amphipathic helix which is essential for antiviral activity [22] . Amphipathic helices have been 75 identified in many eukaryotic, prokaryotic, and viral proteins and are well known for their ability 76
to bind and bend membranes [23, 24] . Another piece of evidence that supports the membrane 77 deformation model of fusion inhibition is that IFITM3 incorporated into enveloped viruses also 78 impairs virion fusion with target cells [25] [26] [27] [28] [29] [30] [31] [32] . Despite this progress, a complete mechanistic 79 view of IFITM3 is lacking because studies describing the impact of IFITM3 on membranes have 80 not included mutants lacking antiviral function. 81
It was previously reported that IFITM3 forms clusters on virus-containing vesicles [33] 82 and that IFITM3 oligomerization promotes restriction of IAV [34] . However, the determinants 83 initially purported to mediate oligomerization (phenylalanines at residues 75 and 78 of the 84 CD225 domain [34] ) were later shown to be unnecessary when oligomerization was measured 85
using a FRET-based approach in living cells [35] . Therefore, the oligomerization of IFITM3 86 appears to be influenced by unknown determinants and its importance to antiviral function is not 87
established. 88
In the current study, we set out to identify a loss-of-function mutation in IFITM3 suitable 89 for mechanistic studies by using a homology-guided approach. The IFITM genes (IFITM1, 90
IFITM2, IFITM3, IFITM5, and IFITM10 in humans) are members of an extended gene family 91 known as the Dispanin/CD225 family (hereafter referred to as CD225 proteins) [36, 37] . 92
Members of this group are characterized by the presence of a CD225 domain but the functions of 93 most remain unknown. However, one member is the subject of numerous studies because it is 94 linked to neurological disorders. Mutations in proline-rich transmembrane protein 2 (PRRT2) 95 result in conditions of involuntary movement, such as paroxysmal kinesigenic dyskinesia, benign 96 familial infantile seizures, and episodic ataxia [38, 39] . PRRT2 is a neuron-specific protein that 97
is localized to pre-synaptic terminals and which inhibits synaptic vesicle fusion [40] [41] [42] . 98
Molecular studies of disease-associated missense mutations in PRRT2 (G305W/R) indicate that 99
it causes loss-of-function, leading to unchecked neurotransmitter release [43] [44] [45] [46] [47] . Interestingly, 100
the homologous residue in human IFITM3 is also subject to rare allelic variation in humans 101
(G95W/R) and this mutation results in partial loss of activity against IAV infection [34] . 102
However, the reason why this site is essential for the respective functions of PRRT2 and IFITM3 103 was unknown. 104
Here, we demonstrate that glycine-95 of human IFITM3 resides within a 91 GxxxG 95 105 motif that is highly conserved among vertebrate IFITM3 orthologs as well as PRRT2. Mutation 106 of glycine-95 rendered IFITM3 less active against entry of IAV and vesicular stomatitis virus 107 (VSV). Using fluorescence lifetime imaging microscopy (FLIM) and Förster resonance energy 108 transfer (FRET) of fluorophore-tagged IFITM3, we found that the 91 GxxxG 95 motif mediates 109 IFITM3 oligomerization in intact cells and that IFITM3 oligomers are mostly dimers. A mutant 110 exhibiting loss of antiviral function (G95L) was deficient for oligomerization, indicating that 111 IFITM3 oligomerization and restriction of virus entry are functionally associated. We leveraged 112 this loss-of-function mutant to identify mechanistic correlates of antiviral function which are 113 dependent upon IFITM3 oligomerization. Using a FLIM-based reporter, we found that IFITM3 114
increased membrane order, as previously suggested, while the G95L mutation failed to do so. 115
These data indicate that membrane stiffening by IFITM3 oligomers is required for antiviral 116 activity. In an effort to further probe the importance of membrane order in the antiviral 117 mechanism, we demonstrate that Amphotericin B (Ampho B) decreases the stiffness of IFITM3-118
containing membranes and prevents virus inhibition. These data provide significant new insight 119 into the mechanism by which IFITM3 inhibits the fusion of diverse pathogenic viruses and 120 reveal that oligomerization is a shared requirement for the distinct anti-fusion functions 121 performed by homologs IFITM3 and PRRT2. 122 123
Results 124 125
Evolution-guided identification of a putative oligomerization motif within CD225 domains 126
A phylogenetic tree of CD225 domains found in the IFITM family (consisting of IFITM1, 127 IFITM2, IFITM3, IFITM5, and IFITM10 in humans) and other CD225 proteins (human PRRT2 128
and TUSC5) are indicative of common ancestry ( Figure 1A) . predicted topology consisting of dual hydrophobic domains and a CIL, but the amino terminus is 138
considerably longer in PRRT2 ( Figure 1B and Supplemental Figure 1A ). When comparing 139 topologies and the protein alignment ( Figure 1C) , we noticed the presence of a GxxxG motif in 140 the CIL of IFITM3 and PRRT2 ( Figure 1B and 1C) which is intact in several other IFITM and 141
CD225 proteins [5] . The GxxxG motif, also known as a glycine zipper, is frequently associated 142
with dimerization of membrane proteins [53, 54] . Most often shown to mediate pairing of 143 hydrophobic transmembrane helices within a bilayer, the motif has also been described to drive 144 oligomerization from cytoplasmic loops or linkers [55] . The GxxxG motif is conserved in 145
IFITM3 of vertebrates, indicating that it may play an important functional role ( Figure 1D ).
Glycine-95 is important for the antiviral functions of IFITM3 148
We generated FLAG-tagged IFITM3 mutants in which glycine-91 and glycine-95 were 149 changed to leucine (G91L and G95L), following an example set by characterization of the 150
GxxxG motif in the human folate transporter [56] . We also produced a G95W mutant because a 151 rare single nucleotide polymorphism known as rs779445843 gives rise to missense mutations 152 (G95W/R) in human populations [57, 58] . Furthermore, G95W in IFITM3 is analogous to a 153 disease-associated polymorphism in PRRT2 (G305W) which results in loss of function [44] .
Upon transfection into human cells, IFITM3 containing G95L or G95W exhibited steady-state 155
protein levels similar to wild-type (WT), but this was not the case for G91A (Supplemental 156 Figure 2A ). This finding may explain why a previous study failed to express an IFITM3 mutant 157 containing a stretch of alanines between positions 90 and 95 [13, 34] . Therefore, we focused on 158 G95L and G95W for further functional characterization. The subcellular localization of IFITM3 159
WT and G95L was found to be similar when confocal immunofluorescence microscopy was 160
performed. As shown previously [3, 59, 60] , protein was detected in early endosomes, late 161 endosomes, and at the plasma membrane (Supplemental Figure 2B) . We generated HEK293T 162 cell lines stably expressing FLAG-tagged IFITM3 WT and mutants to assess antiviral function 163 (Figure 2A ). Challenge of cell lines with IAV demonstrated that, while IFITM3 WT strongly 164
protected cells from infection, the G95L or G95W mutations resulted in a considerable loss of 165 virus restriction ( Figure 2B ). When we tested antiviral function following transient transfection 166 into HEK293T, we found that G95L or G95W resulted in a complete loss of IAV restriction 167 ( Figure 2C and Supplemental Figure 2A ). To confirm that IFITM3 targets the entry step of 168 IAV, we challenged cells with HIV-1 pseudotyped with hemagglutinin (HA, a type 1 viral 169 fusogen) and found that G95L results in a severe loss of pseudovirus restriction ( Figure 2D ).
The G95L and G95W mutations also abrogated restriction of HIV-1 pseudotyped with VSV 171 glycoprotein (a type 3 viral fusogen) in an assay for virus-cell fusion ( Figure 2E ). Therefore, a 172 rational approach identified glycine-95 to be essential for broad inhibition of virus entry by 173 IFITM3.
We and others have previously demonstrated that, in addition to preventing virus entry 175 into naïve target cells, IFITM3 performs another antiviral function in virus-producing (infected) 176 cells by incorporating into virions, reducing viral glycoprotein abundance and function, and 177
reducing the fusogenic potential of virions [25] [26] [27] [28] [29] 32] . Therefore, we also tested the relative 178
impact of IFITM3 WT and IFITM3 G95L on HIV-1 virion infectivity. IFITM3 WT reduced the 179 infectivity of HIV-1 and was accompanied by a defect in viral Envelope (Env) incorporation into 180
virions: processing of the Env gp160 precursor (as determined by gp120/gp160 ratio) was 181 decreased, resulting in relatively low levels of gp120 and gp41, consistent with previous work 182
[ 26, 28, 61] . By comparison, IFITM3 G95L exhibited reduced activity against HIV-1 infectivity 183
and impacted Env processing/incorporation to a lesser extent ( Figure 2F and Supplemental 184 Figure 2C ). Furthermore, IFITM3 G95L itself was incorporated into HIV-1 virions about 50% 185 less than WT IFITM3 (Supplemental Figure 2C) . Therefore, the dual antiviral functions ( Figure 3A and 3C). However, we did not observe decreases in YFP lifetimes when G95L-YFP 206
and G95L-mCherry were examined. To ensure that conclusions drawn from this imaging 207
approach are relevant to IFITM3-mediated antiviral function, we tested the ability for 208
fluorescently-tagged IFITM3 to inhibit IAV. WT IFITM3-mCherry exhibited nearly the same 209 antiviral potency as WT IFITM3-FLAG while WT IFITM3-YFP was slightly less potent. 210
Moreover, versions of fluorescently-tagged IFITM3 encoding G95L exhibited significantly less 211 antiviral activity than their WT counterparts (Supplemental Figure 3A ). Using the same FRET-212 based approach, we found that the G91L mutation partially resulted in higher YFP lifetimes 213 relative to WT IFITM3, indicating that G91L also impairs oligomerization of IFITM3 214 (Supplemental Figure 3B) . Overall, these data provide the first indication that glycine-91 and 215
glycine-95 are important for IFITM3 oligomerization. To quantitatively resolve the specific 216 oligomeric state of IFITM3 under these conditions, we performed a Number and Brightness 217
analysis [63, 64] . This approach is a fluorescence microscopy method capable of measuring the 218 apparent average number of molecules and their brightness in each pixel over time, with 219 brightness being proportional to oligomeric state. Analysis was restricted to IFITM3-mCherry 220 during its diffusion in and out of a static membranous compartment (the plasma membrane). On 221 average, WT IFITM3-mCherry was found to be 2.14 times brighter than mCherry monomers 222
[65] ( Figure 3D ). In contrast, G95L-mCherry brightness was not significantly different than 223 mCherry monomers (averaging 1.30 times brighter). Furthermore, relative to mCherry 224 monomers, both WT IFITM3-mCherry and G95L-mCherry formed assemblies that were up to 225 five times brighter, but WT IFITM3-mCherry demonstrated a greater propensity to form these 226
higher-order oligomers ( Figure 3D ). These data suggest that fluorescently-tagged IFITM3 forms 227 dimers and higher-order oligomers in membranes in a glycine-91-and glycine-95-dependent 228
fashion.
In parallel to our studies of IFITM3 oligomerization in single, living cells, we assayed the 230 ability of IFITM3 pairs tagged with FLAG or myc to co-immunoprecipitate from bulk cell 231
lysates. HEK293T were co-transfected with IFITM3-FLAG and IFITM3-myc followed by 232 FLAG immunoprecipitation, SDS-PAGE, and quantitative immunoblotting. We found that WT 233 IFITM3-myc readily pulled down with WT IFITM3-FLAG, while pull down of G95L-myc with 234
G95L-FLAG was diminished by approximately 50% (Figure 4A and 4B). Therefore, 235
membrane-extracted IFITM3 forms oligomers, but the G95L mutation reduces oligomerization.
We then performed blue native PAGE and immunoblotting to assess the oligomeric state of 237 IFITM3 under non-denaturing conditions. Two populations of IFITM3 oligomers, exhibiting 238
sizes of approximately 300 and 480 kilodaltons, were readily observed for WT and, to a lesser 239 extent, IFITM3 G95L. The largest (480 kilodaltons) was nearly absent for IFITM3 G95L 240 ( Figure 4C ). In contrast to the Number and Brightness analysis ( Figure 3D ), we did not observe 241 dimers, which may result from the conditions under which blue native PAGE was performed 242
here (Supplemental Figure 4A) . Nonetheless, the fact that IFITM3 G95L exhibited a reduced 243 potential for higher-order oligomer formation using this technique is consistent with our 244 experiments using living cells or denaturing conditions. Therefore, glycine-95 is necessary for 245 efficient IFITM3 oligomer formation and oligomerization is necessary for its antiviral function. 246 247 248
While the precise mechanism by which IFITM3 inhibits virus-cell fusion remains 250 unresolved, a salient phenotype of IFITM protein expression is increased membrane order 251
(reduced membrane fluidity) [17, 34] . Therefore, we leveraged our loss-of-function mutant to 252 directly test whether membrane order is functionally associated with inhibition of virus entry by 253
IFITM3. Previous reports of membrane order enhancement by IFITM family members involved 254
the use of a cell-permeable dye known as Laurdan [66] . Here, we assessed membrane order 255 using a recently described sensor known as fluorescent lipid tension reporter (FliptR) [67] [68] [69] .
FliptR is a planarizable push-pull probe that incorporates efficiently into artificial and living cell 257 membranes and whose fluorescence parameters change upon alterations in local lipid packing 258 (order). Specifically, FliptR responds to increasing membrane order at the plasma membrane and 259
at endomembranes by planarization, leading to longer fluorescence lifetimes detected by FLIM 260 [67, 70] . Using HEK293T stably expressing FLAG-IFITM3, we found that WT IFITM3 261 expression led to significantly increased membrane order, consistent with previous reports [14, 262 17] ( Figure 5A ). In fact, the IFITM3-induced enhancement of membrane order was similar to 263
that achieved by addition of soluble cholesterol, while cholesterol depletion with methyl-beta-264
cyclodextrin resulted in profound decreases in membrane order (Supplemental Figure 5A ). In 265 contrast to WT, cells expressing IFITM3 G95L did not exhibit increased membrane order 266 ( Figure 5A ). These data indicate that membrane order enhancement tracks with a functionally-267 competent form of IFITM3 but not with a loss-of-function mutant. To further probe the 268 functional importance of membrane order in the antiviral mechanism of IFITM3, we performed 269 experiments in the presence of Ampho B. This antimycotic polyene compound was previously 270
reported to overcome the antiviral activity of IFITM3, rendering cells stably expressing IFITM3 271
fully permissive to IAV [14] . However, it was unknown how Ampho B counteracts the effects of 272
IFITM3. When we added Ampho B to cells expressing WT IFITM3, we no longer observed 273
increased membrane order ( Figure 5A ). Furthermore, in identically-treated cells, Ampho B 274
prevented restriction of HA-mediated virus entry by IFITM3 ( Figure 5B ). These findings show, 275
for the first time, that the capacity for Ampho B to overcome the antiviral activity of IFITM3 is 276 linked to its ability to decrease membrane order. Therefore, the use of Ampho B as an 277
interrogative tool reinforced the role played by membrane order in the antiviral mechanism of 278 IFITM3. Overall, these data strongly suggest that IFITM3-mediated antiviral activity occurs 279 through oligomerization-dependent membrane stiffening.
Disease-associated G305W impairs oligomerization of PRRT2 282
Since the GxxxG motif is a shared feature of IFITM3 and PRRT2 and that naturally-283 occurring variation at glycine-305 of PRRT2 is predictive of neurological disease, we assessed 284 the oligomerization capacity of WT and mutant PRRT2. As in Figure 3 , we constructed PRRT2 285 fused with YFP or mCherry to create FRET pairs. We observed that co-transfection of PRRT2-286 YFP and PRRT2-mCherry resulted in significant FRET, demonstrating that PRRT2 oligomerizes 287 in living cell membranes ( Figure 6A ). However, FRET was significantly reduced for pairs 288 containing G305W (G305W-YFP and G305W-mCherry) ( Figure 6A ). Furthermore, pairs 289
containing G305W did not exhibit loss in YFP lifetimes relative to their WT counterparts 290 ( Figure 6B ). These data suggest that mutation of glycine-305 in PRRT2 results in loss of protein 291
oligomerization. Therefore, the divergent functions played by homologues IFITM3 and PRRT2 292
in the regulation of fusion processes are controlled by a common determinant. 293 294 295
Discussion 296 297
The physiological importance of IFITM3 in the control of many virus infections in vivo 298
is becoming increasingly apparent [71, 72] . While it has been proposed that membrane 299 remodeling by IFITM3, at the level of membrane order and curvature, protects host cells from 300 virus invasion, functional proof was lacking. However, two recent developments provided a 301 glimpse of how (and when) IFITM3 inhibits virus-cell membrane fusion. First, the identification 302
of an amphipathic helix located in the IM domain of IFITM3 provided a rational explanation for 303
how membrane stiffening and/or bending may occur [22] . Second, IFITM3 has been observed to 304 intercept vesicles carrying inbound virions and to restrict their release into the cytoplasm, while 305
viruses that are insensitive to IFITM3 evade its encounter [31, 73] . Together with the previous 306 demonstrations that the subcellular localization of IFITM3 determines its specificity and potency 307
[30, 74, 75], a "proximity-based" mechanism presents itself in which IFITM3 interacts with and 308 modifies host membranes needed by some viruses to fuse with cells. Importantly, this model 309
accommodates the antiviral effect of IFITM3 inside viral lipid bilayers as well [25, 26, 28, 31, 310 32]. 311
Here, we provide evidence that an additional determinant (protein oligomerization) plays 312 a crucial role within this mechanistic framework. Previously, an alanine scanning approach led to 313 the identification of two residues (phenylalanine-75 and phenylalanine-78) that were important 314
for IFITM3 oligomerization in cell lysates [34] . Importantly, this study did not functionally test a 315 role for glycine-91 or glycine-95 in the oligomerization of IFITM3 because alanine mutagenesis 316 overlapping these residues led to loss of stable protein expression [13, 34] . While it has been 317 confirmed that F75A and F78A mutations disrupt antiviral activity [31, 76] , IFITM3 318 oligomerization was not impacted by these mutations when assessed by FRET in living cells 319
[35]. It remains possible that these two phenylalanines cooperate with the GxxxG motif to 320 mediate IFITM3 oligomerization. However, our data shows that G95L reduces the antiviral 321 potential of IFITM3 as well as its capacity to increase membrane order (reduce membrane 322 fluidity). Furthermore, we show that a compound previously found to abrogate the antiviral 323 function of IFITM3, Ampho B [14, 31], decreases membrane order in IFITM3-expressing cells.
Even though the exact mechanism by which Ampho B impacts mammalian membranes is 325
unclear [14, 77] , these results identify that the membrane stiffening property of IFITM3 is a strict 326
correlate of its antiviral functions in cells and, most likely, in virions. We found that the 327 oligomerization-defective G95L mutant lessened the impact of virion-associated IFITM3 on 328
HIV-1, suggesting that IFITM3 oligomers are needed to maximally reduce virion infectivity. 329
It will be important to assess how oligomerization-defective mutants of IFITM3 (G95L) 330
impact membrane curvature, another reported consequence of ectopic IFITM3 expression in cells 331
[17]. It has been shown that protein oligomerization of the transmembrane protein Mic10 is 332 essential for its capacity to induce curvature in mitochondrial membranes [78] . Furthermore, 333 changes in membrane order are often accompanied by changes in membrane curvature [18] [19] [20] . It 334 is possible that these two alterations to host membranes underlie the restriction of virus fusion by 335 IFITM3. Moreover, amphipathic helices are characterized to interact with and bend membranes 336
[24]. However, the impact that the amphipathic helix of IFITM3 has on membrane order and 337 curvature has not been assessed. Regardless of how the amphipathic helix is responsible for 338
inhibition of virus fusion, our data raise the possibility that oligomerization "activates" the 339 antiviral potential of the amphipathic helix. It is possible that local insertion of multiple 340 amphipathic helices into stretches of membrane is required for inhibition of virus fusion, and 341
IFITM3 oligomers provide a means to fulfill that requirement. 342
In addition to mediating dimerization or oligomerization of transmembrane proteins 343
(homomultimerization), GxxxG motifs have also been described to affect the propensity for 344
interaction with other proteins (heteromultimerization) [53, 79] . Therefore, glycine-95 may also 345 govern which proteins IFITM3 interacts with and to what extent. IFITM3 has been described to 346
bind with IFITM1 and IFITM2, and it is interesting to consider how IFITM heteromultimers may 347
contribute to antiviral protection of the cell [34] . Furthermore, other host proteins have been 348 described to interact, directly or indirectly, with IFITM3. This list includes cholesterol 349
trafficking regulator VAPA and the metalloproteinase ZMPSTE24, two proteins that have been 350 described as essential cofactors for the antiviral effects of IFITM3 [10, 80] . Since the former is 351 associated with the tendency for IFITM3 to cause cholesterol overload in endosomes, the G95L 352 loss-of-function mutant could be used to rule in or rule out VAPA and cholesterol as players in 353 the antiviral mechanism of IFITM3. 354
While glycine-95 is critical for the oligomerization and anti-fusion activity of IFITM3, 355
we show that the homologous site in PRRT2, glycine-305, regulates its oligomerization as well.
The naturally-occurring G305W/R mutations found in patients with neurological dysfunction are 357 known to disrupt PRRT2 activity, and our results here provide novel insight into how loss-of-358 function occurs. Therefore, the presence of a shared GxxxG motif in IFITM3, PRRT2, and some 359 other CD225 family members suggests that an ancestral CD225-containing protein performed an 360 unknown function that required oligomerization. We wonder whether all CD225 proteins play 361 roles in regulating membrane fusion processes in cells-only time and further experiments will 362
tell. However, just as glycine-95 in IFITM3 may mediate both homo-and heteromultimerization, 363
it has been reported that PRRT2 interacts with cellular fusogens known as soluble N-364 ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) Cytofix/Cytoperm (BD), immunostained with anti-IAV NP (AA5H; Abcam), and analyzed on a 400
LSRFortessa flow cytometer (BD). Replication-incompetent HIV-1 pseudotyped with IAV WSN 401
HA and NA was produced by transfecting HEK293T with 2 μg pR8ΔEnv, 1 μg pcRev (NIH 402 AIDS Reagent Resource: 11415), 3 μg Gag-GFP, 1.5 μg of hemagglutinin, and 1.5 μg of 403 neuraminidase from IAV WSN strain, H1N1 (gifts from G. Melikyan). Replication-competent 404
HIV-1 was produced by transfecting HEK293T with 15 μg pNL4-3 and 5 μg empty pQCXIP or 405 pQCXIP encoding IFITM3 WT or the indicated mutant fused with amino-terminal FLAG. 406
Replication-incompetent HIV-1 pseudotyped with VSV-G for virus-cell fusion assays was 407 produced by transfecting HEK293T with 15 μg pNL4-3ΔEnv, 5 μg pCMV4-BlaM-Vpr, and 5 μg 408
pMD2.G (VSV-G). Transfections were performed using the calcium-phosphate method. Briefly, 409
six million HEK293T were seeded in a T75 flask. Plasmid DNA was mixed with sterile H 2 0, 410
CaCl 2 , and Tris-EDTA (TE) buffer, and the totality was combined with Hepes-buffered saline 411
(HBS Odyssey blocking buffer in PBS (Li-COR) and incubated with anti-FLAG M2 (F1804; Sigma) 448
and anti-c-Myc (C3956; Sigma). Secondary antibodies conjugated to DyLight 800 or 680 COR) and the Li-COR Odyssey imaging system were used to reveal specific protein detection. 450
Images were analyzed and assembled using ImageStudioLite (Li-COR immersion. To assess FRET, donor YFP fluorescence was detected with a gallium arsenide 491 phosphide photomultiplier tube (GaAsP PMT) with a 520-550 nm emission window following 492 excitation by a 514 nm laser. Acceptor mCherry fluorescence was detected with a GaAsP PMT 493
detector with a 570-615 nm emission window following excitation by a 561 nm laser. FRET 494 signal (acceptor mCherry fluorescence triggered by excitation of donor YFP) was collected with 495
a GaAsP PMT detector with 570-615 nm emission window after excitation with a 514 nm laser. 496
At least 50 cells per condition were examined in each experiment. Each cell was assigned a 497
FRET index calculated using the FRET and colocalization analyzer plugin for Fiji (ImageJ).
FLIM analysis of donor YFP was performed by excitation with a 950 nm two-photon, pulsed 499 laser (Coherent) tuned at 80 MHz with single photon counting electronics (Becker Hickl) and 500 detection with a HPM-100-40 module GaAsP hybrid PMT (Becker Hickl). Analysis was limited 501
to cells exhibiting 250-1000 photons per pixel to mitigate the effects of photobleaching and low 502 signal to noise ratio. SPCImage NG software (Becker Hickl) was used to acquire the 503 fluorescence decay of each pixel, which was deconvoluted with the instrument response function 504
and fitted to a Marquandt nonlinear least-square algorithm with two exponential models. The 505
mean fluorescence lifetime was calculated as previously described [82] using SPCImage NG. At 506 least 30 cells per condition were analyzed in each experiment. 507 508
Number and Brightness analysis. HEK293T cells were seeded in μ-slide 8 well chambers 509
(Ibidi) (50,000 per well) overnight and transiently with 0.50 μg IFITM3-mCherry using TransIT-510 293 (Mirus). Living cells in Fluorobrite DMEM (Gibco) were imaged with a Zeiss LSM 780 511 confocal microscope using a 63X objective and oil immersion. Regions of interest were limited 512 to portions of cells which were immobile and which focused on plasma membrane fluorescence 513 (intracellular signal from vesicular membranes was excluded). The axial position of a specimen 514 during acquisition was stabilized using the Adaptive Focus Control module. mCherry was 515 excited with a 561 nm laser and detected with a 570-615 emission window. For each cell, 100 516
frames were acquired at a frame rate of 0.385 frames per second with a 9.75 μs pixel dwell time 517
and pixel size of 151.38 nm. Images were always acquired at 256 x 256 pixels such that the pixel 518 size remained 3-4 times smaller than the volume of the point-spread function. Photobleaching of 519 fluorescent proteins during data acquisition was corrected using a detrending algorithm [64] . 520
Twenty cellular regions were examined per condition. Pixel-by-pixel brightness values were 521 calculated in Fiji (ImageJ). 522 523
FLIM for study of membrane order with FliptR. HEK293T stably expressing empty pQCXIP 524 or pQCXIP-WT IFITM3 or the indicated mutant were seeded in μ-slide 8 well chambers (Ibidi) 525
(50,000 per well) overnight and stained with 1 μM FliptR (Spirochrome) for 5 mins according to 526 the manufacturer's protocol. Imaging was performed with a 63X objective under oil immersion 527 on a Leica SP8-X-SMD confocal microscope. When indicated, cells were treated with 100 528 μg/mL soluble cholesterol (C4951, Sigma), 5 mM methyl-cyclo-beta-dextrin (MßCD) (C4555, 529
Sigma), or 1 μM Amphotericin B (A2942, Sigma) for one hour prior to addition of FliptR and 530
imaging. Fluorescence was detected by hybrid external detectors in photon counting mode 531
following excitation by a 488 nm pulsed laser turned to 20 MHz with single photon counting 532 electronics (PicoHarp 300). Analysis was limited to cells with at least 250-1000 photons per 533 pixel to mitigate the effects of photobleaching and low signal to noise ratio. Fluorescence decay 534 of each pixel in FliptR-stained cells was acquired by Symphotime 64 software (Picoquant), and 535 deconvoluted with the instrument response function and fitted to a Marquandt nonlinear least-536 square algorithm with two exponential models. The mean fluorescence lifetime (τ), in addition to 537
individual component lifetimes (long and short lifetimes, τ 1 and τ 2 ), were calculated using 538
Symphotime. At least 30 cells per condition were analyzed in each experiment. co-transfected with EEA1-GFP and empty pQCXIP or IFITM3-FLAG (encoding WT or G95L). 591
Cells were fixed, permeabilized, and immunostained at 48 hours post-transfection with anti-592 IFITM3 and anti-CD63 and cells were analyzed by immunofluorescence confocal microscopy. 593
All images are average Z-stacks from 10 consecutive medial sections. Scale bars, 10 μm. (C) 594
Whole cell lysates and virus-containing supernatants were collected from HEK293T co-595
transfected with the HIV-1 molecular clone NL4.3 and empty pQCXIP, WT IFITM3-FLAG, or 596
the indicated mutant at 48 hours post-transfection. Virus-containing supernatants were 597 ultracentrifuged through sucrose cushions. Both lysates and concentrated, purified virus-598
containing supernatants were subjected to SDS-PAGE and Western blot analysis.
Immunoblotting was performed with anti-gp120, anti-gp41, anti-CA, anti-actin, and anti-FLAG. 600
Levels of IFITM3 (FLAG), gp120, and gp41 were quantified and normalized to levels of CA. 601
For anti-FLAG immunoblotting, the amount of WT IFITM3 in virions was set to 100%. For anti-602 gp120 and anti-gp41, levels observed in the pQCXIP empty vector were set to 100%. used to depict IFITM3-mCherry (either WT or G95L), and filled yellow circles are used to depict 635 IFITM3-YFP (either WT or G95L). Statistical analysis was performed using one-way ANOVA. 636 *, P < 0.05; **, P < 0.001. 637 
(A) As in Figure 5A , except that soluble cholesterol (100 μg/mL) or methyl-beta-cyclo-dextrin (5 677 mM) were added to untransfected HEK293T cells for one hour and washed away prior to 678 addition of 1 μM FliptR and cells were imaged by FLIM. τ 1 was measured for a minimum of 40 679 cells per condition and the results of three independent experiments were pooled. Dots 680 correspond to individual cells. Error bars indicate standard deviation. Statistical analysis was 681 performed using one-way ANOVA. *, P < 0.05; **, P < 0.001. 682 used to depict mCherry, filled red circles are used to depict PRRT2-mCherry (either WT or 691 G305W), and filled yellow circles are used to depict PRRT2-YFP (either WT or G305W). Error 692 bars indicate standard deviation. Statistical analysis was performed using one-way ANOVA. *, P 693 < 0.05; **, P < 0.001 .  694  695  696  697  698  699  700  701  702  703  704  705  706 707 708 