 83/100 130 169
273 291 381
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Elastase digestion releases trimeric E1from
liposomes
EL,2 hr.
Refloat
E1
EL1
EL2
EL4
EL5
B
B
T
T
T
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SFV E1 fusion peptide
82-TGVYPFMWGGAYCFCDSEN-100
E99
C63
C68
C62
C78
C96
c
N100
b
F95
G83
d
D97
V84
C94
Q102
P86
F87
i
Y85
P58
e
S57
A92
M88
P226
j
W89
a
Y93
P232
H230
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Role of cholesterol in SFV infection?
Select for mutants that grow in
cholesterol-depleted insect cells
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Growth in cholesterol-depleted cells
wt/ic
srf-3
srf-4
srf-5
log pfu/ml
time after infection (hrs)
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Location of srf mutations on E1 structure
srf-4
srf-3
srf-5
Domain III Domain I Domain II fusion peptide
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SFV E1 fusion peptide
82-TGVYPFMWGGAYCFCDSEN-100
E99
C63
C68
C62
C78
C96
c
N100
b
F95
G83
d
D97
V84
C94
Q102
P86
F87
i
Y85
P58
e
S57
A92
M88
P226
j
W89
a
Y93
P232
H230
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Role of class II fusion peptide vs. I-J loop
fusion I-J Protease site in homotrimer
yes yes Membrane insertion yes
? Cholesterol regulation ? yes Mutation
blocks fusion G91D H230A
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TBE E fusion peptide
98-DRGWGNHCGLFGKGSI-113
P75
S112
L107
C92
c
d
C74
K93
K110
C105
C116
F108
d
c
I113
R99
i
P247
D98
b
W101
H104
N103
A249
j
H248
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At Einstein Chantal Chanel Prodyot
Chatterjee Don Gibbons Maofu Liao
Brigid Reilly Alyson Urian Xinyong Zhang
Former Anna Ahn Christina Eng Yanping Lu Malini
Vashishtha Marianne Marquardt Tom Phalen
Collaborators Josef Brunner, ETH, Zurich
Fred Cohen, Rush Medical College Holland
Cheng, Karolinska Institute
SFV E1 structure by Félix Rey and colleagues.