the hiv-1 gp120 major variable regions modulate cold-inactivation

1

The HIV-1 gp120 major variable regions modulate cold-inactivation 1

2

Halima Medjahed1,2, Beatriz Pacheco4, Anik Désormeaux1,2, Joseph Sodroski4,5,6 3

and Andrés Finzi1,2,3, # 4

5

1Centre de Recherche du CHUM, 2Department of Microbiology and Immunology, 6

Université de Montréal, Montreal, Quebec, Canada. 3Department of Microbiology 7

and Immunology, McGill University, Montreal, Quebec, Canada. 4Department of 8

Cancer Immunology and AIDS, Dana-Farber Cancer Institute, and Department of 9

Microbiology and Immunobiology, Division of AIDS, Harvard Medical School, 10

Boston, Massachusetts 02215, and 5Department of Immunology and Infectious 11

Diseases, Harvard School of Public Health, Boston, Massachusetts 02215. 12

6Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of 13

Technology and Harvard. 14

#Corresponding author: 15 16 Andrés Finzi 17 Centre de recherche du CHUM (CRCHUM) 18 Address: CRCHUM Saint-Luc Hospital 19 264 Bvd. Rene-Levesque Est, Room 303 20 Montréal, Québec, Canada 21 H2X 1P1 22 Email: [email protected] 23 Phone: 514-890-8000 ext: 35264 24 25

Running Title: gp120 variable regions determine cold-inactivation 26

Word Count Abstract: 92 27

Word Count – Without Acknowledgment: 1,559 28

Word Count – With Acknowledgment: 1,726 29

30

Copyright © 2013, American Society for Microbiology. All Rights Reserved.J. Virol. doi:10.1128/JVI.03124-12 JVI Accepts, published online ahead of print on 23 January 2013

on January 29, 2018 by guesthttp://jvi.asm

.org/D

ownloaded from

http://jvi.asm.org/

2

Abstract 31

32

HIV-1 entry involves the viral envelope glycoproteins (Env) and receptors 33

on the target cell. Receptor binding channels the intrinsic high potential energy of 34

Env into the force required to fuse the membranes of virus and target cell. For 35

some HIV-1 strains, prolonged incubation on ice decreases Env potential energy 36

and results in functional inactivation. By characterizing chimeras between two 37

primary clade C HIV-1 that differ in sensitivity to cold, soluble CD4, and 38

neutralizing antibodies, we found that these properties were largely determined 39

by discrete elements within the gp120 V1V2 and V3 regions. 40

41

42

43

44

45

Key Words: HIV-1, envelope glycoproteins, gp120, cold-inactivation, variable 46

regions, V1, V2, V3, variable loops 47

48

49


.org/D

ownloaded from

http://jvi.asm.org/

3

The envelope glycoprotein (Env gp) complex of human immunodeficiency 50

virus (HIV-1) is composed of the surface glycoprotein gp120 and transmembrane 51

glycoprotein gp41, which are organized into trimeric spikes on the surface of the 52

virus (3, 11, 16). HIV-1 entry is initiated by interaction of gp120 with the CD4 53

receptor. CD4 interaction induces conformational changes in gp120 that allow 54

binding to CCR5 or CXCR4 coreceptors, promoting virus entry (1, 5). The 55

unliganded Env gps exist in a high-potential-energy state. Binding to the 56

receptors induces Env conformational changes that lead to lower-energy states 57

and activate the entry pathway (2, 13). However, it is not yet fully understood 58

how HIV Env channels its intrinsic high potential energy into the force required to 59

fuse the membranes of virus and target cell. Under some circumstances (eg., 60

incubation with sCD4 or some neutralizing Abs), HIV-1 Env undergoes 61

conformational changes that lead to functional inactivation (7). We recently 62

reported that, for some HIV-1 Env isolates, prolonged incubation on ice results in 63

functional inactivation (cold-inactivation) (8). Importantly, cold-inactivation 64

depended on the ability of the HIV-1 gp120 to sample the CD4-bound 65

conformation; for example, changes in Layer 1 of the HIV-1 gp120 inner domain 66

that decreased the spontaneous sampling of the CD4-bound conformation were 67

resistant to cold-inactivation (8). Interestingly, we recently reported that the 68

major variable regions V1, V2 and V3 restrain the unliganded gp120 from 69

«snapping» into the CD4-bound conformation (9). Alteration of a glycosylation 70

site in the V1V2 stem can influence both cold sensitivity and the propensity of 71

Env to transit into the CD4-bound state (6a). These observations suggested the 72

hypothesis that the major gp120 variable regions are able to modulate cold-73

inactivation. 74


.org/D

ownloaded from

http://jvi.asm.org/

4

Here we investigated whether the gp120 variable regions V1, V2 and V3 75

determine the dramatic difference in sensitivity to cold-inactivation of two primary 76

HIV-1 clade C isolates, HIV-1C1086 and HIV-1ZM109 (accession numbers FJ444395 77

and AY424138, respectively) (Figure 1A). We focused our study on these 78

variable regions because they represent the elements that differs the most 79

between these two Env isolates. Importantly, no major differences were 80

observed in the composition of the inner-domain layers or the Phe 43 cavity, 81

previously shown to modulate cold-inactivation (8), or in the CD4-binding site (not 82

shown). Whereas the lengths of the V3 regions are identical and differ by only 83

12%, the V1V2 region is one residue longer in ZM109 (59 residues) but differs 84

significantly in its amino acid sequence, by 45%, when compared to that of 85

C1086 (Figure 1D and E). We also tested whether these variable regions 86

influenced virus sensitivity to CD4-binding site, CD4-induced and quaternary-87

dependent neutralizing Abs. Therefore, we swapped the V1V2, V3 or V1V2V3 88

regions between these two isolates (Figure 1B) and tested their sensitivity to 89

prolonged incubation on ice, as reported (8). Briefly, HIV-1 virions encoding a 90

luciferase reporter (pNL4.3 E-Luc) and bearing wild-type (wt) Env from HIV-1C1086 91

or HIV-1ZM109 or chimeric Envs were normalized by reverse transcriptase and 92

incubated for different amounts of time on ice before being used to infect Cf2Th 93

cells expressing CD4 and CCR5 (10). Luciferase activity was measured 48 h 94

later, as described (6). Viruses bearing wt HIV-1C1086 Env were dramatically 95

sensitive to cold-inactivation, whereas the infectivity of virions bearing wt HIV-96

1ZM109 Env was not affected by cold incubation (Figure 1A and Table 1). Of note, 97

swapping V1V2 from C1086 into the ZM109 backbone slightly decreased 98

infectivity; the reverse was true when the ZM109 V1V2 region was introduced 99


.org/D

ownloaded from

http://jvi.asm.org/

5

into the C1086 backbone. Substitution of V3 from C1086 had a positive effect on 100

the infectivity of ZM109; substitution of the V3 region from ZM109 into C1086 101

slightly decreased infectivity. Nonetheless, all chimeras supported robust HIV-1 102

entry (Figure 1C) and were highly resistant to weakly-neutralizing CD4-binding 103

site Abs such as b12 and VRC03, as well as the CD4-induced 48d Ab (Table 1). 104

Thus, the swaps of the variable regions did not impose a global neutralization-105

sensitive phenotype. Interestingly, the cold-sensitivity of HIV-1C1086 was reduced 106

by substitution of the V1V2 region from the cold-resistant HIV-1ZM109; substitution 107

of the HIV-1ZM109 V3 region did not have this effect (Figure 2A). However, the 108

reciprocal chimeras (i.e. ZM109 with either the V1V2 or V3 region from C1086) 109

exhibited the same cold-resistant phenotype as the parental ZM109. Even 110

substitution of all three variable regions (V1, V2 and V3) from C1086 only slightly 111

enhanced ZM109 sensitivity to cold-inactivation (Figure 2B). These results 112

indicate that variable regions V1, V2 and V3 play a major role in modulating cold-113

inactivation; however, their effects appear to be context-dependent. 114

Changes in the conformation sampled by the HIV-1 Env gps have been 115

shown to modulate sensitivity to both sCD4neutralization (6, 17) and cold-116

inactivation (6a, 7). Therefore, we asked whether the cold-inactivation 117

phenotypes observed for our panel of chimeras correlated with their sensitivity to 118

soluble CD4 (sCD4) or the CD4-binding site antibody VRC01. Recombinant 119

viruses bearing wt or chimeric Env gps were produced, incubated with increasing 120

concentrations of sCD4 or VRC01 for 1 h at 37ºC, and used to infect Cf2Th 121

CD4/CCR5 cells, as indicated above. The cold-sensitive HIV-1C1086 was 122

completely resistant both to sCD4 and VRC01 neutralization (Figure 3A, 4A and 123


.org/D

ownloaded from

http://jvi.asm.org/

6

Table 1), whereas the cold-resistant HIV-1ZM109 was sCD4 and VRC01-sensitive 124

(Figure 3B, 4B and Table 1). Whereas, the V3 region was the major determinant 125

of virus sensitivity for neutralization by sCD4, the V1V2 region determined 126

VRC01 sensitivity. Thus, whereas for these two HIV-1 isolates, cold-inactivation 127

and sCD4 sensitivity did not directly correlate and were determined by different 128

variable regions (Figure 3C), we observed an inverse correlation between cold-129

inactivation and VRC01 sensitivity that appears to be mainly determined by the 130

V1V2 variable region (Figure 4C). 131

132

To further investigate whether sCD4 sensitivity correlated with the ability of 133

the trimeric Env gps to interact with CD4, as previously suggested (14), we 134

introduced a stop codon at position 711 to enhance cell-surface expression of 135

our panel of Env gp chimeras and examined their interaction with CD4-Ig by a 136

cell-based ELISA assay (4, 7) that specifically measures binding of selected 137

ligands to cell-associated trimers (Figure 5A). Of note, truncation of the gp41 138

cytoplasmic tail did not affect the cold-inactivation phenotypes of the chimeras 139

(data not shown). Importantly, the sCD4 sensitivity of our panel of chimeras 140

correlated with the binding of CD4-Ig to the trimeric Env gps on the cell surface 141

(Spearman rank order correlation coefficient = -0.724; P value = 0.0157) (Figure 142

5B). Thus, for this panel of chimeras, the ability of trimeric Env to interact with 143

CD4 predicts sensitivity to sCD4 neutralization. 144

145


.org/D

ownloaded from

http://jvi.asm.org/

7

We then asked whether the differential sensitivity of HIV-1C1086 and HIV-146

1ZM109 to cold might be linked to the integrity of the Env trimer. Indeed, it has 147

been reported that the variable regions V1V2 and V3 are part of the trimer-148

association domain (TAD), a region recently described as being important for 149

trimer stability (12). We therefore evaluated the association of the gp120 and 150

gp41 subunits of each Env trimer by precipitation of radiolabeled envelope 151

glycoproteins from cell lysates and medium with a mixture of sera from HIV-1-152

infected individuals, as previously described (4, 6). Supporting a role of the 153

variable regions V1V2 and V3 and the TAD in the stability of the Env trimer, 154

swapping the V1V2 or V3 alone or in combination decreased the stability of the 155

trimer, irrespectively of the background (Figure 6A). However, this property did 156

not correlate with cold inactivation (Figure 6B). 157

158

Finally, we asked whether cold-inactivation could be linked to the ability of 159

these Env to interact with the broadly-neutralizing, quaternary-dependent 160

PG9/PG16 antibodies that recognize epitopes present in conserved portions of 161

the gp120 variable regions (15). These epitopes are located in the TAD, a region 162

that could potentially influence sensitivity to cold. We produced viral particles 163

expressing the different Env gps from our panel of chimeras and tested their 164

sensitivity to neutralization by the PG9 antibody (Figure 7 and Table 1). HIV-165

1C1086 was resistant to PG9 neutralization whereas HIV-1ZM109 was sensitive. 166

Interestingly, swapping their respective V1V2 regions was sufficient to reverse 167

their phenotypes. A glycan at residue 160 (in V1V2) has been shown to be 168

essential for PG9/PG16 neutralization (15). As HIV-1ZM109 possesses this 169


.org/D

ownloaded from

http://jvi.asm.org/

8

potential N-linked glycosylation site (PNGS) whereas HIV-1C1086 does not, we 170

swapped the residues at position 160 and tested the sensitivity of these mutants 171

to PG9 and PG16 neutralization. Of note, identical results were obtained for PG9 172

and PG16 (not shown) neutralization. As shown in Figure 7, creating a PNGS at 173

position 160 (K160N) in HIV-1C1086 was sufficient to render it sensitive to PG9; 174

however this residue change did not result in increased sensitivity to cold (Figure 175

7C). Alternatively, removal of the PNGS (N160K) in HIV-1ZM109 rendered this 176

virus resistant to PG9 but did not alter its cold-inactivation phenotype (Figure 177

7C). Together these data indicate that cold-inactivation and PG9 neutralization 178

did not correlate (Figure 7D) and that these phenotypes are regulated by different 179

determinants within the gp120 variable regions. 180

181

By characterizing chimeras between two primary clade C HIV-1 that differ 182

in sensitivity to cold, sCD4 neutralization and quaternary-dependent neutralizing 183

Abs, we found that these properties were largely determined by the gp120 V1V2 184

and V3 regions. Whereas cold-inactivation and VRC01 sensitivity where largely 185

determined by the gp120 V1V2 region, the other properties were determined by 186

distinct elements within the variable regions. These results underscore the 187

importance of the gp120 major variable regions in dictating key Env phenotypes, 188

and reveal the independent relationships among sensitivities of HIV-1 to cold, 189

sCD4, and PG9/PG16 neutralization. 190

191

192

193


.org/D

ownloaded from

http://jvi.asm.org/

9

Acknowledgments 194

We thank Yvette McLaughlin and Elizabeth Carpelan for manuscript 195

preparation and Barton Haynes for kindly sharing the HIV-1C1086 Env expressor. 196

The ZM109F.PB4 Env expressor was obtained through the AIDS Research and 197

Reference Reagent Program, Division of AIDS, NIAID, NIH from Drs. C.A. 198

Derdeyn and E. Hunter: ZM109F.PB4, SVPC13. We would like to thank Drs 199

Dennis Burton and Pascal Poignard and IAVI for their generous gift of PG9 and 200

PG16 mAbs; and Drs James Robinson (48d) and Peter Kwong (VRC01) for 201

kindly sharing mAbs. This work was supported by a Canada Foundation for 202

Innovation Program Leader #29866, by a CIHR operating # 257792 and by a 203

FRQS Establishment of young scientist grant #24639 to AF. AF is the recipient 204

of a FRSQ Chercheur Boursier Junior 1 fellowship #24639. This work was also 205

supported by grants from the National Institutes of Health (AI24755 and 206

AI67854), the International AIDS Vaccine Initiative, and a gift from the late 207

William F. McCarty-Cooper. The authors have no conflicts of interest to report. 208

209


.org/D

ownloaded from

http://jvi.asm.org/

10

References 210

1. Alkhatib, G., C. Combadiere, C. C. Broder, Y. Feng, P. E. Kennedy, P. 211

M. Murphy, and E. A. Berger. 1996. CC CKR5: a RANTES, MIP-1alpha, 212

MIP-1beta receptor as a fusion cofactor for macrophage-tropic HIV-1. 213

Science 272:1955-1958. 214

2. Allan, J. S., J. E. Coligan, F. Barin, M. F. McLane, J. G. Sodroski, C. A. 215

Rosen, W. A. Haseltine, T. H. Lee, and M. Essex. 1985. Major 216

glycoprotein antigens that induce antibodies in AIDS patients are encoded 217

by HTLV-III. Science 228:1091-1094. 218

3. Chan, D. C., D. Fass, J. M. Berger, and P. S. Kim. 1997. Core structure 219

of gp41 from the HIV envelope glycoprotein. Cell 89:263-273. 220

4. Desormeaux, A., M. Coutu, H. Medjahed, B. Pacheco, A. Herschhorn, 221

C. Gu, S. H. Xiang, Y. Mao, J. Sodroski, and A. Finzi. 2012. The Highly-222

Conserved Layer 3 Component of the HIV-1 gp120 Inner Domain is 223

Critical for CD4-required Conformational Transitions. J Virol. 224

5. Feng, Y., C. C. Broder, P. E. Kennedy, and E. A. Berger. 1996. HIV-1 225

entry cofactor: functional cDNA cloning of a seven-transmembrane, G 226

protein-coupled receptor. Science 272:872-877. 227

6. Finzi, A., S. H. Xiang, B. Pacheco, L. Wang, J. Haight, A. Kassa, B. 228

Danek, M. Pancera, P. D. Kwong, and J. Sodroski. 2010. Topological 229

layers in the HIV-1 gp120 inner domain regulate gp41 interaction and 230

CD4-triggered conformational transitions. Mol Cell 37:656-667. 231

7. Haim, H., Z. Si, N. Madani, L. Wang, J. R. Courter, A. Princiotto, A. 232

Kassa, M. DeGrace, K. McGee-Estrada, M. Mefford, D. Gabuzda, A. B. 233


.org/D

ownloaded from

http://jvi.asm.org/

11

Smith, 3rd, and J. Sodroski. 2009. Soluble CD4 and CD4-mimetic 234

compounds inhibit HIV-1 infection by induction of a short-lived activated 235

state. PLoS Pathog 5:e1000360. 236

8. Kassa, A., A. Finzi, M. Pancera, J. R. Courter, A. B. Smith, 3rd, and J. 237

Sodroski. 2009. Identification of a Human Immunodeficiency Virus (HIV-238

1) Envelope Glycoprotein Variant Resistant to Cold Inactivation. J Virol. 239

9. Kwon, Y. D., A. Finzi, X. Wu, C. Dogo-Isonagie, L. K. Lee, L. R. Moore, 240

S. D. Schmidt, J. Stuckey, Y. Yang, T. Zhou, J. Zhu, D. A. Vicic, A. K. 241

Debnath, L. Shapiro, C. A. Bewley, J. R. Mascola, J. G. Sodroski, and 242

P. D. Kwong. 2012. Unliganded HIV-1 gp120 core structures assume the 243

CD4-bound conformation with regulation by quaternary interactions and 244

variable loops. Proc Natl Acad Sci U S A 109:5663-5668. 245

10. LaBonte, J. A., T. Patel, W. Hofmann, and J. Sodroski. 2000. 246

Importance of membrane fusion mediated by human immunodeficiency 247

virus envelope glycoproteins for lysis of primary CD4-positive T cells. J 248

Virol 74:10690-10698. 249

11. Liu, J., A. Bartesaghi, M. J. Borgnia, G. Sapiro, and S. Subramaniam. 250

2008. Molecular architecture of native HIV-1 gp120 trimers. Nature 251

455:109-113. 252

12. Mao, Y., L. Wang, C. Gu, A. Herschhorn, S. H. Xiang, H. Haim, X. 253

Yang, and J. Sodroski. 2012. Subunit organization of the membrane-254

bound HIV-1 envelope glycoprotein trimer. Nature structural & molecular 255

biology. 256

13. Myszka, D. G., R. W. Sweet, P. Hensley, M. Brigham-Burke, P. D. 257

Kwong, W. A. Hendrickson, R. Wyatt, J. Sodroski, and M. L. Doyle. 258


.org/D

ownloaded from

http://jvi.asm.org/

12

2000. Energetics of the HIV gp120-CD4 binding reaction. Proc Natl Acad 259

Sci U S A 97:9026-9031. 260

14. Thali, M., U. Olshevsky, C. Furman, D. Gabuzda, J. Li, and J. 261

Sodroski. 1991. Effects of changes in gp120-CD4 binding affinity on 262

human immunodeficiency virus type 1 envelope glycoprotein function and 263

soluble CD4 sensitivity. J Virol 65:5007-5012. 264

15. Walker, L. M., S. K. Phogat, P. Y. Chan-Hui, D. Wagner, P. Phung, J. L. 265

Goss, T. Wrin, M. D. Simek, S. Fling, J. L. Mitcham, J. K. Lehrman, F. 266

H. Priddy, O. A. Olsen, S. M. Frey, P. W. Hammond, S. Kaminsky, T. 267

Zamb, M. Moyle, W. C. Koff, P. Poignard, and D. R. Burton. 2009. 268

Broad and potent neutralizing antibodies from an African donor reveal a 269

new HIV-1 vaccine target. Science 326:285-289. 270

16. Weissenhorn, W., A. Dessen, S. C. Harrison, J. J. Skehel, and D. C. 271

Wiley. 1997. Atomic structure of the ectodomain from HIV-1 gp41. Nature 272

387:426-430. 273

17. Xiang, S. H., P. D. Kwong, R. Gupta, C. D. Rizzuto, D. J. Casper, R. 274

Wyatt, L. Wang, W. A. Hendrickson, M. L. Doyle, and J. Sodroski. 275

2002. Mutagenic stabilization and/or disruption of a CD4-bound state 276

reveals distinct conformations of the human immunodeficiency virus type 1 277

gp120 envelope glycoprotein. J Virol 76:9888-9899. 278

279

280

281


.org/D

ownloaded from

http://jvi.asm.org/

13

FIGURE LEGENDS 282

Figure 1. Differential sensitivity to cold of two primary clade C Env 283

isolates. (A) Recombinant HIV-1 bearing wt C1086 or ZM109 Env 284

gps were incubated on ice for different amounts of time. At the 285

indicated time points, aliquots were removed and frozen at -80°C. 286

After completion of the longest incubation, all samples were thawed 287

and infectivity on Cf2Th-CD4/CCR5 cells was measured. Data is 288

representative of results from at least three independent 289

experiments, performed in quadruplicate. (B) Schematic 290

representation of the chimeric Env gps variants used in this study. 291

Sequences derived from C1086 and ZM109 Env gps are 292

represented in white or gray, respectively. (C) The infection of 293

Cf2Th-CD4/CCR5 cells by 10,000 RT units of virus containing each 294

Env gp variant was measured. Data are representative of results 295

from at least three independent experiments, performed in 296

quadruplicate. (D) Primary sequence alignment of variable regions 297

1 (V1) and 2 (V2) from two primary HIV-1 clade C isolates, HIV-298

1C1086 and HIV-1ZM109 (accession numbers FJ444395 and 299

AY424138, respectively). (E) Alignment of the variable region 3 300

(V3). The shading highlights residues that are conserved. 301

302

303


.org/D

ownloaded from

http://jvi.asm.org/

14

Figure 2. Effect of swapping variable regions V1V2 and V3 on cold-304

inactivation. Cold-inactivation sensitivity of recombinant HIV-1 305

bearing wt C1086, ZM109 or chimeric Env gps was measured in the 306

C1086 (A) or ZM109 backbone (B), as described in the legend of 307

Figure 1 A. Data are representative of results from at least three 308

independent experiments, performed in quadruplicate. 309

310

Figure 3. Effect of swapping variable regions V1V2 and V3 on sCD4 311

neutralization. The sCD4-sensitivity of recombinant HIV-1 bearing 312

wt C1086, ZM109 or chimeric Env gps was measured in the C1086 313

(A) or ZM109 backbone (B). Recombinant HIV-1 bearing wt 314

C1086, ZM109 Env gps or chimeric Env gps were incubated with 315

serial dilutions of sCD4 at 37°C for 1 h before infection of Cf2Th-316

CD4/CCR5 cells. Forty-eight h later cells were lysed and infectivity 317

measured by luciferase. Infectivity at each dilution of sCD4 tested is 318

shown as the percentage of infection seen in the absence of sCD4. 319

Data are representative of results from at least three independent 320

experiments performed in quadruplicate, with means and standard 321

deviations indicated. (C) sCD4 neutralization does not correlate with 322

cold-inactivation. Sensitivity to cold-inactivation of recombinant HIV-323

1 bearing wt C1086, ZM109 or chimeric Env gps (presented in 324

Figure 1B) as well as PNGS 160 variants (Table 1) was measured 325

as described in the legend of Figure 1A. The half-life (T1/2) of each 326

virus incubated on ice was calculated from data presented in Figure 327


.org/D

ownloaded from

http://jvi.asm.org/

15

2 and correlated by the Spearman rank correlation method (using 328

SigmaPlot software) with the sCD4-neutralization IC50 (presented in 329

A and B and Table 1). 330

331

Figure 4. Effect of swapping variable regions V1V2 and V3 on VRC01 332

neutralization. The VRC01-sensitivity of recombinant HIV-1 333


C1086 (A) or ZM109 backbone (B). Recombinant HIV-1 bearing wt 335

C1086, ZM109 Env gps or chimeric Env gps was incubated with 336

serial dilutions of VRC01 at 37°C for 1 h before infection of Cf2Th-337

CD4/CCR5 cells. Forty-eight h later cells were lysed and infectivity 338

measured by luciferase. Infectivity at each dilution of VRC01 tested 339

is shown as the percentage of infection seen in the absence of 340

VRC01. Data are representative of results from at least four 341

independent experiments performed in quadruplicate, with means 342

and standard deviations indicated. (C) VRC01 sensitivity inversely 343

correlates with cold-inactivation. Sensitivity to cold-inactivation of 344

recombinant HIV-1 bearing wt C1086, ZM109 or chimeric Env gps 345

(presented in Figure 1B) as well as PNGS 160 variants (Table 1) 346

was measured as described in the legend of Figure 1A. The half-life 347

(T1/2) of each virus incubated on ice was calculated from data 348

presented in Figure 2 and correlated by the Spearman rank 349

correlation method (using SigmaPlot software) with the VRC01-350

neutralization IC50 (presented in A and B and Table 1). 351


.org/D

ownloaded from

http://jvi.asm.org/

16

352

Figure 5. CD4 binding predicts sCD4 neutralization. We first measured the 353

ability of our cell-based ELISA assay to discriminate between cell-354

associated Env gps versus shed gp120 by inducing shedding. 355

Cells expressing HIV-1YU2 Env gps lacking their cytoplasmic tail 356

were used to enhance cell-surface expression. Env-expressing 357

cells were incubated with concentrations of sCD4 previously shown 358

to induce significant gp120 shedding (4, 6). Cell-associated Env 359

was then detected with the outer-domain recognizing 2G12 360

antibody (A). Data is representative of at least three independent 361

experiments done in quadruplicate +/- SD. Please note that upon 362

sCD4 addition, the signal of 2G12 decreases in a manner 363

dependent on sCD4 dose, indicating that shed gp120 is removed 364

during the washing steps and does not reassociate with the cell 365

membrane. (B) The IC50 of sCD4-neutralization (presented in 366

Figure 3 and Table 1) was inversely correlated with the ability of 367

trimeric Env gps variants to interact with CD4-Ig by cell-based 368

ELISA. The P value and the Spearman rank correlation coefficient 369

are presented. 370

371

Figure 6. Swapping the variable regions V1V2 and V3 affects gp120-372

trimer association, which does not correlate with cold-373

inactivation. (A) Cell lysates and supernatants (SN) of 35S-labeled 374

cells transiently expressing the HIV-1C1086 or HIV-1ZM109 wild-type or 375


.org/D

ownloaded from

http://jvi.asm.org/

17

chimeric Env gps were precipitated with a serum mixture from HIV-376

1-infected individuals. The precipitated proteins were loaded onto 377

SDS-PAGE polyacrylamide gels and analyzed by autoradiography 378

and densitometry. Trimer stability is a measure of the ability of the 379

mutant gp120 molecule to remain associated with the envelope 380

glycoprotein complex on the expressing cell, relative to that of the 381

wild-type envelope glycoproteins. The association index was 382

calculated as previously described (4, 6). Data shown represent 383

the average +/- SD of at least two independent experiments. (B) 384

The half-life (T1/2) of each virus incubated on ice was calculated 385

from data presented in Figure 2 and correlated with the trimer 386

stability index (presented in A). The P value and the Spearman rank 387

correlation coefficient are presented. 388

389

Figure 7. Effect of variable loops V1V2 and V3 or a PNGS at position 160 390

on PG9 neutralization. PG9-sensitivity of recombinant HIV-1 391


C1086 (A) or ZM109 backbone (B). Recombinant HIV-1 bearing wt 393

C1086, ZM109 or Env gps variants were incubated with serial 394

dilutions of PG9 at 37°C for 1 h before infection of Cf2Th-395

CD4/CCR5 cells. Forty-eight hours later, cells were lysed and 396

infectivity measured by luciferase. Infectivity at each dilution of PG9 397

tested is shown as the percentage of infection seen in the absence 398

of PG9. (C) Cold-inactivation profiles of PNGS mutants (at position 399


.org/D

ownloaded from

http://jvi.asm.org/

18

160) in C1086 and ZM109 Env gps. Data are representative of 400

results from at least three independent experiments performed in 401

quadruplicate, with means and standard deviations indicated. (D) 402

Lack of correlation between PG9 neutralization and cold-403

inactivation. Sensitivity to cold-inactivation of recombinant HIV-1 404

bearing wt C1086, ZM109 or chimeric Env gps (presented in Figure 405

1B), as well as PNGS 160 variants, was measured as described in 406

the legend of Figure 1A. (A) The half-life (T1/2) of each virus 407

incubated on ice was calculated from data presented in Figure 2 408

and correlated with the IC50 for PG9 neutralization (presented in A 409

and B). The P value and the Spearman rank correlation coefficient 410

are presented. 411

412

413


.org/D

ownloaded from

http://jvi.asm.org/

19

Table 1. Phenotypes of variable regions (V1V2 and V3) chimeras between 414

two primary clade C HIV-1 Env isolates. 415

The phenotypes of the wt and chimeric Env gps were determined as described in 416

the text. Results represent the mean values derived from at least two 417

independent experiments performed in quadruplicate. Less than 20% deviation 418

from the reported mean value was typically observed. 419

420

421


.org/D

ownloaded from

http://jvi.asm.org/

20

Table 1. Phenotypes of variable region (V1V2 and V3) chimeras between Envs 422

from two primary clade C HIV-1 isolates. 423

424

425

426

Chimera CD4-Ig binding

Cold Inactivation

(T1/2)

Neutralization sCD4 (IC50)

Neutralization VRC01 (IC50)

Neutralization VRC03 (IC50)

Neutralization b12 (IC50)

Neutralization 48d (IC50)

ZM109 wt

1.0 › 48 h 2.0 µg/mL 1.0 µg/mL ›10 µg/mL ›10 µg/mL ›10 µg/mL

C1086 wt

0.4 6.0 h ›10 µg/mL 3.0 µg/mL ›10 µg/mL ›10 µg/mL ›10 µg/mL

ZM109(C1086V1V2)


ZM109(C1086V3)

0.4 › 48 h 10 µg/mL 1.0 µg/mL ›10 µg/mL ›10 µg/mL ›10 µg/mL

ZM109(C1086V1V2V3)

0.6 18 h 7.0 µg/mL 1.5 µg/mL ›10 µg/mL ›10 µg/mL ›10 µg/mL

ZM109 N160K


C1086(ZM109V1V2)

0.6 12 h ›10 µg/mL 2.5 µg/mL ›10 µg/mL ›10 µg/mL ›10 µg/mL

C1086(ZM109V3)

0.9 6 h 1.5 µg/mL 3.0 µg/mL ›10 µg/mL ›10 µg/mL ›10 µg/mL

C1086(ZM109V1V2V3) 0.7 9.0 h 4.0 µg/mL 2.0 µg/mL ›10 µg/mL ›10 µg/mL ›10 µg/mL

C1086 K160N

0.9 6.0 h ›10 µg/mL 5.0 µg/mL 9.5 µg/mL ›10 µg/mL ›10 µg/mL


.org/D

ownloaded from

http://jvi.asm.org/


.org/D

ownloaded from

http://jvi.asm.org/

the hiv-1 gp120 major variable regions modulate cold-inactivation

Documents