Signal Peptide of HIV-1 Envelope Modulates Glycosylation Impacting Exposure of V1V2 Epitopes

HIV-1 envelope (Env) is a trimer of gp120-gp41 heterodimers, synthesized from a precursor gp160 that contains an ER-targeting signal peptide (SP) at its amino-terminus. Each trimer is swathed by ∼90 N-linked glycans, comprising complex-type and oligomannose-type glycans, which play an important role in determining virus sensitivity to neutralizing antibodies. We previously examined the effects of single point SP mutations on Env properties and functions. Here, we aimed to understand the impact of the SP diversity on glycosylation of virus-derived Env and virus neutralization by swapping SPs. Analyses of site-specific glycans revealed that SP swapping altered Env glycan content and occupancy on multiple N-linked glycosites, including the conserved N156 and N160 glycans in the V1V2 region at the Env trimer apex. Virus neutralization was also affected, especially by antibodies against the V2i, V2p and V2q epitopes. Likewise, SP swaps affected the recognition of soluble and cell-associated Env by antibodies targeting distinct V1V2 configurations. These data highlight the contribution of SP sequence diversity in shaping the Env glycan content and its impact on the configuration and accessibility of V1V2 epitopes on Env. Author Summary HIV-1 Env glycoprotein is produced by a precursor gp160 that has a signal peptide at its N-terminus. The SP is highly diverse among the HIV-1 isolates and no two SP are same. This study presents site-specific analyses of N-linked glycosylation on HIV-1 envelope glycoproteins from infectious viruses produced with different envelope signal peptides. We show that signal peptide swapping alters the envelope glycan shield, including the conserved N156 and N160 located in the V1V2 region on the trimer apex, to impact Env recognition and virus neutralization by antibodies, particularly those targeting the the V1V2 region. The data offer crucial insights into the role of signal peptide in the interplay between HIV-1 and antibodies and its potential utility to control Env glycosylation in the development of Env-based HIV-1 vaccine.


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of gp120 (lower band) in REJO-MW was also reduced by 90% (Fig 3C). The poor 266 incorporation of cleaved functional Env into REJO-MW correlated with low infectivity of 267 this virus. 268 We subsequently assessed the impact of SP switch on REJO Env glycosylation 269 by lectin-probed Western blotting (Fig 3D). Comparable WT and SP-swapped total Env 270 bands were detected by anti-gp120 mAbs. However, differences were observed with 271 lectins (GNA and AAL) (Fig 3D). Compared with WT, the lower band of REJO-NL4.3 272 reacted more strongly with GNA (2.5-fold) and AAL (1.7-fold). Both GNA and AAL showed 273 reduced binding to the upper band of REJO-AA05 and increased binding to the lower 274 band of REJO-MW. The LC-MS/MS analysis was able to detect 36% (10/28) and 46% 275 (13/28) of glycosites present on the virion-derived Envs of REJO-WT and REJO-AC02 276 respectively. Glycan compositions on seven glycosites detected in both Envs are 277 compared ( Fig 3E). The data showed altered glycan occupancy at N160 and N188 on the 278 V1V2 apex, as well as at N289 in the C2 region. While 62% of N160 was occupied by 279 oligomannose in REJO-WT, it was 100% decorated with complex-type glycans on REJO-280 AC02. The oligomannose occupancy of N188 was 13% in REJO-WT but increased to 281 50% in REJO-AC02. In REJO-WT N289 was 100% oligomannose but became 100% 282 complex in REJO-AC02. The N362 and N460 glycans were not altered. Thus, as seen 283 with CMU06, SP swapping of REJO also altered Env glycan composition with a significant 284 impact on the two V1V2 glycosites detected in the study. 285 Similar to CMU06, virus sensitivity to neutralization was also altered for SP-286 swapped REJO vs WT; however, the effect was most pronounced for REJO-AC02, 287 REJO-AA05 and REJO-NL4.3 which became more resistant to most of the V2i mAbs 288 15 tested (Fig. 4A, B). REJO with AA05, AC02, and NL4.3 SPs also became more resistant 289 to V2q mAb PG9 and to lesser extent PGT145 (Fig. 4A, B). In contrast, the REJO-MW 290 sensitivity to V2i and V2q mAbs was minimally altered. REJO-MW, however, was more 291 sensitive to V3 mAb 2219 and CD4bs mAbs VRC01 and 3BNC117. 292 To investigate the effect of SP swaps on REJO Env antigenicity, we assessed mAb 293 binding to virus-derived gp120 (Fig. 4C) and Env expressed on 293T cells (Fig. 4D). 294 Reduced binding of all V2i mAbs was seen with REJO-AC02. In contrast, binding of V3 295 mAbs, CD4bs mAb NIH45-46, and CD4-IgG to REJO-AC02 was unaltered. REJO-MW 296 was excluded because of low gp120 content. When we examined mAb binding to native 297 Env on 293T cells, a different pattern was observed. Lower binding of V2i mAbs and 298 CD4bs mAbs was observed for one or more SP-swapped Envs. Swapping MW SP onto 299 REJO Env almost abrogated the binding of trimer specific V2q mAb PGDM1400 and 300 reduced the binding of V2i mAbs 697 and 2158, without affecting the binding of V2p mAb 301 CH59. As compared with REJO-WT, REJO-MW also displayed reduced reactivity with 302 CD4bs mAbs NIH45-46 and 3BNC117, and V3 glycan-specific PGT121. In contrast, 303 REJO-AC02, REJO-AA05, and REJO-NL4.3 had lower reactivity mainly with V2i mAbs 304 (697 and/or 2158). PGT151 mAb, which binds specifically to the cleaved Env trimer, 305 showed increased binding to REJO-AC02, REJO-AA05, and REJO-NL4.3, while binding 306 to REJO-WT and REJO-MW was comparable. Hence, like CMU06 SP swapping, REJO 307 SP exchanges also modified mAb interactions with different formats of REJO Env, with 308 the most common effects of reduced mAb recognition against V1V2. 309 Strain-specific impact of SP swapping on SF162 310 16 To assess the consequences of SP exchanges on another virus strain, we generated 311 SF162 viruses with WT and SP-swapped Envs (Fig. 5A). SF162 is a tier-1A 312 neutralization-sensitive, chronic, clade B isolate. All SP-swapped and WT SF162 viruses 313 displayed comparable levels of infectivity and Env incorporation (Fig. 5B, C). The lectins 314 GNA and AAL reactivity demonstrated moderate changes of oligomannose and fucose-315 bearing complex glycans on SP-swapped Envs vs WT (Fig. 5D). However, in comparison 316 with CMU06, the same SP exchanges did not make SF162 more resistant to V2i mAbs. 317 The SP-swapped viruses were slightly more sensitive to neutralization by some V2i mAbs 318 and CD4bs mAbs (Fig. 5E). Compared to WT, SF162-MW especially were more sensitive 319 to V2i mAbs 1357 and 1361 and CD4bs mAbs VRC01 and 3BNC117. SF162-398F1 and 320 SF162-CH119, on the other hand, were more resistant to MPER gp41 mAb 2F5. 321 Neutralization by V3 mAbs was not markedly changed. 322 Figures S4 and S5 summarize the differential effects of SP exchanges on neutralization 323 phenotypes that depend on the Env backbones. These data illuminate the pronounced 324 effects on neutralization by V1V2 mAbs: the SP swaps reduced neutralization sensitivity 325 of CMU06 and REJO but had opposing effects on SF162. MW SP increased 326 neutralization sensitivity to many V2i, V2q, V3, and CD4bs mAbs, which varied depending 327 on the Env backbones. 328

Host-cell dependence of SP impact on glycosylation and neutralization 329
Because glycosylation is host cell-dependent (61, 62), we investigated the effect 330 of SP exchanges on CMU06 produced in primary CD4 + T cells. PBMC-derived SP-331 swapped CMU06 viruses displayed a radically distinct neutralization pattern from 293T-332 produced counterparts (Fig 6 A-B, Fig S4,  neutralization <50% against CMU06-WT, and the SP-swapped viruses were even more 341 resistant than WT ( Fig 6B). 342 SP swapping also altered the antigenicity of Env expressed on primary CD4 + T 343 cells. The changes were more drastic on SP-swapped CMU06 Envs expressed on CD4 + 344 T cells (Fig. 6C) vs 293T cells (Fig. 2D). V2i mAbs bound CMU06-398F1, CMU06-CH119, 345 and CMU06-271.1 less than CMU06-WT, but binding to CMU06-MW was unaltered. 346 These SP-swapped viruses also showed reduced binding with V3 mAbs (2219 and 2557), 347 and CD4bs mAbs (NIH45-46 and 3BNC117), although binding of PGT151 and PGT121 348 was comparable. Collectively, the data show that SP swapping altered the antigenicity of 349 native Env present on virions and cells, albeit in an Env strain-and host cell-dependent 350 manner. Notwithstanding these variabilities, SP swapping clearly affects the V1V2 351

epitopes. 352
We further determined glycosylation changes on SP-swapped Envs from PBMC-353 produced CMU06 using lectin-probed Western blot. Unlike 293T-derived viruses (Fig  354   S2C), we detected predominantly the lower gp120 bands with anti-gp120 mAbs (Fig 7A), 355 suggesting host cell-specific differences in gp120/gp41 processing. Therefore, we 356 18 analyzed GNA and AAL binding to the lower bands only. Moderate differences were 357 observed in GNA and AAL binding to SP-swapped vs WT CMU06 ( Fig 7A). However, the 358 binding patterns were distinct from those seen with 293T-derived CMU06 (S2C Fig).  Interestingly, the prominent change at N160 on the V1V2 apex was consistently 369 noted in SP-swapped CMU06 produced in PBMCs and 293T cells ( Fig. 1 and 7, S6 Fig). N160 had a decreased oligomannose content upon 398F1 SP swapping, confirming that 371 glycan alterations were incurred by 398F1 SP swap regardless of the producer cells. 372 We also assessed glycans that were affected by both host cells and SPs; these 373  Notably, N156 on the V1V2 apex displayed only oligomannose glycans in PBMC-derived 377 CMU06-WT, but had a mix of oligomannose and complex glycans in 293T-derived 378 CMU06-WT. On PBMC-derived CMU06-271.1, N156 also was completely occupied with 379 19 oligomannose, whereas on 293T-derived counterpart it was mostly unoccupied. N156 380 had reduced oligomannose contents on CMU06-398F1 vs CMU06-WT, when produced 381 in PBMC or 293T cells. Another example is illustrated by N88 on the gp120-gp41 382 interface, which was completely unoccupied in PBMC-derived CMU06-WT, but 82% 383 unoccupied in 293T-derived WT. In contrast, N88 was populated exclusively by complex 384 glycans in CMU06-398F1 and CMU06-271.1 from PBMC, although it was 50% and 80% 385 unoccupied in 293T-derived counterparts. 386 The N398 glycan changes depended on host cells only and are independent of 387 SPs. N398 was 100% occupied by complex glycans on all three PBMC-derived viruses, 388 these were reduced to 22-36% in 293T-derived viruses. 389 Few glycosites were not identified in all samples, allowing only partial comparison.

HIV-1 Env glycosylation is influenced by viral and cellular factors. This study 402
demonstrates the effect of Env SP, a highly variable albeit understudied viral element, on 403 glycosylation and consequently on Env properties and virus phenotypes. Introducing SPs 404 from different virus strains to the same Env backbone altered proportions of 405 oligomannose, complex, and unoccupied glycans on multiple glycosites. Interestingly, 406 exchanging CMU06 SP with 398F1 SP reduced the oligomannose content of N156 and 407 N160 glycosites on V1V2 at the Env spike apex essential for the V2q-class bNAbs. This 408 effect was observed in both PBMC and 293T-produced CMU06-398F1 viruses. Swapping 409 the AC02 SP onto the REJO Env similarly reduced the oligomannose content of N160 410 from 62% on WT to 0% on REJO-AC02. Consistent with glycan changes, these SP 411 exchanges affected Env recognition and virus neutralization by mAbs targeting V1V2 412 epitopes. Thus, although SP is not a part of the mature Env, it is a determinant that affects 413

Env glycosylation and antibody recognition. 414
Among the three classes of V1V2 mAbs, V2i and V2q mAbs were prominently 415 affected by SP swaps, as evidenced by increased or reduced resistance of many SP-416 swapped tier-2 CMU06 and REJO vs their respective WT to neutralization by these mAbs. 417 The effect nonetheless varied depending on SPs and host cells producing the virus. 418 Reasons for altered sensitivity to V2i mAb-mediated neutralization are not fully 419 understood. The V2i epitopes are often occluded on native pre-fusion trimers of tier-2 420 viruses (22) and become exposed to mAbs only after prolonged virus-antibody incubation 421  crosses the ER and the Golgi apparatus, the high-mannose structure is trimmed and 495 subsequently elaborated with hybrid-and complex-type glycans. In view of the observed 496 effect of SP swapping, we suggest that the extent to which the glycan on a particular 497 glycosite is processed is decided while Env is an ER resident with its SP still tethered. SP 498 may subtly affect the compactness of Env folding, which consequently imposes or 499 releases structural constrains to enzymes that generate hybrid or complex glycans in the 500 Golgi. Nonetheless, the steps at which the SP sequence dictates the glycan content of 501 Env are unknown. The Env SP is cleaved post-translationally and this delayed SP 502 cleavage is seen across HIV-1 subtypes ensuring low Env expression on virions (5, 6, 503 74). In addition to the positively charged n-region, residues in the hydrophobic SP region 504 hydrophobic, and leucine residues (S1 Fig); the significance of each element needs to be 536 better understood, considering that SP exchange is a common strategy to promote 537 26 secretion of recombinant Env vaccines and SP selection is critical to generate glycomes 538 faithfully representing those of native Envs. 539 In summary, this study demonstrates an important role of HIV-1 Env SP in 540 influencing Env glycan content. By introducing SP from a particular HIV-1 strain, we can 541 modulate the relative proportion of unoccupied, high-mannose, and complex glycans on 542 specific glycosites, including N156 and N160 on V1V2 at the apex of Env spike. SP swaps 543 also can alter Env recognition and virus neutralization by mAbs, especially mAbs against 544 V1V2. Data from this study have significant implications for vaccine development: SP is 545 a critical component that must be rationally selected and incorporated into the design of 546 Env-based HIV-1 vaccine. 547 27

Materials and methods 548
Plasmids 549 The infectious molecular clones (IMC) were generated by cloning the CMU06 and 550 infection, PBMC were activated by incubation in RPMI-intereukin-2 (IL-2) growth medium 592 containing 10 μg of phytohemagglutinin (PHA) (PHA-P) per ml. The RPMI-IL-2 growth 593 29 medium was RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf 594 serum, L-glutamine (2 mM), penicillin (100 U/ml) and streptomycin (100 μg/ml) and 20 U 595 of recombinant IL-2 per ml. After overnight incubation with PHA, cells were washed and 596 cultured with IL-2 for additional 2 to 3 days. All cultures were maintained in 5% CO2 597 incubators at 37°C. PBMCs were exposed to virus (50ng p24 of 293T-derived stock) 598 overnight and then washed to remove the viral inoculum. Virus-containing supernatant 599 was harvested on day 7 and day 10. Virus infectivity and p24 contents were measured 600 as above. used to detect Env. The mAb 91-5D (1µg/ml) was used to detect Gag p24. Biotinylated 635 GNA, and biotinylated AAL were each used at 2µg/ml. Lectin binding was detected with 636 HRP-neutravidin (1:1500 for 1 hr RT). All dilutions were made in Superblock T20 Buffer. 637 Membranes were developed with Clarity Western ECL substrate and scanned by 638 ChemiDoc Imaging Systems (Bio-Rad Laboratories). Purified recombinant gp120 and 639 31 p24 proteins were also loaded at a known concentration as controls (data not shown). 640 Band intensities were quantified using the Image Lab Software Version 5.0. 641 gp120 -mAb binding assay. The relative binding of mAbs to gp120 from WT and SP-642 swapped viruses was measured by a sandwich ELISA. Half-area high-binding ELISA 643 plates were coated with sheep anti-C terminal gp120 Abs (1µg/ml in PBS), blocked with 644 2% bovine serum albumin (BSA) in PBS, and incubated with 1% Triton X100-virus lysates 645 containing 20 ng/ml Env (quantitated by Western blots). Serially diluted mAbs (0.01-646 10μg/ml) were then added for 2 hours, and the bound mAbs were detected with alkaline 647 phosphatase-conjugated goat anti-human IgG and p-nitrophenyl phosphate substrate. 648 The optical density (OD) was read at 405nm using BioTek PowerWave HT Microplate 649

Spectrophotometer. 650
Cell-associated Env binding assay. Assay to detect antibody binding to cell surface-651 expressed Env was performed as described (98)  Env-stained cells, were quantified. Background MFI, as determined from cells stained 670 without primary antibodies was subtracted from all Env-mAb pairs. 671 For testing mAb binding to Env expressed on primary CD4+ T cells, the 672 experiments were conducted as above with the following modifications: CD4+ T cells 673 were isolated from PBMCs (isolated from Leukopaks) using an EasySep Human CD4+ T 674 cell Enrichment Kit. PHA-activated CD4+ T cells were infected with WT or SP-swapped 675 viruses (500 ng p24/million cells) by spinoculation at 1200×g for 2 hours. The virus 676 inoculum was replaced with fresh medium (RPMI 1640 media supplemented with 10% 677 FBS with 20 U/mL IL-2) and the cells were further incubated for 7 days at 37°C. Cells 678 were harvested for mAb staining as above. 679 Mass Spectrometry: Analysis for site-specific glycosylation was performed as in (99). 680 Briefly, 293T-and PBMC-derived infectious virus stocks were concentrated by sucrose 681 cushion centrifugation. Concentrated (250X) virus preparations were loaded on SDS-682 PAGE gel (7.5%) and the separated Env bands were excised to use directly for mass 683 spectrometry (MS). 684

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The SDS-PAGE gel bands were washed with 100% acetonitrile and water three 685 times. A proteomics-based strategy was used to assess the degree of glycan processing 686 and the degree of site-occupancy of each glycosite (99). The proteins in the gel bands 687 were denatured and alkylated by 10 mM dithiothreitol (DTT) and 55 mM iodoacetamide 688 in 25 mM ammonium bicarbonate, respectively. The resulting proteins were digested with 689 the combination of trypsin and chymotrypsin at an enzyme/substrate ratios of 1:15 (w/w) 690 and 1:10 (w/w) respectively in 25 mM ammonium bicarbonate. Sequential treatment with 691 two endoglycosidases was then performed to introduce novel mass signatures for 692 peptides that contain glycans of high-mannose types and complex-type glycans (99). 693 First, the Env peptides were digested with Endo H to cleave high-mannose (and hybrid) 694 glycans between the innermost GlcNAc residues, leaving a GlcNAc attached to the Asn 695 (N+203). The subsequent PNGase F treatment removed the remaining complex-type 696 glycans, and in the process converted Asn to Asp, resulting in a +0.984 Da mass shift 697 (N+1) (100, 101). For peptides with unoccupied glycosites, these treatments produce no 698 mass shift (N+0). Using this strategy, liquid chromatography-mass spectrometry (LC-699 MS/MS) data were acquired for each sample. Peptides were identified using SEQUEST. 700 The abundance of each peptide was determined by the sum of the peak areas from all 701 identified charge states (63). 702 The 293T-derived Env samples were analyzed on a Q-Exactive mass 703 spectrometer. De-glycosylated peptides were separated on a Dionex Ultimate 3000 704