of 34
1
Electron tomography visualization of HIV
-
1 fusion with target cells using
fusion
inhibitor
s
to trap the
pre
-
hairpin
intermediate
Authors:
Mark S. Ladinsky
1
, Priyanthi N.P. Gnanapragasam
1
,
Zhi Yang
1
, Anthony P. West, Jr.
1
,
Michael S,
Kay
2
, Pamela J. Bjorkman
1,*
1
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena,
California,
91125,
USA.
2
Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, UT,
84112,
USA
*corresponding author email:
bjorkman@caltech.edu
.
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was not certified by peer review) is the author/funder. It is made available under a
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doi:
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2
Abstract
Fusion of HIV
-
1
with
the membrane of its
target
cell
,
an obligate
first step in
virus
infectivity
,
is
mediated by binding of the viral
envelope (Env)
spike protein to its receptor
s
,
CD4
and
CCR5/CXCR4
,
on the cell surface.
The process of viral fusion
appears to be
fast
compared
with
viral egress
and has not been visualized by electron microscopy
(EM)
.
To capture fusion event
s
for EM, the
process must be slowed or stopped by trapping Env
-
receptor binding at an
intermediate stage.
Here we describe
using
fusion
inhibitors to trap
HIV
-
1 virions attached to
target cells by Envs in an extended pre
-
hairpin intermediate state. E
lectron tomography
revealed
HIV
-
1 virions bound to TZM
-
bl cells by 2
-
4 narrow spokes
, with slightly more spokes present when
evaluated
with mutant virions that
lacked the Env cytoplasmic tail
.
These
results
represent the
first direct visualization of the hypothesized pre
-
hai
rpin intermediate and
improve
our
understanding of Env
-
mediated HIV
-
1 fusion and infection of host cells.
Introduction
T
he first step of HIV
-
1 entry into a host target cell
, fusion between the viral and target cell
membranes, is mediated by the viral envelope spike protein (Env).
HIV
-
1 Env
is a trimeric
glycoprotein comprising
three gp120 subunits
that contain host receptor binding sites
and three
gp41 subunits
that include
the fusion peptide and membrane
-
spanning regions.
Binding of the
primary
receptor CD4 to gp120 triggers conformational changes that expose a binding site for
co
-
receptor
(CCR5 or CXCR4). Coreceptor binding
results in
further
conformational changes
within g
p41 that
promote
release of the hydrophobic fusion peptide, its insertion into the host
cell membrane, and subsequent fusion of the host cell and viral membrane bilayers
[1]
.
Structural studies relevant to understanding Env
-
mediated
membrane
fusion include
X
-
ray and
single
-
particle cryo
-
EM
structures of soluble
native
-
like Env trimers in the closed
(pre
-
fusion)
conformation
[2]
, CD4
-
bound open trimers in which the co
-
receptor binding site on the third
hypervariable loop (V3)
of gp120
is exposed by V1V2 loop rearrangement
[3
-
6]
, a gp120
monomeric core
-
CD4
-
CCR5 complex
[7]
, and
a
post
-
fusion
gp41
six
-
helical bundle formed by
a
n
a
-
helical trimeric coiled coil from the
gp41
N
-
trimer region surrounded by three helices from
the C
-
peptide region
[8, 9]
(
Figure
1
a
)
.
Prior to membrane fusion and formation of the post
-
fusion gp41 helical bundle, the viral and host cell membranes are
hypothe
sized to be
linked by
an extended
pre
-
hairpin
intermediate in which insertion of the gp41 fusion peptide into the host
cell membrane exposes the N
-
trimer
(HR1)
region of gp41
[10]
.
F
ormation
of the six
-
helical
bundle and subsequent fusion
can be inhibited by targeting the
N
-
trimer region
with
C
-
peptide
-
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3
based inhibitors
; e.g.,
the fusion inhibitor T20 (enfuvirtide [Fuzeon])
[11]
,
T1249,
a more
pote
nt
derivative of T20
[12]
,
and a highly potent
trimeric
D
-
peptide
(CPT31)
[13]
,
or with
anti
-
gp41
antibodies such as D5
[14]
(
Figure
1
a
)
.
Visualizing the
pre
-
hairpin
intermediate that joins the host and viral membranes has not b
een
straightforwar
d. Despite 3
-
D imaging by
electron tomography (ET)
of HIV
-
1 infection of cultured
cells
[15
-
18]
and tissues
[19
-
22]
, viruses caught in the act of fusion have not been
unambiguously
found
.
In our
ET imaging
of HIV
-
1
infected humanized mouse tissues, we have
identified
hundreds of
budding virions at various stages of egress
and
thousands of free mature
and immature virions
[19
-
22]
, but not a single example
of
a virus attached to a host cell via a
pre
-
hairpin
intermediate or in the process of fusing its membrane with the target cell membrane.
The
absence
of observed viral fusion events
might
be explained if fusion is a fast proc
ess
compared with viral budding;
thus when cells or tissues are immobilized for EM
or ET, the
relatively slow process of viral budding
would be
more easily
captured
compared with
the faster
process of fusion
.
We assume that
fusion could
t
heoretically
be observed
if a virus were caught
at exactly the right time,
but this
might require exa
mining
thousands or m
illions of images
.
Here we report
visualizing the
pre
-
hairpin
intermediate by ET after
treatment of
HIV
-
1
exposed
target cells with inhibitors of six
-
helix bundle formation that bind the N
-
trimer region of gp41 that
is exposed
during the fusion process
.
Using opti
mally
preserved samples for ET with a nominal
resolution ~7 nm
, we found
>100
examples of H
IV
-
1 virions linked to
TZM
-
bl
target cells by 2
-
4
narrow
rods
of density
(spokes)
in inhibitor
-
treated samples, but none in untreated or control
-
treated samples. The approximate dimensions of the majority of the
spokes
matched models of
gp41
-
only
pre
-
hairpin
intermediates in which the
Env
gp120 subunit had been shed.
The
average number of
observed
spokes
connecting a virion to a target cell increased
slightly when
using
a virus
containing an Env with
a cytoplasmic tail
deletion, suggesting that the increased
lateral mobility of cytoplasmic tail
-
deleted Env
s
in the viral membrane
[23, 24]
allowed more
Envs to join the interaction with the target cell. We discuss
the implications of these studies for
understanding HIV
-
1 Env
-
mediated membrane fusion and
how these results differ
from
a
previous ET study of the “entry claw” that is formed upon HIV
-
1 or SIV interactions with target
cells
[25]
.
Results
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4
Experimental design
.
A previous study used ET to visualize HIV
-
1 and SIV virions in contact
with target cells
after
promoting a temperature
-
arrested state
[26]
in which viru
ses can remain
attached to cells prior to fusion
[25]
.
For
that
study, t
arget cells were incubated with virus at 4
̊C
to allow binding but not fusion, warmed to 37
̊C
, and then fixed after incubations ranging from
15 min to 3 h
[25]
.
At all time points after warming, viruses were found attached to target cells
by a cluster of 5
-
7
rod
s,
each ~100 Å long and ~100 Å
wide.
The fact that the attachme
nt
structure was not found when the viruses and target cells were
incubated in the presence of
C34,
a gp41
N
-
trimer
targeting
C
-
peptide
inhibitor
related to T20
[25]
,
suggests that the
rod
structure that was
trapped during the temperature
-
a
rrested state did not involve the
pre
-
hairpin
intermediate
.
We
hypothesized
that addition of an HIV
-
1
fusion inhibitor
that binds to the exposed gp41 N
-
trimer after host cell receptor and coreceptor binding would slow or stop virus
-
host cell
membrane
fusion such that we could visualize
pre
-
hairpin
intermediate structure
s
by ET
(
Figure
1
b
)
.
In addition,
incubating with
a
fusion inhibitor
at 37
̊C
obviated the need for
a
4
̊C
incubation
of virus and target cells
, which we reasoned was desirable
since low temperatures alter
membrane
fluidity
[27
-
29]
,
which
could
affect one or more steps in membrane fusion
.
Since
target cells for HIV
-
1 are several microns in height, much thicker than the 0.5
-
1 μm limit for cryo
-
ET
[30]
, we used stained, plastic
-
embedded samples that coul
d be cut into 300
-
400 nm sections
using a microtome, and then
examined the samples in 3
-
D using
ET.
Furthermore,
because
attached virions
were
rare,
it was an advantage that radiation damage is minimal in plastic
sections
[31]
, thus
more cells
could
be assayed in plastic sections than
in
samples prepared by
cryo
-
ET methods (e.g.
,
by
examining thin leading edges of cells or using
focused
-
ion
-
beam
milling
[32]
to prepare a
sufficiently
thin
sample
), thus allowing for statistically
-
significant
observations of virion attachment events.
We prepared
samples by
ligh
t fixation followed by
high
-
pressure freezing/freeze substitution fixation (HPF
-
FSF) instead of
the
traditional
chemical
fixation
protocol used previously
[25]
because HPF vitrifies cells at ~10,000°/sec, stopping all
cellular movemen
t within msec
and allowing optimal preservation of ultrastructural features
[33
-
36]
. By contrast,
chemical fixation immobilizes
elements in the cell at different rates, and
m
ovement and
rearrangement of transmembrane proteins may continue even in the presence
of aldehyde fixatives
[37
-
39]
. Following HPF
-
FSF, samples were
plastic embedded and stained
with uranyl acetate and lead citrate as described in our previous ET studies of HIV
-
1 in infected
tissues
[20
-
22]
.
Since
biosafety requirement
s
for the current study
necessitated
the use of
HIV
-
1
pseudoviruses instead of infectious HIV
-
1, we
verified
that the ultrastructure of HIV
-
1
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5
pseudoviruses
, including
approximate
numbers and
dimensions of
Env trimer spikes and the
presence of
collapsed
(in mature virions) versus C
-
shaped (in
immature virions)
cores
[15, 40
-
42]
, was preserved during the fixation, embedding, and staining procedures
(Supplementary
Figure 1), consistent with our
previous publications
involving ET of infectious HIV
-
1 in tissue
samples
[20
-
22]
.
These results are
also
consistent
with previous direct comparisons of
tomograms of
stained and
plastic
-
embedded
versus unstained and
cryopreserved SIV virions
[25]
.
W
e
characterized three
fusion
inhibitor
s
of different sizes
and potencies
that target the exposed
gp41
N
-
trimer region of the
pre
-
hairpin
intermediate
for attempts to visualize the
pre
-
hairpin
intermediate
: T1249
-
Fc, a C
-
peptide
based
inhibitor
that
we
linked to human Fc (MW =
65
kDa),
D5 IgG (MW = 150 kDa)
[14]
, and CPT31, a high
-
affinity D
-
peptide inhibitor linked to
cholesterol
(MW =
9
kDa)
[13, 43]
(
Figure
1
a
;
Supplementary
Figure
2
a
)
.
We measured their
neutralization
potencies
using in vitro
HIV
-
1
pseudovirus
neutralization assay
s
[44]
against the
SC4226618 and 6535 viral strinas
.
We found potencies ranging from
50% inhibitory
concentration (
I
C
50
)
values of
~
0.13 ng/mL
for CPT31 to
40
μg/mL for D5 IgG
(
Supplementary
Figure
2
a
)
. T1249
-
Fc exhibited intermediate
potencies
(
IC
50
s =
0.99
μg/mL
;
17 μg/mL
)
(
Supplementary
Figure
2
a
)
,
higher than IC
50
s measured
for T1249 peptide alone, consistent
with
limited
steric accessibility
resulting in decreased
potencies for larger
fusion inhibitor
s
[45]
.
We conducted ET experiments by first
incubating
TZM
-
bl cells, a HeLa cell line that stably
expresses
high levels of
human CD4
and coreceptors
CCR5
and CXCR4
[46]
,
wit
h 130 μg/mL
of
inhibitor (either
T1249
-
Fc
, D5, or CPT31)
and
~
5000
TCID
50
/mL
of HIV
-
1 pseudovirus
at 37
̊C
for 2, 4, or 48 hours
, followed
by HPF, FSF, plastic embedding, sectioning,
and visualization by
ET
.
In order to verify that results were not dependent upon
a particular
viral strain, we
conducted experiments using
pseudoviruses derived from
two
primary isolate
HIV
-
1
strains:
SC4226618
(Tier 2) and
6535
(Tier 1B)
[47]
, chosen for their sensitivity to the
fusion inhibitor
s
(
Supplementary
Figure
2
a) and because we
had both wild
-
type and Env cytoplasmic tail
-
deleted
forms of the
6535
pseudovirus
.
To
identify
attached virions
by EM
(
Figure
2,
Supplementary
Figure
3)
, t
he peripheries of
TZM
-
bl
cells were scanned to
locate
roughly spherical
objects
with
diameters ~100 nm that were
near
a
cell surface.
Regions of interest
were then examined at
higher magnification
and
at
tilts of
0 ̊
,
35 ̊
and
-
35°
to
verify that
the objects
were spherical, as
expected for an HIV
-
1 virion.
Potential virions were then observed through a defocus series to
detect core structures found inside authentic virions: i.e., a bullet
-
shaped core in mature HIV
-
1
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6
and a C
-
shaped core in immature HIV
-
1
[40, 42]
. Once verified
as a virion
,
tilt series for 3
-
D
reconstructions were collected
.
Control experiments in which pseudovirus
and TZM
-
bl cells
were incubated without inhibitor, with an irrelevant IgG, or with a low concentration of inhibitor
,
were prepared and analyzed in the same way.
TZM
-
bl cells are contaminated with ecotropic murine leukemia virus
[48]
, which does not affect
their use for HIV
-
1 in vitro neutral
ization assays
[49]
.
In
our
surveys of TZM
-
bl cells incubated in
the presence o
r
absence of
fusion inhibitor
s, we
occasionally observed
budding
MLV
virions
(
Supplementary
Figure
4
)
. As MLV serves as
a
control for
non
-
specific inhibition in
TZM
-
bl
based
HIV
-
1
in vitro neutralization assays
[50]
, the
fusion inhibitor
s used in our experiments are
known to have no effect on MLV fusion
, thus contaminating MLV virions were not captured
during fusion in our experiments.
En
try inhibitor
-
treated virions are attached to target cells by 2
-
3 narrow spokes
.
In
surveys of TZM
-
bl cells treated with
either
SC4226618 or 6535 pseudovirus
and 130 μg/mL of
any of the three
fusion inhibitor
s, we found virions that were attached to the
surface of a TZM
-
bl
cell by several (
usually 2 or 3
) narrow densities.
To verify that the attached virions result
ed
from
treatment with
a fusion inhibitor
, we
analyzed
control experiments in which we incubated TZM
-
bl
cells and pseudovirus with either no in
hibitor, with an irrelevant Fc
-
containing protein (Z004, an
anti
-
Zika virus IgG
[51]
), or with the T1249
-
Fc inhibitor at a concentration equivalent to 0.01x of
its
neutralization potency (i.e., its
IC
50
value
)
(
Supplementary
Figure
2
a
). In examinations
of >100 cells, we found no attached virions and also very few virions that were within
a distance
that could accommodate attachment. We
used ET to examine
the few cases (<20) in which we
found virions adjacent to cells, which confirmed that none of the virions were attached to a host
cell membrane (
Supplementary
Figure
5
).
The approximate
dimensions of the
densities found for virions attached to TZM
-
bl cells in the
presence of
a fusion inhibitor
were 11
-
15 nm in length and ~
2
-
4
nm in width (
Figure
3
), thus we
refer to the densities as “spokes” to distinguish them from the wider densities described as
“rods” in the previous ET study of virions attached to cells
[25]
.
In some samples incubated with
the
largest inhibitor (
D5 IgG
)
, we
occasionally found densities adjacent to one or more spokes
that could represent the bound IgG (Fi
gure
2d
).
We found
virions
attached with spokes to
10
-
30%
of TZM
-
bl cells that were examined, usually
located
on
the
thin leading edges of cells.
Most cells showed only one attached virion per 400 nm section, but occasional cells exhibited 3
-
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7
5 attached virions.
The
attachment sites were generally flat, as opposed to the target cell
exhibiting a concave surface correspond
ing to the
circumference
of the virion as previously
described
[25]
,
with dista
nces of
~
7
nm to
~
15
nm between spokes.
The majority of attached
virions were mature, as identified by their bullet
-
shaped cores
(
Figure
s
2, 3
), but a minor subset
of attached virions
(~5%)
were immature.
Figure
2
shows
overview images and
2
-
D
tom
ographic slic
es from
3
-
D
reconstructions of
fusion inhibitor
-
treated
virions
attached
to
target
cells
; see also
a
gallery of examples in
Supplementary
Figure
6
.
Attachment densities were
sometimes located in different planes so they are not always visible in any given 2
-
D
tomographic slice, thus we identified them in 3
-
D as shown in
Supplementary
Movie 1.
Despite using inhibitors of different sizes and at
concentrations varying from 3.2
-
to 10
6
-
fold
above their IC
50
values for neutralization of the SC4226618 pseudovirus (
Supplementary
Figure
2a), we found no systematic differences in the numbers of attached virions per cell, their
locations on TZM
-
bl cells,
or the numbers and dimensions of spokes per attached virion. In
addition,
no systematic differences were observed
as a function of which
fusion inhibitor
or
pseudovirus was included in the incubation or the length of the 37
̊C
incubation (
Figure
3d;
Extend
ed Data
Table
1
). Although a 48
-
hour incubation at 37
̊C
should result in
a substantial
loss of virus infectivity, the T1249
-
Fc incubation for 48 hours condition yielded an equivalent
number of attached virions (
Figure
3d
) and similar spoke
structures as the 2
-
and 4
-
hour
incubations
(
Supplementary
Figure
6
)
. This finding is rationalized by calculations and infectivity
measurements showing that the 48
-
hour incubation conditions contained several million
infectious virions (
Supplementary
Figur
e
2
b
), enough to account for the observed attached
virions.
A cytoplasmic tail deletion virus forms attachment structures with more spokes.
T
he
finding of only 2
-
3
spokes
per attached virion
implies
that not all of the ~14 Envs per
HIV
-
1
virion
[52
-
56]
are involved in the fusion process.
Indeed,
Env trimers
that did not participate in
attachment
were
sometimes
observed
in tomographic slices (
Figure
4
).
We hypothesized that
more Envs
might
join in the attachment site
if we used
a virus in which Envs could exhibit
increased
lateral mobility
i
n the viral membrane
to allow Env diffusion
to the attachment
site.
Increased Env mobility in the viral membrane has been described in viruses with an
Env
cytoplasmic tail deletion
that eliminates
interactions with the viral matrix protein
[23, 24]
.
T
o
evaluate
potential effects of increased lateral mobility
, we
used
a pseudovirus
containing an Env
with
a
cytoplasmic tail deletion
(6535
-
D
CT
) and compared its attachment sites with target cells
.
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8
when incubated with the
T1249
-
Fc
fusion inhibitor
to those of wild
-
type 6535
and SC4226618
pseudovirus
es
when incubated
under the same conditions
(
Figure
5
)
.
We first confirmed that attachment sites formed by wild
-
type 6535 and
SC4226618
pseudoviruses were in
distinguishable in terms of their
locations and numbers of observed
spokes
(
Figure
3
)
. We
then
gathered data for 6535
-
D
CT
attachment sites, finding a small, but
statistically significant (
p
=
3
x
10
-
4
)
increase
in
the mean number of spokes:
3.
9
+/
-
1.2
(
n
=
1
5
)
spokes for 6535
-
D
CT
as compared to 2.4 +/
-
0.6 (
n
= 116
) spoke
s for wild
-
type pseudoviruses
(
Figure
s
3d
, 5
)
.
Discussion
Despite a wealth of information
about
the
pre
-
and post
-
fusion structures of HIV
-
1 Env
and
Env
’s
interactions with host receptors
[2
-
9, 54]
,
the architecture of the virus
-
host cell contact
and
the structure of Env
during the act of fusion ha
ve
remained elusive.
Because HIV
-
1 virions have
not
been visualized during
the act of
fusion, we assumed that viral fusion is a fast process that
has yet to be captured
by
electron microscopy
imaging of
either cultured cells or tissues.
In
order to
visualize
the hypothesized
pre
-
hairpin
intermediate structure
in which the gp41
transmembrane region and the fusion peptide link the viral and target cell membranes
[10]
, we
incubated
HIV
-
1
fusion inhibitor
s that bind to the N
-
trimer region of gp41 that is exposed upon
Env
binding to the host receptor and coreceptor
on
target cells.
We chose to use
diverse
fusion
inhibitor
s to trap the
hypothesized pre
-
hairpin intermediate structure linking the viral and host
cell membranes (
Figure
1a) and to avoid a temperature jump protocol
[25]
that
could result in unanticipated effects on membranes; for example,
membrane trafficking events
can be altered or stopped at decreased, non
-
physiological temperatures, and structures
associated with them, such as Golgi cisternae, are perturbed and noticeably altered relative to
physiological conditions
[57]
.
We chose three
fusion inhibitor
s with
strong, medium, and weak neutralization potencies
(CPT31
,
T1249
-
Fc, D5 IgG, respectively)
,
incubated them at 37
̊C
with virus and target cells for
variable times, and examined them in 3
-
D by ET.
For
all experimental conditions, we found
virions linked to the surface of target cells by several (mean =
2.4
+/
-
0.
5
;
n
=
116
) n
arrow
spokes (
Figure
3
d
), thus all conditions contained sufficient
numbers of
virions and inhibitor
s
to
form
stable
attachments to target cells. By contrast, in control experiments with either no
inhibitor, an irrelevant protein, or inhibitor added at a concentration equal to 1% of its
.
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9
neutralization IC
50
value, we did not observe virions
attached to target cells. Base
d on
current
understanding of
how
fusion inhibitor
s prevent fusion
[10, 58]
(
Figure
1
a,b
)
, we
conclude
that
the
attachment densities represent Env trimers in
the
pre
-
hairpin
intermediate conformation that
link
viral
and ta
rget cell membranes (
Figure
1
a
).
Having
trapped
virions in the act of fusion
, we can
use
information
gathered from tomograms to
address mechanistic and structural details of fusion between viral and target cell membranes.
First, the results suggest that only 2 to 3 (and occasionally 4) Envs participate in the reaction
with target cells that is inhibited
by
a fusion inhibitor
.
This number represents a minority of the
Env trimers on HIV
-
1, even considering that HIV
-
1 includes a unusually low number of spike
s
per virion:
an
average
of
~
14 with a range from
4
35
[52
-
56]
(By comparison, similarly
-
sized
influenza A virions contain
~450 spikes
[59]
)
.
STED microscopy
studies
suggested
that HIV
-
1
spikes rearrange from a random distribution on immature vir
ions
to
a cluster
on mature virions
[60, 61]
.
However,
interspike distance distributions on mature virions
derived from
independent
cryo
-
ET
reconstructions
revealed
random
spike
distributions rather than
a single cluster of
spikes
[53, 54]
.
O
ur finding
of
only
2
-
3
spokes
per virion
-
target cell attachment site (
Figure
3
d
Extended Data
Table 1
)
is also consistent with random spike distributions on
mature
virions
since more
attachment
spokes would
be expected if Envs
are
clustered
. In addition, although
free
Env trimers
on HIV
-
1 virions
are difficult to
identify conclusively
by ET
using stained,
plastic
-
embedded samples
, we sometimes found
Env
spikes on the opposite
surface
of
attached
mature
virions
as
the
attachment structures
(
Figure
4
c,d
)
, consistent with the
assumption that only two or three Envs are usually close enough to each other to participate in
binding to host cell receptors and that slow lateral diffusion of HIV
-
1 Envs within the virion
bilayer prohibits recruitment of additional
Envs into the attachment structure
.
The ectodomains of Env trimers on immature virions are not expected to exhibit conformational
changes that
would
prevent binding to CD4 and coreceptor. Indeed, immature virions can fuse
with targets, although less eff
iciently than mature virions
[62]
.
Consistent with Env
trimers
on
immature virions
binding
to receptors
on
target cells and
forming
pre
-
hairpin
intermediate
structures,
we
occasionally
found immature
virions
attached to target cells
that
were
incubated
with
a fusion inhibitor
. Immature virions comprised <5% of the datasets recorded.
A
ttached
immature virions
exhibited
comparable
numbers and locations of
spokes
as
we found for
attached
mature virions (
Figure
4
),
consistent with
a similar distribution of Envs
on mature and
immature
virions
. Although
measurements of
Env mobility within the membranes of mature and
.
CC-BY-NC-ND 4.0 International license
was not certified by peer review) is the author/funder. It is made available under a
The copyright holder for this preprint (which
this version posted April 30, 2020.
.
https://doi.org/10.1101/2020.04.29.068775
doi:
bioRxiv preprint