1
Electron tomography visualization of HIV
-
1 fusion with target cells using
fusion
1
inhibitor
s
to trap the
pre
-
hairpin
intermediate
2
3
Authors:
Mark S. Ladinsky
1
, Priyanthi
N.P. Gnanapragasam
1
,
Zhi Yang
1
, Anthony P. West, Jr.
1
,
4
Michael S,
Kay
2
, Pamela J. Bjorkman
1,*
5
6
1
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena,
7
California,
91125,
USA.
8
9
2
Department of Biochemistry, University
of Utah School of Medicine, Salt Lake City, UT, 84112,
10
USA
11
12
*corresponding author email:
bjorkman@caltech.edu
13
14
2
Abstract
15
Fusion of HIV
-
1
with
the membrane of its
target
cell
,
an obligate
first step in
virus
infectivity
,
is
16
mediated by binding of the viral
envelope (Env)
spike protein to its receptor
s
,
CD4
and
17
CCR5/CXCR4
,
on the cell surface.
The process of viral fusion
appears to be
fast
compared
with
18
viral egress
and has not been visualized
by
E
M
.
To captur
e fusion event
s
, the process must be
19
curtailed
by trapping Env
-
receptor binding at an intermediate stage.
We have
used
fusion
20
inhibitors to trap
HIV
-
1 virions attached to target cells by Envs in an extended pre
-
hairpin
21
intermediate state. E
lectron tomograp
hy
revealed HIV
-
1 virions bound to TZM
-
bl cells by 2
-
4
22
narrow spokes
, with slightly more spokes present when
evaluated
with mutant virions that
23
lacked the Env cytoplasmic tail
.
These
results
represent the first direct visualization of the
24
hypothesized
pre
-
hairpin
intermediate of
HIV
-
1 Env
and
improve
our understanding of Env
-
25
mediated HIV
-
1 fusion and infection of host cells.
26
27
Introduction
28
T
he first step of HIV
-
1 entry into a host target cell
, fusion between the viral and target cell
29
membranes, is mediated by the viral envelope spike protein (Env).
HIV
-
1 Env
is a trimeric
30
glycoprotein comprising
three gp120 subunits
that contain host receptor binding sites
and three
31
gp41 subunits
that include
the fusion peptide and membrane
-
spanning regions.
Binding of the
32
primary
receptor CD4 to gp120 triggers conformational changes that expose a binding site for
33
co
-
receptor
(CCR5 or CXCR4). Coreceptor binding
results in
further
conformational changes
34
within g
p41 that
promote
release of the hydrophobic fusion peptide, its insertion into the host
35
cell membrane, and subsequent fusion of the host cell and viral membrane bilayers
[1]
.
36
37
Structural studies relevant to understanding Env
-
mediated
membrane
fusion include
X
-
ray and
38
single
-
particle cryo
-
EM
structures of soluble
native
-
like Env trimers in the closed
(pre
-
fusion)
39
conformation
[2]
, CD4
-
bound open trimers in which the co
-
receptor binding site on the third
40
hypervariable loop (V3)
of gp120
is exposed by V1V2 loop rearrangement
[3
-
6]
, a gp120
41
monomeric core
-
CD4
-
CCR5 complex
[7]
, and
a
post
-
fusion
gp41
six
-
helical bundle formed by
42
a
n
-
helical trimeric coiled coil from the
gp41
N
-
trimer region surrounded by three helices from
43
the C
-
peptide region
[8, 9]
(
Figure
1
a
)
.
Prior to membrane fusion and formation of the post
-
44
fusion gp41 helical bundle, the viral and host cell membranes are
hypothe
sized to be
linked by
45
an extended
pre
-
hairpin
intermediate in which insertion of the gp41 fusion peptide into the host
46
cell membrane exposes the N
-
trimer
(HR1)
region of gp41
[10]
.
F
ormation
of the six
-
helical
47
bundle and subsequent fusion
can be inhibited by targeting the
N
-
trimer region
with
C
-
peptide
-
48
3
based inhibitors
; e.g.,
the fusion inhibitor T20
(enfuvirtide [Fuzeon])
[11]
,
T1249,
a more
pote
nt
49
derivative of T20
[12]
,
and a
highly potent
trimeric
D
-
peptide
(CPT31)
[13]
,
or with
anti
-
gp41
50
antibodies such as D5
[14]
(
Figure
1
a
)
.
51
52
Visualizing the
pre
-
hairpin
intermediate that joins the host and viral membranes has not b
een
53
straightforward. Despite 3
-
D imaging by
electron tomography (ET)
of HIV
-
1 infection of cultured
54
cells
[15
-
18]
and tissues
[19
-
22]
, viruses caught in the act of fusion have not been
55
unambiguously
found
.
In our
ET imaging
of HIV
-
1
–
infected humanized mouse tissues, we have
56
identified
hundreds of
budding virions at various stages of egress
and
thousands of free mature
57
and immature virions
[19
-
22]
, but not a single example
of
a virus attached to a host cell via a
58
pre
-
hairpin
intermediate or in the process of fusing its membrane with the target cell membrane.
59
The
absence
of observed viral fusion events
might
be explained if fusion is a fast proc
ess
60
compared with viral budding;
thus when cells or tissues are immobilized for EM or ET, the
61
relatively slow process of viral budding
would be
more easily
captured
compared with
the faster
62
process of fusion
.
We assume
that
fusion could
t
heoretically
be observed
if a virus were caught
63
at exactly the right time,
but this
might require examining
thousands or m
illions of images
.
64
65
Here we report
visualizing the
pre
-
hairpin
intermediate by ET after
treatment of
HIV
-
1
–
exposed
66
target cells with inhibitors of six
-
helix bundle formation that bind the N
-
trimer region of gp41 that
67
is exposed
during the fusion process
.
Using opti
mally
preserved samples for ET with a nominal
68
resolution ~7 nm
, we found
>100
examples of H
IV
-
1 virions linked to
TZM
-
bl
target cells by 2
-
4
69
narrow
rods
of density
(spokes)
in inhibitor
-
treated samples, but none in untreated or control
-
70
treated samples. The approximate dimensions of the majority of the
spoke
[23]
s
matched
71
models of gp41
-
only
pre
-
hairpin
intermediates in which the
Env
gp120 subunit had been shed.
72
The average number of
observed
spokes
connecting a virion to a target cell increased
slightly
73
when
using
a virus
containing an Env
with
a cytoplasmic tail deletion, suggesting that the
74
increased lateral mobility of cytoplasmic tail
-
deleted Env
s
in the viral membrane
[24
-
26]
a
llowed
75
more Envs to join the interaction with the target cell. We discuss
the implications of these
76
studies for understanding HIV
-
1 Env
-
mediated membrane fusion and
how these results differ
77
from
a previous ET study of the “entry claw” that is formed upon H
IV
-
1 or SIV interactions with
78
target cells
[27]
.
79
80
81
Results
82
4
Experimental design
.
A previous study used ET to visualize HIV
-
1 and SIV virions in contact
83
with target cells
after
promoting a temperature
-
arrested state
[28]
in which viruses can remain
84
attached to cells prior to fusion
[27]
.
For
that
s
tudy, t
arget cells were incubated with virus at 4
̊C
85
to allow binding but not fusion, warmed to 37
̊C
, and then fixed after incubations ranging from
86
15 min to 3 h
[27]
.
At all time points after warming, viruses were found attached to target cells
87
by a cluster of 5
-
7
“
rod
s,
”
each ~100 Å long and ~100 Å
wide.
The fact that the attachment
88
structure was not found when the viruses and target cells were
incubated in the presence of
89
C34,
a gp41
N
-
trimer
–
targeting
C
-
peptide
inhibitor
related to T20
[27]
,
suggests that the
rod
90
structure that was
trapped during the temperature
-
a
rrested state did not involve the
pre
-
hairpin
91
intermediate
.
92
93
We
hypothesized
that addition of an HIV
-
1
fusion inhibitor
that binds to the exposed gp41 N
-
94
trimer after host cell receptor and coreceptor binding would slow or stop virus
-
host cell
95
membrane
fusion such that we could visualize
pre
-
hairpin
intermediate structure
s
by ET
(
F
igure
96
1
b
)
.
We characterized three
fusion inhibitor
s of different sizes and potencies that target the
97
exposed gp41 N
-
trimer region of the pre
-
hairpin intermediate for attempts to visualize the pre
-
98
hairpin intermediate: T1249
-
Fc, a C
-
peptide
–
based inhibitor that we linked to human Fc (MW =
99
65 kDa), D5 IgG (
MW = 150 kDa)
[14]
, and CPT31, a high
-
affinity D
-
peptide inhibitor linked to
100
cholesterol (MW = 9 kDa)
[13, 45]
(
Figure
1a;
Figure 1
-
Supplementary Figure
1a
). We
101
measured their
neutralization
potencies
using in vitro HIV
-
1 pseudovirus neutralization assay
s
102
[46]
against the SC4226618 and 6535 viral strains
. We found potencies ranging from 50%
103
inhibitory concentration (IC
50
) va
lues of
~
0.13 ng/mL for CPT31 to
≥
40 μg/mL for D5 IgG
(Figure
104
1
–
Supplementary Figure 1a
). T
1249
-
Fc exhibited intermediate
potencies
(IC
50
s =
0.99 μg/mL
;
105
17 μg/mL
)
(Figure 1
–
Supplementary Figure 1a
)
,
higher than IC
50
s measured for T1249 peptide
106
alone, c
onsistent with limited steric accessibility resulting in decreased potencies for larger
107
fusion inhibitor
s
[47]
.
108
109
I
ncubating with
a fusion inhibitor
at 37
̊C
obviated the need for
a
4
̊C
incubation
of virus and
110
target cells
, which we reasoned was desirable
since low temperatures alter membrane
fluidity
111
[29
-
31]
,
which
could
affect one or more steps in membrane fusion
.
Since target cells for HIV
-
1
112
are several microns in height, much thicker than the 0.5
-
1 μm limit for cryo
-
ET
[32]
, we used
113
stained, plastic
-
embedded samples that coul
d be cut into 300
-
400 nm sections using a
114
microtome, and then
examined the samples in 3
-
D using
ET.
Although ET of stained, plastic
-
115
embedded sections results in lower resolution than
cryo
-
ET
,
the minimal effects of radiation
116