Science - USA (2022-01-07)

Gc function in membrane fusion, we estab-
lished an assay to follow syncytia formation of
cells expressing the CCHFV glycoproteins at
their surface upon low-pH treatment. We used
this assay to test single point mutations at the
interface between domains I and III (at the

turn of the postfusion hairpin) to explore their

functional effect. Alanine substitutions of

two conserved residues abrogated low-pH–

triggered cell-cell fusion: His^1479 on domain

III, which makes a salt bridge with Glu^1113 of

domain I, and Trp^1068 in the N-terminal tail,

which projects into a pocket at the domain I/II

boundary (Fig. 2, D to E). Gc-derived linear

peptides spanning the N-terminal tail (amino

acids 1041 to 1060 and 1061 to 1080) around

the functionally important Trp^1068 residue

robustly react with CCHFV-positive human

106 7 JANUARY 2022•VOL 375 ISSUE 6576 science.orgSCIENCE

GnGn

F900

W766

bb

cc

dd

ii

jj

A901

P774

V776

N769

N764

Y739

Y762

11

44

55

22

MPRLV Gn/Gc pre-fusion

1

5

6

3

2

W1191

W1199

M1362

T1166

V1201

bb

cc

dd

ii

jj

N1194

mm

W1365

W1197

R1189

T1206

CCHFV Gc post-fusion

ij

Lo

op

cdLoop

bc

Lo

op

1

4

5

2

F900

W766

bb

cc

dd jjii

A901

P774

V776

N769

N764

Y739

Y762

MPRLV Gc post-fusion

cdLoop

bc

Lo

op

ij

Lo

op

1

5

6

(^23)
W1199 W1191
M1362
bb
cc
dd ii
jj
N1194
W1365
W1197
R1189
T1206
HCHC LCLC
CCHFV Gc / ADI-37801 complex
200
100
0
CCHFV
Ortho-
nairo
bc Loop cd Loop ij Loop
MPRLV
Ortho-
hanta
200
100
Area (Å 0
2 )
Area (Å
2 )
1
H 2 O
L3L3
H3H3
W1191
V1201
H1187
W1197
N1194 W1199
T1203
G1202
T1166
bc Loop
cd Loop
T57
S56
S54
Y53
S100B
M97
Y35
Y50
Y52
S100
Y99
Y94
S93
W32
L1L1
H2
90°/x H1H1
90°/z
BB EE
CC
D’D’
AA
FF
11
9
12
+-
11 GG
33
E1113
H1479
CC (^00) DD 00 EE 00 FF^00
kkllaa
7
D1139 E1232
K1414
H1398
S1141
I1234
P1067
W1068
EE
BB
CC
D’D’
A
FF
11
9
12
+-
11 GG
33
E1113
H1479
CC (^00) DD 00 EE 00 FF^00
kkllaa
7
D1139
E1232
K1414
H1398
S1141
I1234
P1067
W1068
W1191AW1197AW1199AM1362AW1365
A
H1398AH1479
A
Wild typW1068A
e
N1563A
Acidic pH
Neutral pH
100
0
150
% Relative
cell-cell fusion
50
% Gc surfaceexpression
0
20
40
Empty vector

---

ns

---

ns ns ns ns ns ns ns ns ns
A
B D
C
F G
E
0.5
0.4
0.3
0.2
0.1
0.0
0 500 1000 1500 2000
33.3
11.1
3.70
1.23
BLI shift (nm) 0.41
pH 7.5 Gc (^1579) WT (nM)
0.5
0.4
0.3
0.2
0.1
0.0
0 500 1000 1500 2000
Time (s)
33.3
11.1
3.70
1.23
BLI shift (nm) 0.41
pH 5.5 Gc (^1579) WT (nM)
kon
2.6 x10^5 M-1s-1
koff
2.3 x10-5 s-1
KD
89 pM
kon
3.4 x10^5 M-1s-1
koff
4.5 x10-5 s-1
KD
133 pM
Fig. 2. ADI-37801 binds HMIS residues required for Gc-driven syncytia
formation.(A) The CCHFV HMIS of the postfusion trimer (left) and in complex
with ADI-37801 (right). In the left panel, W1191, W1197, and W1199, which were
mutated to obtain the crystals, have been modeled for clarity. (B) Hantavirus
fusion loops in the postfusion trimer forming the HMIS (left; PDB ID: 6y68, MPRLV
structure) and in the prefusion Gn-Gc heterodimer, where the HMIS is not formed
(right; PDB ID: 6y62) ( 15 ). (C) Fusion loop sequences of CCHFV Gc with consensus
sequence logo for theOrthonairovirus(top) andOrthohantavirus(bottom) genera.
Thebarchartshowstheexposedsurfaceareaperresidueinpre-(hantavirusGc)
and postfusion (CCHFV and hantavirus Gc) structures. The accessible and buried
surface per residue are represented in gray and black, respectively. Nonpolar
residues, black; acidic, red; basic, blue; cysteines, green. (D) CCHFV Gc-induced
syncytia formation by wild-type and indicated mutant Gc at neutral and acidic pH.
The transfected cell surface expression is shown for each mutant below. (E) Details
of two alternative conformations of the N-tail and a pH-sensitive salt bridge
between domains I and III. The helical conformation (top) is dominant, whereas
theb-hairpin (bottom) is well defined in only two of the six polypeptide chains in the
asymmetric unit of the monoclinic crystals obtained at pH 7.5. The view is the
same as in Fig. 1C. (F) Interface between the ADI-37801 CDRs and the Gc fusion
loops. The antibody heavy and light chain CDRs are colored blue and gray,
respectively. CCHFV Gc is colored orange (cdloop) and yellow (bcloop). Polar
interactions are denoted by dashed lines. (G) Biolayer interferometry (BLI)
sensorgrams showing binding kinetics of CCHFV Gc^1579 to ADI-37801 at pH 7.5
(top) or pH 5.5 (bottom). WT, wild-type;KD, dissociation constant;kon, on rate
constant;koff, off rate constant. ns, not significant. (P>0.05);,P< 0. 0 1 ; *,P<

0. 0 0 1 ; a n d ,P< 0.0001). Single-letter abbreviations for the amino acid residues are
   as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu;
   M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.

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