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HIV-1 escape from a peptidic anchor inhibitor by 1 envelope glycoprotein spike stabilization 2
3 Dirk Eggink1, Steven W. de Taeye1,#, Ilja Bontjer1,#, Per Johan Klasse2, Johannes P.M. 4
Langedijk3,#, Ben Berkhout1, and Rogier W. Sanders1, 2* 5 6 7
1 Laboratory of Experimental Virology, Department of Medical Microbiology, 8 Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center 9 of the University of Amsterdam, the Netherlands 10 2 Department of Microbiology and Immunology, Weill Medical College of Cornell 11 University, New York, United States of America 12 3 Pepscan Therapeutics BV, Lelystad, the Netherlands 13 # Current address: Crucell Holland BV, Leiden, the Netherlands 14 15 #These authors contributed equally 16 17 18 Running title: VIR165-dependent HIV-1 19 20 Keywords: HIV-1, resistance, entry inhibitors, envelope glycoprotein 21 22 23 Address correspondence to: *Rogier Sanders, Laboratory of Experimental 24 Virology, Department of Medical Microbiology, Center for Infection and Immunity 25 Amsterdam (CINIMA), Academic Medical Center of the University of Amsterdam, 26 Meibergdreef 15, 1105 AZ Amsterdam, the Netherlands. Phone: +31-20-5667859 27 Fax: +31-20-6916531 E-mail: [email protected] 28
29
JVI Accepted Manuscript Posted Online 21 September 2016J. Virol. doi:10.1128/JVI.01616-16Copyright © 2016, American Society for Microbiology. All Rights Reserved.
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ABSTRACT (max. 250 words) 30
The trimeric HIV-1 envelope glycoprotein spike (Env) mediates viral entry 31
into cells using a spring-loaded mechanism that allows for the controlled insertion 32
of the Env fusion peptide into the target membrane, followed by membrane fusion. 33
Env is the focus of vaccine research aimed at inducing protective immunity by 34
antibodies as well as efforts to develop drugs that inhibit the viral entry process. 35
The molecular factors contributing to Env stability and decay need to be better 36
understood in order to optimally design vaccines and therapeutics. We generated 37
escape viruses against VIR165, a peptidic inhibitor that binds the fusion peptide of 38
the gp41 subunit and prevents its insertion into the target membrane. 39
Interestingly, a number of escape viruses acquired substitutions in the C1 domain 40
of the gp120 subunit (A60E, E64K and H66R) that rendered these viruses 41
dependent on the inhibitor. These viruses could only infect target cells when 42
VIR165 was present after CD4 binding. Furthermore, the VIR165-dependent 43
viruses were resistant to soluble CD4-induced Env destabilization and decay. 44
These data suggest that VIR165-dependent Env proteins are kinetically trapped in 45
the unliganded state and require the drug to negotiate CD4-induced 46
conformational changes. These studies provide mechanistic insight into the action 47
of the gp41 fusion peptide and its inhibitors, and provide new ways to stabilize 48
Env trimer vaccines. 49
50
IMPORTANCE (max. 150 words) 51
Because of the rapid development of HIV-1 drug resistance new drug 52
targets need to be continuously explored. The fusion peptide of the envelope 53
glycoprotein can be targeted by anchor inhibitors. Here we describe virus escape 54
from the anchor inhibitor VIR165. Interestingly, some escape viruses became 55
dependent on the inhibitor for cell entry. We show that the identified escape 56
mutations stabilize the ground state of the envelope glycoprotein and should thus 57
be useful in the design of stabilized envelope-based HIV vaccines. 58
59
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INTRODUCTION 60
With over 35 million people currently being infected, HIV-1 remains a major health 61
problem. Although progress has been made in the development of antiviral drugs, 62
the potential of the virus to acquire resistance remains an issue. Neither a cure nor 63
an effective vaccine is available. HIV-1 enters target cells by using its envelope 64
glycoprotein (Env) spikes on the virus surface. Env is the target for drugs that 65
inhibit the viral entry process and for broadly neutralizing antibodies (bNAbs) that 66
researchers aim to induce with Env-based vaccines. 67
Env is a trimeric complex consisting of three gp41 transmembrane subunits 68
and three non-covalently attached gp120 subunits. The entry process is initiated 69
by binding of gp120 to the CD4 receptor. This interaction induces conformational 70
changes in Env, revealing the binding site for a chemokine co-receptor, generally 71
CXCR4 or CCR5. Additional conformational changes in gp41 involve two heptad 72
repeat regions (HR1 and HR2) in gp41 and the fusion peptide (FP) (1). The FP 73
might be partially exposed in the pre-fusion, native state of Env. In a complex of 74
the Env trimer with the FP-targeting bNAb VRC34 residues 512-519 are in contact 75
with the bNAb, suggesting that they might be solvent exposed. In contrast, residues 76
520-527 are buried within the protein (2). During the conformational changes that 77
lead to membrane fusion a trimeric coiled coil consisting of the three HR1 domains 78
is extended and the FP is inserted into the target cell membrane. Next, the three 79
HR2 domains associate with the HR1 coiled coil, resulting in the formation of a 80
stable six-helix bundle which juxtaposes the viral and cellular membranes and 81
provides the free energy for membrane fusion (3-5). The structure of an Env 82
trimer was recently solved, providing the first detailed images of this intricate 83
molecular machine (6-10). 84
Despite the weak nature of the gp120-gp41 interactions, the presence of 85
gp120 is crucial to maintain gp41 in its metastable, spring-loaded state before 86
encountering a susceptible cell. gp120 is likely to orchestrate the sequential 87
conformational changes in gp41 that culminate in membrane fusion, and 88
premature release or shedding of gp120 renders gp41 inactive (11-14). A number 89
of residues in the C1 and C5 domains of gp120 were shown to be important for the 90
interaction with gp41 (12, 13, 15-18). These domains are part of a 7-stranded β-91
sandwich with three loops directed towards the target cell, thereby organizing the 92
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inner domain of gp120 in three structurally mobile layers (19-21). Layer 1 and 93
layer 2 contribute to the non-covalent interactions between gp120 and gp41 and 94
stabilize the binding of CD4, thereby linking receptor binding to the fusion 95
machinery in gp41 (22). Layer 3 helps to expose the initial site of contact with CD4 96
and provides the interaction between the gp120 inner and outer domains (23). 97
Env is the target of entry inhibitors and two such inhibitors, maraviroc, 98
which prevents co-receptor binding, and enfuvirtide/fuzeon, which prevents six-99
helix bundle formation, have been approved for clinical use. The VIRIP peptide and 100
its derivatives (e.g. VIR165, VIR353, VIR576, Fig 1A, (24)) form another class of 101
entry inhibitors termed anchor inhibitors that bind to the FP (the “anchor”) and 102
prevent its insertion into the target membrane (24). Although mutations in FP can 103
provide resistance to VIRIP-derivative VIR165 (unpublished) and limit the clinical 104
benefit of the related inhibitor VIR576, escape studies with another anchor 105
inhibitor, VIR353, selected for resistance mutations outside the binding site in the 106
C4 and C5 domain of gp120 and in HR1 and the loop domain of gp41 (25, 26). 107
Here we describe virus escape studies with the VIRIP-derivative VIR165. 108
We selected resistance mutations and describe the underlying molecular 109
mechanisms. Interestingly, we identified a number of escape mutations in the C1 110
domain of the gp120 subunit of Env (A60E, E64K, and H66R) that rendered the 111
virus dependent on the drug for CD4-induced conformational changes and viral 112
entry. The VIR165-dependent viruses were more thermostable and remarkably 113
resistant to CD4-induced virus inactivation. These results have relevance for 114
understanding HIV entry and the roles of CD4-binding and FP action, as well as for 115
entry-targeting therapeutics and Env-based vaccine design. 116
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MATERIALS AND METHODS 118
119
Reagents 120
Reagents were obtained as gifts, or purchased, from the following sources: William 121
Olson (Progenics Pharmaceuticals) provided soluble CD4 (sCD4) and CD4-IgG2. 122
AMD3100 was a generous gift from Dr D. Schols (Rega Institute, Leuven 123
University). Peptides VIR165 (LEAIPCSIPPCFAFNKPFVF), and T20 124
(YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF) were synthesized as 125
described previously (27, 28). 126
127
Selection of VIRIP-resistant HIV-1 variants 128
For the selection of VIR165-resistant viruses, 1 x 106 SupT1 cells were infected 129
with 1.5 ng CA-p24 wild-type (WT) HIV-1LAI molecular clone. Six independent 130
cultures were started to initiate evolution with a concentration of 3.8 μg/ml 131
VIR165, which corresponds to the IC50 of WT HIV-1LAI. Cultures were split twice 132
weekly and when HIV-induced cytopathic effects and/or increased CA-p24 133
production were apparent, virus replication was maintained by passage of cell-free 134
culture supernatant onto uninfected SupT1 cells with a 2-fold increased VIR165 135
concentration. Initially we started with passaging 100 μl cell free supernatant onto 136
fresh cells. We gradually decreased the volume of supernatant in subsequent 137
passages, from 100 μl in the second passage to a minimum of 10 μl. Cells and 138
supernatant samples were taken at regular time points and stored at -80°C. Cell 139
culturing, transfections and CA-p24 determination was performed as previously 140
reported (29). After 2½ months of culture, DNA was extracted from infected cells 141
using the QIAamp DNA mini kit (Qiagen, Valencia, CA), and the complete proviral 142
env gene was PCR amplified using primers 1 (5'-143
ATAAGCTTAGCAGAAGACAGTGGCAATG-3') and 2 (5'-GCAAAATCCTTTCCAAGCCC-144
3') and sequenced. Amino acid numbering is based on the HXB2 isolate. 145
146
Construction of HIV-1LAI molecular clones 147
The full-length molecular clone of HIV-1LAI (pLAI) (30) was used to produce WT 148
and mutant viruses. The plasmid pRS1 was used to introduce mutations as 149
described previously (27, 31) and the entire env gene was verified by DNA 150
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sequencing. Mutant env genes in pRS1 were cloned back into pLAI as SalI-BamHI 151
fragments. Each virus variant was transiently transfected in C33A cells by calcium 152
phosphate precipitation as previously described (32) or by transfection in 153
HEK293T cells using lipofectamine 2000 (Invitrogen) according to the 154
manufacturers instructions. The virus containing supernatant was harvested 3 155
days post-transfection and stored at -80°C and the virus concentration was 156
quantitated by capsid CA-p24 ELISA as described previously (29). 157
158
Infectivity and IC50 determination 159
The TZM-bl reporter cell line (33, 34) stably expresses high levels of CD4 and HIV-160
1 co-receptors CCR5 and CXCR4 and contains the luciferase and β-galactosidase 161
genes under the control of the HIV-1 long-terminal-repeat promoter. The TZM-bl 162
cell line was obtained through the NIH AIDS Research and Reference Reagent 163
Program, Division of AIDS, NIAID, NIH (John C. Kappes, Xiaoyun Wu, and Tranzyme 164
Inc. [Durham, NC]). One day prior to infection, 17 x 103 TZM-bl cells per well were 165
plated in a 96-well plate in Dulbecco's modified Eagle's medium containing 10% 166
fetal bovine serum and penicillin-streptomycin (both at 100 units/ml) and 167
incubated at 37°C with 5% CO2. Virus (5.0 ng CA-p24) was pre-incubated for 30 168
min at room temperature with serial dilutions of antisera or entry inhibitor (HIVIg, 169
sCD4, CD4-IgG2, AMD3100, T20 or VIR165). This mixture was added to the cells in 170
the presence of 400 nM saquinavir (Roche, Mannheim, Germany) to block 171
secondary rounds of infection and 40 µg/ml DEAE in a total volume of 200 µl. 172
DEAE was added to enhance HIV-1 infection of TZM-bl reporter cells. This did not 173
affect the sensitivity to the inhibitors tested (data not shown) (35-37). Two days 174
post-infection, the medium was removed and cells were washed once with 175
phosphate-buffered saline (50 mM sodium phosphate, pH 7.0, 150 mM NaCl; PBS) 176
and lysed in reporter lysis buffer (Promega, Madison, WI). Luciferase activity was 177
measured using a luciferase assay kit (Promega, Madison, WI) and a Glomax 178
luminometer according to the manufacturer's instructions (Turner BioSystems, 179
Sunnyvale, CA). All infections were performed in duplicate in at least two 180
independent experiments. Uninfected cells were used to correct for background 181
luciferase activity. The infectivity of each mutant without inhibitor was set at 182
100%. Nonlinear regression curves were determined using sigmoidal dose-183
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response curves (variable slope, bottom constraint = 0) and 50% inhibitory 184
concentrations (IC50) were calculated using Prism software version 5.0. When full 185
inhibition was not yet reached at the highest concentration, sigmoidal curves were 186
modeled using extrapolation. Fold differences compared to WT were calculated 187
and were considered resistant when the calculated IC50 was over three fold higher 188
than that of WT (35, 36). 189
The relative infectivity of mutant viruses was calculated compared to HIV-1LAI. 190
Infections were performed in quadruple and luciferase activity without inhibitor 191
was measured corrected for background luciferase activity. Infectivity of HIV-1LAI 192
WT was set at 100% and the relative infectivity of the mutants was calculated. 193
194
Virus thermostability experiments 195
WT and mutant viruses were incubated for 1 h in the presence or absence of 0.5 196
μg/ml sCD4 at a temperature range of 37°C to 50°C using a thermocycler before 197
testing their residual infectivity on TZM-bl reporter cells as above. Because the 198
A60E, E64K and H66R mutants were not infectious in the absence of drug, VIR165 199
(10 or 30 μg/ml) was added after incubation in the presence or absence of sCD4 200
and present during infection of TZM-bl reporter cells. The midpoint of thermal 201
denaturation (Tm) was defined as the temperature at which 50% residual 202
infectivity was observed. 203
204
Virus decay experiments 205
WT and mutant viruses were incubated at 37°C in the presence or absence of 1.0 206
ug/ml sCD4 (final concentration during infection 0.5 μg/ml) for 0, 2, 4, 8, 16, 24, 32 207
and 96 hours before testing their infectivity on TZM-bl reporter cells as above. As 208
mutants A60E, E64K and H66R are not infectious in the absence of drug, VIR165 209
(10 or 30 μg/ml) was added during infection of TZM-bl reporter cells. Nonlinear 210
regression curves were determined and half-lifes (t½) were calculated using Prism 211
software version 5.0. 212
213
Statistical analysis. 214
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All statistical comparisons were performed by the unpaired t test (two tailed) 215
using Prism software version 5.0 and indicated in figures with asterisks: * P < 0.05; 216
** P < 0.005. 217
218
RESULTS 219
220
VIR165 escape occurs in C1 of gp120 and HR1 of gp41 221
To investigate whether HIV-1LAI is able to escape from VIR165 (Fig. 1A), we 222
maintained six independent cultures of wild-type (WT) HIV-1LAI that were 223
passaged on SupT1 cells in the presence of increasing concentrations of VIR165. 224
The initial VIR165 concentration was 3.8 μg/ml (1.7 μM; corresponding to the IC50 225
against WT HIV-1LAI). Over the course of 2½ months (20 passages, Fig 1B), the 226
VIR165 concentration was gradually increased up to 250 μg/ml (110 μM), 64-fold 227
the IC50 for WT HIV-1LAI. The complete viral env gene of the proviral DNA was 228
subsequently sequenced. Each culture contained one or two amino acid 229
substitutions in Env (Table 1). We mapped these mutations on the Env trimer 230
structure (Fig. 1C, D) (6-8, 38). Surprisingly, most acquired amino acid 231
substitutions mapped to the C1 domain of gp120: V42I, A58V, A60E, E64K and 232
H66R. Except for V42I, these residues are located in layer 1 of the inner domain of 233
gp120 (19, 20), within the disulfide bonded loop between C54 and C74 (39, 40). 234
It’s worth noting that residue 42 is in close contact with residue 523 via extensive 235
side chain interactions (9). Residue 523 has been shown to be one of the contact 236
residues of VIR165 (24). We also observed substitutions in HR1 of gp41 (A558T 237
and Q577R). We note that Q577R was previously linked to resistance against the 238
3rd generation HR2-based fusion inhibitor T2635 (41). All positions identified in 239
these escape cultures are highly conserved: V42: 99.1%; I42 0.17%, A58: 99.6%; 240
V58: 0.02%, A60: 87.5%; E60: not found, E64: 99.8%; K64: 0.05% (found in two 241
sequences of which one contains multiple frameshifts and stop-codons), H66: 242
99.9%; R66: 0.048% (in two sequences from different subtypes), A558: 99.9%; 243
T558: 0.048%, Q577: 99.3%; R577: 0.36% (Los Alamos database (42)). We 244
observed different substitutions compared to those found in escape studies with 245
VIR353, in which resistance mutations in the C4 and C5 domain of gp120 and in 246
HR1 and the loop domain of gp41 were selected (25, 26). No escape mutations 247
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were observed in FP, which is consistent with the lack of such mutations in escape 248
studies using the VIRIP-derivative VIR353 (24, 26), probably because FP-249
mutations pose a large penalty to fitness (43). 250
251
Substitutions in gp41 HR1 confer VIR165-resistance 252
To confirm the importance of the selected substitutions in providing 253
resistance against VIR165, we constructed molecular clones of HIVLAI with the HR1 254
substitution A558T or Q577R. The T20 resistant mutant V549A was included as a 255
control (28). Single cycle infection experiments were performed using the TZM-bl 256
reporter cell-line in the presence of serial dilutions of VIR165 (Fig. 2A, Table 2). 257
HR1 mutants A558T and Q577R showed 10-fold and >10-fold VIR165-resistance, 258
respectively, compared to WT. The control virus V549A did not show a significant 259
change in sensitivity to VIR165. In the absence of drug, the A558T and Q577R 260
substitutions caused a drop in viral infectivity in the single cycle infection 261
experiments in the TZM-bl reporter cell-line (35% and 20% of WT, respectively), 262
indicating that resistance comes with a significant loss of Env protein function and 263
viral fitness (Fig. 2D), consistent with what was observed upon viral escape from 264
the fusion inhibitor T2635 (41). 265
266
Substitutions in gp120 C1 confer VIR165-resistance or -dependence 267
We next evaluated the contribution of the selected C1 mutations V42I, 268
A58V, A60E, E64K, and H66R to VIR165-resistance. The D62N mutant in C1 was 269
generated to serve as a control. The H66N substitution, which was previously 270
described in a cold-resistant HIV-1 variant and shown to affect the interaction with 271
CD4, was also included (44, 45). Substitutions V42I and A58V caused 4-fold and 8-272
fold VIR165-resistance (Fig. 2B, Table 2) with little impact on viral fitness (Fig. 273
2D). More intriguing was the effect of the A60E, E64K and H66R substitutions. 274
Although these substitutions are located very close to the resistance mutation 275
A58V, they yielded a strikingly different phenotype. In the absence of VIR165, 276
these variants were barely infectious or not infectious at all (Fig. 2D). However, 277
they became infectious with increasing VIR165 concentrations (Fig. 2C). Thus, 278
these mutants display a VIR165-dependent phenotype. Drug dependence is a 279
relatively rare phenomenon in HIV-1 drug resistance. T20- or retrocyclin (RC101)-280
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dependent viruses have been described that involve a combination of substitutions 281
in the HR1 binding site of the inhibitors and in HR2 (46, 47). The T20-dependent 282
virus V549A/N637K did not show cross-resistance or -dependence to VIR165 (Fig. 283
2A, Table 2). The control D62N and H66N substitutions did not confer VIR165-284
resistance or -dependence, and did not affect infectivity (Fig. 2B, C, Table 2). 285
To test whether VIR165-dependent variants could be inhibited by a high 286
dose of VIR165, we repeated the dose-inhibition experiments with VIR165 287
concentrations up to 300 μg/ml (Fig. 2E). Clear drug-induction was observed 288
reaching maximum infectivity at approximately 10 (A60E, E64K) and 30 (H66R) 289
μg/ml VIR165. However, all three VIR165-dependent variants were inhibited at 290
higher concentrations. We determined IC50 values of 60 μg/ml for A60E and E64K 291
and 100 μg/ml for H66R, translating in approximately 37-fold and 58-fold 292
resistance compared to WT. 293
Using the optimal VIR165-concentration for stimulation of each VIR165-294
dependent virus variant we reassessed the maximal infectivity of these viruses. We 295
already showed a nearly complete loss of infectivity of A60E, E64K and H66R in 296
the absence of drug (Fig. 2C, D) and we now compared WT virus infectivity without 297
VIR165 with the maximum infectivity of the dependent viruses in the presence of 298
VIR165 (10 μg/ml for A60E and E64K, and 30 μg/ml for H66R) (Fig. 2F). Infectivity 299
of WT virus was fully inhibited in the presence of 10 μg/ml VIR165. Mutants E64K 300
and H66R exhibited only ~10% infectivity in the presence of VIR165, while the 301
A60E mutant could be activated by VIR165 to achieve ~50% infectivity compared 302
to WT virus without VIR165. Thus, VIR165-dependence comes at a considerable 303
loss of viral fitness, in particular for the E64K and H66R variants. 304
305
Modeling of VIR165 occupancy explains the bell-shaped dose-response curves 306
The enhancement and inhibition of VIR165-dependent HIV-1 infection was 307
modeled mathematically in order to assess whether enhancing or inhibitory effects 308
could be explained by differential FP occupancy levels by VIR165 (1, 2 or 3 VIR165 309
peptides per Env trimer). Different mathematical VIR165 occupancy models were 310
fitted to the experimentally obtained bell-shaped stimulation-inhibition data (Fig. 311
3). The model functions were derived from the binomial theorem, as described 312
(48). They postulate incremental effects of trimer ligation by VIR165. No 313
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assumptions were made about total numbers of trimers or minimal thresholds of 314
functional trimers required for infectivity. Discrepancies between the modeled 315
curves and the experimental data can be partly attributed to this 316
oversimplification (48, 49). 317
The occupancy of VIR165 on a protomer was expressed as 318
p=(C/Kd)/(1+C/Kd), where C is the VIR165 concentration and Kd its dissociation 319
constant, on the simplifying assumption that neither positive nor negative 320
cooperativity between the three protomers of a trimer upon VIR165 binding 321
occurs. For the WT virus an inhibition model was applied that describes the 322
infectivity, I, as being proportional to the fraction of completely unoccupied 323
trimers: I=A(1-3p+3p2-p3), where A is a constant reflecting the maximum 324
amplitude, i.e., the maximum infectivity. An alternative model, which assumes that 325
inhibition occurs only when three VIR165 molecules are bound per trimer, was 326
also fitted to the data for comparison: I=Ap3. 327
For the VIR165-dependent viruses the experimental data were fitted to 328
three models. The first model posits that VIR165 occupancy of one or two 329
protomers per trimer allows infection, whereas occupancy of all three or none 330
does not: I = A(3p-3p2). The second model assumes the induction of infection by 331
exactly one VIR165 molecule per trimer: I = A(3p-6p2+3p3), and the third 332
postulates that infectivity requires exactly two VIR165 molecules per trimer: I = 333
A(3p2-3p3). The models were fitted to the inhibition data by non-linear regression. 334
The models postulating complete inhibition of WT Env function by a single 335
VIR165 peptide or by three peptides per trimer both fitted well to the 336
experimental data (R2=0.964 and R2=0.961, respectively). The model for induction 337
of infectivity of the VIR165-dependent mutants by one VIR165 peptide per trimer 338
fitted better to the experimental data than the other two models (R2=0.937-0.993 339
for enhancement by one VIR165 per trimer; R2=0.934-0.953 for enhancement by 340
one or two VIR165 per trimer; and R2=0.887-0.984 for enhancement by two 341
VIRIP165 per trimer). The models that fitted best to the experimental data are 342
shown in Fig. 3. 343
In conclusion, the mathematical modeling strongly supports a mechanistic 344
scenario in which partial occupancy of VIR165-dependent Env trimers by VIR165 345
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enables infection, whereas occupancy of all three binding sites, which is reached at 346
high VIR165 concentrations, blocks infection. 347
348
VIR165-resistant and -dependent viruses are more sensitive to T20 349
Since all selected escape mutations are located outside the putative VIR165 350
binding site, it is possible that the kinetics of one or more steps of the entry 351
process are affected, thereby limiting the window of opportunity for the drug to 352
act (50-54). To probe whether and how the entry process was affected by the 353
VIR165-resistance and -dependence substitutions, we analyzed the sensitivity to 354
reagents that interfere with well-defined entry steps. As mutants A60E, E64K and 355
H66R are not infectious in the absence of drug, VIR165 (10 μg/ml) was always 356
present when these mutants were evaluated. First, we tested sensitivity to 357
polyclonal Ig from HIV-positive individuals (HIVIg). Neither VIR165-resistant nor 358
VIR165-dependent viruses showed altered sensitivity to HIVIg compared to WT 359
virus, indicating that these viruses did not have a generally altered sensitivity to 360
antibodies. Furthermore, the presence of VIR165 during infection of the dependent 361
variants did not alter the overall neutralization sensitivity (Table 3). 362
The windows of opportunities for VIR165 and HR2-based inhibitors 363
overlap and some mutations that confer resistance to HR2-based inhibitors confer 364
cross-resistance to VIR165 e.g. Q577R. To investigate whether the reverse was also 365
true, we tested all VIR165-resistant viruses (V42I, A58V, A558T and Q577R) and 366
two VIR165-dependent viruses (E64K and H66R) for T20-sensitivity. We did not 367
observe cross-resistance to T20. On the contrary, all VIR165-resistant and 368
dependent variants were more susceptible to the fusion inhibitor T20 (3-fold to 8-369
fold), except for Q577R (Table 3) which was not unexpected as this mutant was 370
also selected in fusion inhibitor resistance studies (41). The control T20-resistance 371
mutation V549A in HR1 (55), displayed 54-fold resistance to T20 and the D62N 372
change did not affect T20-sensitivity. Interestingly, similar observations were 373
made in a previous study, in which a combination of VIR353 resistance mutations 374
also resulted in enhanced sensitivity to T20 (26). These data suggest that the T20-375
sensitive step in entry (transition from pre-hairpin intermediate to six-helix 376
bundle), is probably slower in VIR165-resistant and -dependent virus variants, 377
resulting in an increased window of opportunity for HR1-targeting fusion 378
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inhibitors. These data confirm that VIR165 and T20 inhibit a non-identical viral 379
entry step. 380
381
VIR165-dependent virus is resistant to inhibition by soluble CD4 but more 382
sensitive to inhibition by AMD3100 383
Soluble CD4 (sCD4) induces an activated Env state that is able to mediate 384
virus entry for a limited time after which Env rapidly decays into a functionally 385
inactive form (12, 56, 57). Alterations in Layer 1 in the C1 domain (e.g., H66A and 386
W69L) have been shown to affect this decay process and are thought to stabilize 387
the sCD4-activated Env intermediate, slowing its transition into a functionally 388
inactive Env form (44, 45, 57, 58). We wondered whether VIR165 resistance and 389
dependence mutations affected the interaction of Env with sCD4. We therefore 390
first performed neutralization studies with sCD4 as well as the CD4 mimetic CD4-391
IgG2. Both sCD4 and CD4-IgG2 can neutralize WT HIV-1 infection effectively (Table 392
3). Most VIR165-resistant viruses showed similar sensitivities to sCD4 and CD4-393
IgG2 compared to WT and the D62N variant. V42I was slightly less sensitive to 394
CD4-IgG2 (3-fold), but not to sCD4, while A558T was slightly more sensitive to 395
sCD4 but not to CD4-IgG2. Interestingly, the VIR165-dependent mutants E64K and 396
H66R were both less sensitive to neutralization by sCD4 (4-fold and 13-fold) and 397
CD4-IgG2 (3-fold and 6-fold). 398
The effect of mutations in either C1 or HR1 on the conformational changes 399
associated with CD4 binding could also have an effect on the affinity for the co-400
receptor. We therefore tested the sensitivity of WT virus and variants for 401
inhibition of the CXCR4 antagonist AMD3100 (Table 3). Whereas VIR165-402
resistance mutations in the C1 had no effect on sensitivity to AMD3100 inhibition, 403
resistance mutations within gp41 caused a small increase in AMD3100 sensitivity. 404
VIRIP dependency mutations E64K and H66R rendered the virus 3- to 5-fold more 405
sensitive to AMD3100, suggesting an enhanced dependence on CXCR4 for 406
proceeding with conformational changes towards fusion. 407
408
VIR165-dependent viruses are less prone to CD4-induced decay 409
We hypothesized that VIR165-dependent viruses might have an altered Env 410
stability and require VIR165 to trigger conformational changes following CD4 411
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binding. Soluble CD4 induces an activated Env state that is short-lived in the 412
absence of a co-receptor and target membrane (57, 58). Substitutions at position 413
66 have been reported to decrease this CD4-induced decay (44, 45, 58). We 414
therefore tested the decay rates of WT, VIR165-resistant and VIR165-dependent 415
viruses by incubating them at physiological temperature (37˚C) for different time 416
intervals, followed by assessment of their remaining infectivity on TZM-bl cells. 417
For the VIR165 dependent viruses, VIR165 was added before the transfer to TZM-418
bl cells (Fig. 4A,B, Table 4). WT HIV-1LAI decayed with a half-life (t½) of 11.3 h, 419
which is in the range of values measured for JR-CSF (t½ = 18.9 h) and ADA (t½ = 9.0 420
h) (59). VIR165-resistant virus was less stable (A58V: t½ = 8.2 h), and so where 421
VIR165-dependent viruses (A60E: t½ = 9.4 h; E64K: t½ = 9.5 h; H66R: t½ = 8.2 h). 422
We next tested viral decay in the presence of sCD4. To avoid interference 423
with the subsequent infection, a sub-neutralizing concentration of sCD4 was used. 424
Again, VIR165 was added to the VIR165 dependent viruses before transferring the 425
viruses to TZM-bl cells. In the presence of sCD4, WT virus decayed twice as quickly 426
(1.9-fold, Δt½ = -5.2 h), indicative of CD4-induced deactivation (Fig. 4A,C, Table 4). 427
The VIR165-resistant virus (A58V), which was among the least stable viruses in 428
the absence of sCD4, also decayed more rapidly in the presence of sCD4: 1.5-fold 429
Δt½ = -2.6 h). In contrast, the VIR165-dependent variants, in particular the H66R 430
variant, remained relatively stable in the presence of sCD4 (E64K: 1.2-fold, Δt½ = -431
1.5 h; H66R: 1.1-fold. Δt½ = -0.3 h; and to a lesser extent A60E: 1.3-fold, Δt½ = -2.4 432
h). Because an H66N mutant was reported to be resistant to virus inactivation by 433
cold (44, 45), we repeated these experiments at 0˚C. None of the VIR165-434
dependent viruses were resistant to cold inactivation, but the VIR165-dependent 435
viruses were more resistant to sCD4-induced decay at 0˚C (data not shown), 436
similar to what we observed at 37˚C. We therefore conclude that VIR165-437
dependent Env variants are less prone to CD4-induced decay. 438
439
VIR165-dependent viruses have increased thermostability in the presence of 440
sCD4 441
We also tested the thermostability of WT, VIR165-resistant and -dependent 442
viruses by incubating them for 1 h at escalating temperatures, followed by testing 443
the residual infectivity on TZM-bl reporter cells (in the presence of VIR165 for the 444
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dependent variants). WT virus had a midpoint of thermal denaturation (Tm) of 445
44.6˚C (Fig. 5A, B, Table 4), which is in the same range to what has been published 446
for HIV-1ADA (Tm = 40.5˚C), HIV-1JR-CSF (Tm = 44.6˚C) and HIV-1LAI (Tm = 42.0˚C) 447
(59, 60)(D. Leaman & M. Zwick, personal communication). The VIR165-resistant 448
virus had a similar Tm as WT virus (A58V: Tm = 44.8˚C), and so had the VIR165-449
dependent viruses (A60E: Tm = 44.7˚C; E64K: Tm = 44.6˚C H66R: Tm = 44.7˚C) (Fig. 450
5A, B, Table 4). Thus, we did not major differences in the themostability of the test 451
viruses. 452
To study whether the addition of sCD4 would affect the thermostability, we 453
also performed these experiments in the presence of (sub-neutralizing 454
concentrations of) sCD4. Addition of sCD4 destabilized WT HIV-1LAI and VIR165-455
resistant virus considerably (WT: ΔTm = -2.7˚C; A58V: ΔTm = -2.7˚C) (Fig 5A, C, 456
Table 4). In contrast, the VIR165-dependent viruses were less affected by the 457
addition of sCD4 (A60E: ΔTm = -1.4˚C; E64K: ΔTm = -0.9˚C; H66R: ΔTm = -0.5˚C) (Fig 458
5C, Table 4). We conclude that VIR165-dependent viruses, in particular the H66R 459
variant, are more thermostable and less prone to undergo CD4-induced 460
conformational changes that destabilize the Env spike. 461
462
DISCUSSION 463
VIRIP and VIRIP-derivatives are members of a class of peptide fusion inhibitors 464
termed anchor inhibitors and are thought to inhibit the HIV-1 entry process by 465
binding to the hydrophobic fusion peptide of gp41. A previous study used the 466
VIRIP analogue VIR353 to select resistance mutations in the HR1 and loop domain 467
of gp41 subunit and substitutions in the C4 and C5 domains of the gp120 subunit 468
(26). Here, we performed in vitro HIV-1 evolution experiments to select resistance 469
against the VIRIP analogue VIR165, one of the most potent VIRIP-derivatives. 470
Again, escape mutations were not selected in the putative FP binding site of the 471
peptide, but rather in the HR1 domain of gp41 and the C1 domain of gp120. 472
Moreover, we found that some C1 mutations conferred drug-dependence. 473
How can we mechanistically explain these escape mutations that occur 474
outside the putative binding site of VIR165? An alternative escape mechanism to 475
“decreased drug affinity” is “decreased drug opportunity” (46, 61, 62). In this 476
scenario, the drug-target affinity per se is unaltered, but the opportunity for the 477
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drug to access the target site is restricted, for example because the target site is 478
exposed for a shorter period of time. This mechanism has been described for HIV-1 479
fusion inhibitors (61, 63). We think that such a window mechanism can also 480
account for the VIR165-resistance mutations. Part of the binding site for VIR165 481
(residues 513-518) might might be partially exposed in the pre-fusion state. It is 482
likely that CD4 and possibly co-receptor induced conformational changes allow 483
additional contact residues 520-523 and 525-531 to become available for VIR165 484
binding (2, 9, 24). We propose that the C1 and HR1 substitutions change the 485
kinetics of Env conformational changes, thereby decreasing the time that the FP is 486
accessible. The observation that a resistance mutation against the third-generation 487
HR2-based fusion inhibitor T2635 that did not occur in the drug binding site 488
(Q577R), provided cross-resistance to VIR165, is consistent with this hypothesis 489
(41). In fact, the Q577R substitution was selected in both T2635 (41) and VIR165-490
escape studies (Table 2). Since VIR165 and HR2-based inhibitors target 491
overlapping, although not identical, intermediate Env states, it makes sense that 492
some mutations can provide cross-resistance by changing the kinetics of this entry 493
step. We have not studied the C1 resistance mutations V42I and A58V in much 494
detail, but the A58V mutant was less stable and more prone to CD4-induced decay, 495
suggesting that it is more metastable than the WT Env spike. This might be 496
consistent with enhanced fusion kinetics and shortened FP exposure. Residue 42 is 497
in close contact with residue 523, which is one of the contact residues of Env with 498
VIR165. Alterations of the side-chain interactions between residues 42 and 523 499
might affect the exposure of the critical VIR165 contact residue 523. The selection 500
of VIR353 resistance mutations in the C4 and C5 domain of gp120 and in HR1 and 501
the loop domain of gp41, i.e. outside the inhibitor binding, might be explained by a 502
similar mechanism, especially as these substitutions also resulted in enhanced 503
sensitivity to fusion inhibitor T20 (25, 26). 504
The substitutions that confer VIR165-dependence might be explained by a 505
“decrease drug opportunity” mechanism. Drug-dependence can be classified as a 506
special form of resistance, since the virus is able to replicate in the presence of the 507
drug, but the resistance mutations cause a severe replication defect in the absence 508
of drug, which can only be overcome by addition of the drug. This phenomenon has 509
been described only for a limited number of cases, although drug stimulatory 510
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effects and drug-dependence were reported for several HIV-1 inhibitors targeting 511
the Gag, Protease and Env proteins (46, 47, 64-66). For Env, enhanced fusion 512
kinetics can render HIV-1 resistant to fusion inhibitors (decreased opportunity), 513
but ‘hyperfusogenic’ Env variants can become dependent on the drug to avoid a 514
premature HR1-HR2 collapse in the absence of a target membrane (46, 47, 62). In 515
such cases the drug acts as a safety pin to prevent premature conformational 516
changes, but drug dissociation is eventually needed to allow such conformational 517
changes to occur in the presence of a target cell, leading to membrane fusion and 518
virus entry (46). VIR165-dependence is mechanistically distinct from T20-519
dependence. The VIR165 presence is not required during virus production, but 520
during an intermediate step in viral entry. We speculate that VIRIP-dependent 521
viruses have a hyperstable gp41-C1 interaction that cannot be sufficiently 522
weakened by CD4-binding and as a consequence CD4-induced conformational 523
changes in the gp120-gp41 complex are blocked. Since the FP is located at the 524
interface of gp120 and gp41, it is conceivable that binding of a peptide to the FP 525
affects the gp120-gp41 interactions. We propose that these dependent viruses 526
require VIR165 to act as a “wedge” between gp120 and gp41 to facilitate the CD4-527
induced conformational changes that allow the formation of the complete HR1(1), 528
followed by co-receptor binding and ultimately gp41-C1 dissociation and 529
membrane fusion. In this scenario, VIR165 might bind to the exposed contact 530
residues in the N-terminal part of the FP and ‘pull-out’ the rest of the fusion 531
peptide and thereby force the required conformational changes that allow 532
continuation of the entry process. In agreement with this, the VIR165-dependent 533
viruses are more stable and resistant to sCD4-induced decay. Recently, we used 534
the E64K and H66R substitutions to stabilize recombinant soluble (SOSIP) trimers 535
in the closed pre-fusion state. These substitutions facilitated the stabilization of 536
SOSIP trimers from 4 different isolates: BG505 (clade A), B41 and AMC008 (both 537
clade B), ZM197M (clade C). Hydrogen-Deuterium Exchange (HD-X) and other 538
studies demonstrated that these substitutions stabilize SOSIP trimers by blocking 539
CD4-induced conformation changes consistent with the virological observations 540
described here (67). Whereas T20-dependence involves enhanced fusion kinetics 541
that need to be tempered by T20 from the moment Env appears at the cell/virus 542
surface (61, 62, 68), VIR165-dependence involves reduced fusion kinetics 543
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necessitating the presence of VIR165 as a catalyst for Env conformational changes 544
following CD4 binding. 545
If one assumes that this resistance phenotype involves a decreased window 546
of opportunity, then this might represent a paradox with delayed fusion kinetics. 547
How can reduced fusion kinetics decrease the window of opportunity for VIR165 548
to inhibit? The fact that VIR165-dependent virus is more sensitive to HR2-based 549
fusion inhibitors that target a similar stage during entry also appears paradoxical. 550
This can only be explained if the steps in which VIR165 and HR2-based inhibitors 551
act are separated in time. CD4 binding induces a short-lived activated state that 552
probably also exposes the FP. The FP is probably only fully exposed very shortly, 553
after CD4 binding and until FP is inserted in the target membrane. VIR165 554
exclusively targets this step, whereas HR2-based fusion inhibitors that may also 555
act during this stage will remain active when the FP is inserted into the target cell 556
membrane, up to the time that the six-helix bundle is formed. The windows of 557
opportunity for VIR165 and HR2-based inhibitors are therefore overlapping but 558
not identical. 559
Inhibition of VIR165-dependent viruses required a high dose of inhibitor. 560
The bell-shaped activation-inhibition curves could be mathematically explained by 561
a model in which VIR165-occupancy of one or two FPs per Env trimer could allow 562
for entry, while occupation of all three FPs would result in inhibition. It has indeed 563
been proposed that not all three FPs are required for fusion (69) (MJ. Root, 564
personal communication). This is consistent with a model in which VIR165-565
dependent Env is activated when one or two VIR165 molecules separate gp120 566
from gp41, while the remaining unbound FP(s) can insert into the target 567
membrane and mediate membrane fusion. 568
There is an interest in generating stable soluble mimics of the native Env 569
trimer for vaccine and structural studies. Efforts to generate such mimics have 570
been hampered by the metastable nature of the native Env spike. We have 571
reported a number of stabilization tricks, resulting in the stable trimer mimic 572
BG505 SOSIP.664 gp140 (18, 70-75). Additional modifications that kinetically trap 573
BG505 SOSIP.664 trimers in the unliganded state have recently been described (9, 574
38, 76, 77). We have shown that the VIR165-dependence mutations are useful for 575
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further stabilization of recombinant different Env trimers to lock them in the 576
unliganded ground-state and improve their properties as immunogens (67). 577
578
ACKNOWLEDGEMENTS 579
We are grateful to Stef Heynen for technical assistance. We thank Michael Root for 580
sharing unpublished observations. 581
582
FUNDING 583
This work was supported by the Aids Fonds Netherlands, grants #2005021, 584
#2008013, and by National Institutes of Health Grants P01 AI82362, P01 585
AI110657, and UM1 AI100663 (Scripps CHAVI-ID). R.W.S. is a recipient of a Vidi 586
grant from the Netherlands Organization for Scientific Research (NWO) and a 587
Starting Investigator Grant from the European Research Council (ERC-StG-2011–588
280829-SHEV). P.J.K. is supported by NIH grant R37 AI36082.The funders had no 589
role in study design, data collection and analysis, decision to publish, or 590
preparation of the manuscript. 591 592
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51. Tolstrup M, Selzer-Plon J, Laursen AL, Bertelsen L, Gerstoft J, Duch M, Pedersen FS, Ostergaard L. 2007. 735 Full fusion competence rescue of the enfuvirtide resistant HIV-1 gp41 genotype (43D) by a prevalent 736 polymorphism (137K). AIDS 21:519-521. 737
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53. Reeves JD, Gallo SA, Ahmad N, Miamidian JL, Harvey PE, Sharron M, Pohlmann S, Sfakianos JN, Derdeyn 742 CA, Blumenthal R, Hunter E, Doms RW. 2002. Sensitivity of HIV-1 to entry inhibitors correlates with 743 envelope/coreceptor affinity, receptor density, and fusion kinetics. Proceedings of the National Academy 744 of Sciences of the United States of America 99:16249-16254. 745
54. Platt EJ, Durnin JP, Kabat D. 2005. Kinetic factors control efficiencies of cell entry, efficacies of entry 746 inhibitors, and mechanisms of adaptation of human immunodeficiency virus. Journal of virology 79:4347-747 4356. 748
55. Shafer RW, Schapiro JM. 2008. HIV-1 drug resistance mutations: an updated framework for the second 749 decade of HAART. AIDS Rev 10:67-84. 750
56. Chertova E, Bess Jr JW, Jr., Crise BJ, Sowder IR, Schaden TM, Hilburn JM, Hoxie JA, Benveniste RE, Lifson 751 JD, Henderson LE, Arthur LO. 2002. Envelope glycoprotein incorporation, not shedding of surface 752 envelope glycoprotein (gp120/SU), Is the primary determinant of SU content of purified human 753 immunodeficiency virus type 1 and simian immunodeficiency virus. J Virol 76:5315-5325. 754
57. Haim H, Si Z, Madani N, Wang L, Courter JR, Princiotto A, Kassa A, DeGrace M, McGee-Estrada K, 755 Mefford M, Gabuzda D, Smith AB, 3rd, Sodroski J. 2009. Soluble CD4 and CD4-mimetic compounds 756 inhibit HIV-1 infection by induction of a short-lived activated state. PLoS Pathog 5:e1000360. 757
58. Haim H, Strack B, Kassa A, Madani N, Wang L, Courter JR, Princiotto A, McGee K, Pacheco B, Seaman 758 MS, Smith AB, 3rd, Sodroski J. 2011. Contribution of intrinsic reactivity of the HIV-1 envelope 759 glycoproteins to CD4-independent infection and global inhibitor sensitivity. PLoS pathogens 7:e1002101. 760
59. Agrawal N, Leaman DP, Rowcliffe E, Kinkead H, Nohria R, Akagi J, Bauer K, Du SX, Whalen RG, Burton 761 DR, Zwick MB. 2011. Functional stability of unliganded envelope glycoprotein spikes among isolates of 762 human immunodeficiency virus type 1 (HIV-1). PloS one 6:e21339. 763
60. Leaman DP, Zwick MB. 2013. Increased functional stability and homogeneity of viral envelope spikes 764 through directed evolution. PLoS pathogens 9:e1003184. 765
61. Ray N, Blackburn LA, Doms RW. 2009. HR-2 mutations in human immunodeficiency virus type 1 gp41 766 restore fusion kinetics delayed by HR-1 mutations that cause clinical resistance to enfuvirtide. J Virol 767 83:2989-2995. 768
62. Baldwin C, Berkhout B. 2008. Mechanistic studies of a T20-dependent human immunodeficiency virus 769 type 1 variant. J Virol 82:7735-7740. 770
63. Bai X, Wilson KL, Seedorff JE, Ahrens D, Green J, Davison DK, Jin L, Stanfield-Oakley SA, Mosier SM, 771 Melby TE, Cammack N, Wang Z, Greenberg ML, Dwyer JJ. 2008. Impact of the Enfuvirtide Resistance 772 Mutation N43D and the Associated Baseline Polymorphism E137K on Peptide Sensitivity and Six-Helix 773 Bundle Structure. Biochemistry doi:10.1021/bi702509d. 774
64. Aberham C, Weber S, Phares W. 1996. Spontaneous mutations in the human immunodeficiency virus 775 type 1 gag gene that affect viral replication in the presence of cyclosporins. J Virol 70:3536-3544. 776
65. Menzo S, Monachetti A, Balotta C, Corvasce S, Rusconi S, Paolucci S, Baldanti F, Bagnarelli P, Clementi 777 M. 2003. Processivity and drug-dependence of HIV-1 protease: determinants of viral fitness in variants 778 resistant to protease inhibitors. AIDS 17:663-671. 779
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66. Baldwin C, Berkhout B. 2007. HIV-1 drug-resistance and drug-dependence. Retrovirology 4:78. 780 67. de Taeye SW, Ozorowski G, Torrents de la Pena A, Guttman M, Julien JP, van den Kerkhof TL, Burger JA, 781
Pritchard LK, Pugach P, Yasmeen A, Crampton J, Hu J, Bontjer I, Torres JL, Arendt H, DeStefano J, Koff 782 WC, Schuitemaker H, Eggink D, Berkhout B, Dean H, LaBranche C, Crotty S, Crispin M, Montefiori DC, 783 Klasse PJ, Lee KK, Moore JP, Wilson IA, Ward AB, Sanders RW. 2015. Immunogenicity of Stabilized HIV-1 784 Envelope Trimers with Reduced Exposure of Non-neutralizing Epitopes. Cell 163:1702-1715. 785
68. Reeves JD, Gallo SA, Ahmad N, Miamidian JL, Harvey PE, Sharron M, Pohlmann S, Sfakianos JN, Derdeyn 786 CA, Blumenthal R, Hunter E, Doms RW. 2002. Sensitivity of HIV-1 to entry inhibitors correlates with 787 envelope/coreceptor affinity, receptor density, and fusion kinetics. Proc Natl Acad Sci U S A 99:16249-788 16254. 789
69. Salzwedel K, Berger EA. 2009. Complementation of diverse HIV-1 Env defects through cooperative 790 subunit interactions: a general property of the functional trimer. Retrovirology 6:75. 791
70. Binley JM, Sanders RW, Master A, Cayanan CS, Wiley CL, Schiffner L, Travis B, Kuhmann S, Burton DR, 792 Hu SL, Olson WC, Moore JP. 2002. Enhancing the proteolytic maturation of human immunodeficiency 793 virus type 1 envelope glycoproteins. J Virol 76:2606-2616. 794
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72. Ringe RP, Sanders RW, Yasmeen A, Kim HJ, Lee JH, Cupo A, Korzun J, Derking R, van Montfort T, Julien 798 JP, Wilson IA, Klasse PJ, Ward AB, Moore JP. 2013. Cleavage strongly influences whether soluble HIV-1 799 envelope glycoprotein trimers adopt a native-like conformation. Proceedings of the National Academy of 800 Sciences of the United States of America 110:18256-18261. 801
73. Sanders RW, Derking R, Cupo A, Julien JP, Yasmeen A, de Val N, Kim HJ, Blattner C, de la Pena AT, 802 Korzun J, Golabek M, de Los Reyes K, Ketas TJ, van Gils MJ, King CR, Wilson IA, Ward AB, Klasse PJ, 803 Moore JP. 2013. A next-generation cleaved, soluble HIV-1 Env trimer, BG505 SOSIP.664 gp140, expresses 804 multiple epitopes for broadly neutralizing but not non-neutralizing antibodies. PLoS pathogens 805 9:e1003618. 806
74. Klasse PJ, Depetris RS, Pejchal R, Julien JP, Khayat R, Lee JH, Marozsan AJ, Cupo A, Cocco N, Korzun J, 807 Yasmeen A, Ward AB, Wilson IA, Sanders RW, Moore JP. 2013. Influences on trimerization and 808 aggregation of soluble, cleaved HIV-1 SOSIP envelope glycoprotein. Journal of virology 87:9873-9885. 809
75. Sanders RW, van Gils MJ, Derking R, Sok D, Ketas TJ, Burger JA, Ozorowski G, Cupo A, Simonich C, Goo L, 810 Arendt H, Kim HJ, Lee JH, Pugach P, Williams M, Debnath G, Moldt B, van Breemen MJ, Isik G, Medina-811 Ramirez M, Back JW, Koff WC, Julien JP, Rakasz EG, Seaman MS, Guttman M, Lee KK, Klasse PJ, 812 LaBranche C, Schief WR, Wilson IA, Overbaugh J, Burton DR, Ward AB, Montefiori DC, Dean H, Moore 813 JP. 2015. HIV-1 VACCINES. HIV-1 neutralizing antibodies induced by native-like envelope trimers. Science 814 349:aac4223. 815
76. Kwon YD, Pancera M, Acharya P, Georgiev IS, Crooks ET, Gorman J, Joyce MG, Guttman M, Ma X, 816 Narpala S, Soto C, Terry DS, Yang Y, Zhou T, Ahlsen G, Bailer RT, Chambers M, Chuang GY, Doria-Rose 817 NA, Druz A, Hallen MA, Harned A, Kirys T, Louder MK, O'Dell S, Ofek G, Osawa K, Prabhakaran M, Sastry 818 M, Stewart-Jones GB, Stuckey J, Thomas PV, Tittley T, Williams C, Zhang B, Zhao H, Zhou Z, Donald BR, 819 Lee LK, Zolla-Pazner S, Baxa U, Schon A, Freire E, Shapiro L, Lee KK, Arthos J, Munro JB, Blanchard SC, 820 Mothes W, Binley JM, et al. 2015. Crystal structure, conformational fixation and entry-related 821 interactions of mature ligand-free HIV-1 Env. Nat Struct Mol Biol 22:522-531. 822
77. Guenaga J, Dubrovskaya V, de Val N, Sharma SK, Carrette B, Ward AB, Wyatt RT. 2016. Structure-Guided 823 Redesign Increases the Propensity of HIV Env To Generate Highly Stable Soluble Trimers. J Virol 90:2806-824 2817. 825
78. Garces F, Lee JH, de Val N, Torrents de la Pena A, Kong L, Puchades C, Hua Y, Stanfield RL, Burton DR, 826 Moore JP, Sanders RW, Ward AB, Wilson IA. 2015. Affinity Maturation of a Potent Family of HIV 827 Antibodies Is Primarily Focused on Accommodating or Avoiding Glycans. Immunity 43:1053-1063. 828
829 830
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TABLES 831 832 Table 1. Mutations selected in VIR165 escape studies 833 834
835 1Env numbering according to HXB2 sequence, gp41 numbering indicated in brackets 836
*Mixed sequences detected 837
838
C1 HR1 Env position 421 58 60 64 66 558 (47) 577 (66)
Culture 1
A58A/V* H66R
2 Q577R
3 E64K/E
* A558T/A
* 4 A558T 5 V42I E64K
6 A60E/A
*
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Table 2. VIR165-resistance in single cycle infection assays 839
VIR1651
Virus Mutant IC50 (μg/ml)
Fold resistance
WT 2.7 1.0 V42I 10.6 3.8 A58V 21.2 7.7 A60E 602 37 C1 D62N 3.2 1.2 E64K 602 37 H66N 3.2 1.1 H66R 1002 58 A522V 2.2 0.8 HR1 A558T 27.33 9.93 Q577R
A549A A549A/N637K
>30 3.5 2.4
>10 1.3 0.9
840 1Fold -resistance values indicated in bold represent resistance of 3-fold or more compared to WT 841 virus. 842 2VIR165-dependent viruses, IC50 values estimated based on Fig. 2E. 843 3Estimated IC50 and fold-resistance values as no complete sigmoidal dose-response curve was 844 obtained. 845
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Table 3. Sensitivity of VIR165-resistant and -dependent mutants to a series of antibodies, entry and fusion inhibitors 846
HIV-Ig2 sCD42 CD4-IgG22 AMD31002 T203
Virus mutant IC50 n-fold4 IC50 n-fold IC50 n-fold IC50 n-fold IC50 n-fold
WT 2.40 1.0 2.30 1.0 2.29 1.0 1.85 1.0 54.9 1.0 V42I 4.90 2.0 2.57 1.1 6.98 3.1 2.02 1.1 68.9 1.3 A58V 2.58 1.1 3.01 1.3 2.14 0.9 1.96 1.1 14.7 0.3 D62N 3.08 1.3 2.10 0.9 3.76 1.6 2.08 1.1 53.3 1.0 E64K1 2.76 1.2 9.84 4.3 7.88 3.4 0.29 0.2 22.0 0.4 H66R1 1.84 0.8 30.30 13.2 14.42 6.3 0.58 0.3 16.5 0.3 A558T 2.40 1.0 0.99 0.4 2.07 0.9 1.26 0.7 6.7 0.1 A549A ND ND ND ND 2983 54.3 A549A/N637K ND ND ND ND 1426 26.0 Q577R ND ND ND ND 71.4 1.3
847 1 Because VIR165-dependent mutants are not infectious in the absence of VIR165, 10 μg/ml VIR165 was present for the A60E, E64K and H66R mutants 848 2 Values presented in μg/ml 849 3 Values presented in ng/ml 850 4 Fold-resistance (n-fold) compared to WT. Values indicated in bold represent 3-fold or more resistance compared to WT; values indicated in bold italic represent 3- fold 851 or more sensitivity compared to WT 852
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Table 4. Stability of VIR165-resistant and -dependent mutants in the absence or presence of sCD4 853
854 855 856 857 858 859 860
1 Average values are shown from at least three independent experiments, each performed in duplo 861 with SEM values shown in brackets. 862
863
Decay at 37°C Thermostability t1/2 (h) t1/2 (+sCD4) Δt1/2 Tm (°C) Tm (°C) ΔTm (°C)
+sCD4 +sCD4 WT 11.3 (0.9) 1 6.1 (0.3) -5.2 (1.0) 44.6 (0.3) 41.9 (0.2) 2.7 (0.3)
A58V 8.2 (1.1) 5.6 (0.6) -2.6 (0.5) 44.8 (0.2) 42.1 (0.1) 2.7 (0.2) A60E 9.4 (0.5) 7.0 (0.4) -2.4 (0.6) 44.7 (0.2) 43.3 (0.1) 1.4 (0.1) E64K 9.5 (1.4) 8.0 (1.2) -1.5 (0.9) 44.6 (0.2) 43.6 (0.1) 0.9 (0.1) H66R 8.2 (1.3) 7.9 (1.1) -0.3 (0.5) 44.7 (0.2) 44.2 (0.4) 0.5 (0.3)
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FIGURE LEGENDS 864 865 Figure 1. Escape of HIV-1LAI form anchor inhibitor VIR165. (A) Sequence of the 866
natural peptide VIRIP and the more potent and stable derivatives VIR165 and 867
VIR353, both cyclized by a disulfide bond, and the di-peptide VIR576. The p in 868
VIR353 indicates the introduction of a D-proline. (B) Step-wise increase of the 869
VIR165 concentration during passage experiments. Cultures were passaged twice a 870
week for 2½ months with increasing concentrations of VIR165. (C) Linear 871
representation of HIVLAI gp160 showing positions of VIR165 escape mutations in C1 872
and HR1, as well as previously identified escape mutations to VIRIP analogue 873
VIR353 (26). (D) The residues involved in VIR165-resistance and -dependence were 874
mapped on the structure of the BG505 SOSIP.664 trimer structure containing the 875
complete gp41 interactive domain (PDB accession code 5CEZ (78)) using Pymol 876
(DeLano Scientific; http://pymol.sourceforge.net). The residues are shown as 877
spheres on a cartoon model of one of the protomers, with gp41 in sand and gp120 in 878
green. A surface model of the two other protomers is shown in white and grey. 879
Residues at position 42, 58, 558 and 577 which are involved in VIR165-resistance, 880
are indicated in red. The control position 62 is indicated in cyan, and residues 60, 64 881
and 66, which are involved in VIR165-dependence, are depicted in yellow. For 882
comparison, residues involved in resistance to VIRIP analogue VIR353 are indicated 883
in blue (26). The right panel shows a detail of the region that includes residues 58, 884
60, 64 and 66, all situated in layer 1 of gp120. Gp41 is depicted in orange, gp120 is 885
in green, with layer 1 in magenta and layer 2 in blue. 886
887
Figure 2. HIV-1LAI VIR165 escape variants can be resistant to or dependent on VIR165. 888
Single cycle infection experiments were performed as described in the material and 889
methods section. Inhibition of HIV-1LAI variants containing (A) VIR165-resistance 890
mutations in gp41, (B) VIR165-resistance mutations in the C1 domain, (C) VIR165-891
dependence mutations within C1. A D62N control virus, the T20-resistant variant 892
V549A, and the T20-dependent virus V549A/N637K (46) were included as control 893
viruses. (D) Infectivity of VIR165-resistant and -dependent viruses in the absence of 894
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VIR165 in a single cycle infection assay relative to WT virus. The virus mutants 895
indicated with an (*) were VIR165-dependent, i.e. they were not infectious in the 896
absence of VIR165. (E) VIR165-dependent virus variants are inhibited at high 897
VIR165 concentrations. Single cycle infection experiments were performed using 898
concentrations of VIR165 up to 300 μg/ml revealing a bell-shaped dose-response 899
curve. (F) Maximum infectivity of VIR165-dependent mutants relative to WT in the 900
presence of VIR165. The infectivity of VIR165-dependent viruses was obtained in 901
the presence of VIR165 (indicated with an *) at 10 μg/ml VIR165 (A60E and E64K), 902
or 30 μg/ml (H66R). WT infectivity was measured in the absence of VIR165 (WT) or 903
in the presence of 10 μg/ml VIR165 (WT*). 904
Figure 3. Modeling of trimer occupancy by VIR165 explains the bell-shaped dose-905
response curves of VIR165-dependent viruses. The enhancement and inhibition of WT 906
and VIR165-dependent viruses was modeled mathematically to assess whether 907
enhancing or inhibitory effects of VIR165 could be associated with occupancy levels 908
of 1, 2, or 3 VIR165 molecules per trimer. Different mathematical occupancy models 909
were fitted to the experimentally obtained bell-shaped stimulation-inhibition data 910
of Fig. 2E. The best fit models are shown. 911
912
Figure 4. VIR165-dependent viruses are less prone to CD4-induced decay. (A) 913
Representative experiment of at least three independent experiments in which 914
VIR165-resistant and VIR165-dependent viruses were incubated at physiological 915
temperature (37˚C) in the absence or presence of sCD4, for different time intervals, 916
followed by assessing the remaining infectivity on TZM-bl cells in the absence of 917
VIR165 for WT virus or in the presence of 10 or 30 μg/ml VIR165 for the dependent 918
variants. (B) Half life (t1/2) for WT and VIR165 resistant and dependent mutants, 919
measured in at least three independent experiments performed in duplo. (C) The 920
difference in t1/2 when incubated in the absence or presence of sCD4 during 921
incubation at 37˚C. Statistical significance is indicated with asterisks: * P < 0.05; ** P 922
< 0.005. 923
924
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Figure 5. VIR165-dependent viruses show increased thermostability in the presence of 925
sCD4. (A) The thermostability of WT, VIR165-resistant and VIR165-dependent 926
viruses was tested by incubating them for 1 h at escalating temperatures, followed 927
by testing the remaining infectivity on TZM-bl reporter cells in the absence of 928
VIR165 (WT) or the presence of 10 or 30 μg/ml VIR165 (dependent variants). A 929
representative experiment is show of at least three independent experiments. (B) 930
The midpoints of thermal denaturation (Tm) of WT and mutant viruses measured in 931
at least three independent experiments performed in duplo. (C) Changes in Tm in the 932
presence of sCD4. Statistical significance is indicated with asterisks: ** P < 0.005; *** 933
P < 0.0005. 934
935
Figure 6. Hypothetical model of the inhibiting and enhancing modes of action of 936
VIRIP-derived peptides of WT and VIR165-dependent viruses. 937
(A) The FP becomes fully exposed upon binding of the CD4 receptor and co-receptor 938
and is inserted into the target membrane, followed by subsequent fusion of the two 939
membranes. (B) VIR165 binds to the FP in the short-lived intermediate state that is 940
induced by CD4-binding, thereby inhibiting insertion of the FP into the target 941
membrane. (C) In VIR165-dependent viruses a hyperstable gp120-gp41 interaction 942
cannot be sufficiently weakened by CD4-binding and CD4-induced conformational 943
changes in the gp120-gp41 complex and subsequent entry steps are blocked. (D) 944
VIR165 in sub-saturating amounts acts as a “wedge” between gp120 and gp41, 945
destabilizing the gp120-gp41 interaction and facilitating CD4-induced 946
conformational changes that allow subsequent entry steps. We do not know 947
whether in this scenario VIR165 remains associated with the one or two FP(s) that 948
is occupies and only the free FP(s) insert into the cell membrane, or whether it 949
dissociates to allow all three FPs to insert. (E) High concentrations of VIR165 result 950
in occupancy of all three FPs of the Env trimer, effectively inhibiting infection. 951
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LEAIPMSIPPEVKFNKPFVF VIRIPLEAIPCSIPPCFAFNKPFVF VIR165
LEAIPCSIPpCFLFNKPFVF VIR353
LEAIPCSIPPEFLFGKPFVF(x2) VIR576
BA
S S
Figure 1
C
D
S S
90°58
64
66
60
0 5 10 15 202
4
8
16
32
64
128
256
IC50 WT HIV-1LAI
time (passages)
VIR
165
(µg/
ml)
V1 V2 C2 V3 C3 V4 C4 V5 C5 C1 HR1 HR2 TM 131# 511#470#460#418#385#331#296#196#157#
FP
42#58#60#
64#
66# 563#577#
gp120# gp41#
558433 489545 570 612
62
S612
V570
V489
A433
A558
V42
A60
E64H66E62
A558Q577
A58
V42
A60E64
H66E62
S612
V489
A433
A558
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WTWT*
A60E*
E64K*
H66R*
0
25
50
75
100
125
150
Max
imum
infe
ctiv
ity (%
WT)
A B C
D
Figure 2
E F 01030
WTV42
IA58
VA60
ED62
NE64
KH66
NH66
RA51
7VA55
8T
Q577R
0
25
50
75
100
125
150
Max
imum
infe
ctiv
ity (%
WT)
* * *
(μgVIR165/ml)
0.001 0.01 0.1 1 10 100 10000
25
50
75
100
125
150WT
E64KH66R
A60E
[VIR165 (µg/ml)]
Rel
ativ
e In
fect
ivity
(% m
ax)
WTWT*
A60E*
E64K*
H66R*
106
107
108
Luci
fera
se u
nits
01030(μgVIR165/ml)
0.1 1 10 1000
25
50
75
100
125
150
D62N
WTV42IA58V
[VIR165 (µg/ml)]
Rel
ativ
e In
fect
ivity
(%)
0.1 1 10 1000
25
50
75
100
125
150
A60ED62NE64KH66R
WT
[VIR165 (µg/ml)]
Rel
ativ
e In
fect
ivity
(%)
0.1 1 10 1000
25
50
75
100
125
150 WT V549A
Q577RA558TV549A/N637K
[VIR165 (µg/ml)]
Rel
ativ
e In
fect
ivity
(%)
0.1 1 10 100 10000
25
50
75
100
125
150 BG505-WTBG505-E64KBG505-H66R
[VIR165] µg/ml
Rel
ativ
e In
fect
ivity
(% m
ax)G
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Figure 3
WT A60E
E64K H66R
R2 0.964 R2 0.964
R2 0.937 R2 0.993
0.001 0.010 0.100 1 10 100 10000
25
50
75
100
125
150
[VIR165 (µg/ml)]
Rel
ativ
e In
fect
ivity
(% m
ax)
0.001 0.010 0.100 1 10 100 10000
25
50
75
100
125
150
[VIR165 (µg/ml)]
Rel
ativ
e In
fect
ivity
(% m
ax)
0.001 0.010 0.100 1 10 100 10000
25
50
75
100
125
150
[VIR165 (µg/ml)]
Rel
ativ
e In
fect
ivity
(% m
ax)
0.001 0.010 0.100 1 10 100 10000
25
50
75
100
125
150
[VIR165 (µg/ml)]
Rel
ativ
e In
fect
ivity
(% m
ax)
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0.1 1 10 1000
25
50
75
100
125
time (hrs)
Rel
ativ
e In
fect
ivity
(%)
0.1 1 10 1000
25
50
75
100
125
time (hrs)
Rel
ativ
e In
fect
ivity
(%)
0.1 1 10 1000
25
50
75
100
125
time (hrs)
Rel
ativ
e In
fect
ivity
(%)
0.1 1 10 1000
25
50
75
100
125
time (hrs)
Rel
ativ
e In
fect
ivity
(%)
0.1 1 10 1000
25
50
75
100
125
no sCD4+ sCD4
time (hrs)
Rel
ativ
e In
fect
ivity
(%)
Figure 4
A
WT A58V A60E
E64K H66R
WT A58V A60E E64K H66R0
1
2
3
4
5
6
7
-Δt 1/
2 (ho
urs,
+sC
D4)
* ***
B
C*
WT A58V A60E E64K H66R
6
8
10
12
14
t 1/2 (
hour
s)
*
on April 4, 2018 by guest
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Figure 5
B
C
A
WT A58V A60E
E64K H66R
36 41 46 510
25
50
75
100
125
temp (°C)
Rel
ativ
e In
fect
ivity
(%)
36 41 46 510
25
50
75
100
125
temp (°C)
Rel
ativ
e In
fect
ivity
(%)
36 41 46 510
25
50
75
100
125
temp (°C)
Rel
ativ
e In
fect
ivity
(%)
36 41 46 510
25
50
75
100
125
temp (°C)
Rel
ativ
e In
fect
ivity
(%)
36 41 46 510
25
50
75
100
125
no sCD4+ sCD4
temp (°C)
Rel
ativ
e In
fect
ivity
(%)
WT A58V A60E E64K H66R0.0
0.5
1.0
1.5
2.0
2.5
3.0
ΔT m
(°C
)+s
CD
4 co
mpa
red
to n
o sC
D4 ** ***
***WT A58V H66R A60E E64K
43.0
43.5
44.0
44.5
45.0
T m (°
C)
on April 4, 2018 by guest
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Figure 6
A
B
C
D
E
CD4Cell
Virus
VIR165
CXCR4
WT
WT+VIR165
VIR165-dependent
VIR165
VIR165Dependent+highVIR165
VIR165
VIR165-dependent+lowVIR165
post-fusionstateNativepre-fusionstate CD4-boundstate Intermediate1 Intermediate2
on April 4, 2018 by guest
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Dow
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