The HIV Env Signal Peptide Impacts the Glycosylation and Antigenicity of gp120 Jason Yolitz1,2, Catherine Schwing1, Claudia Cicala1, Danlan Wei1, Fatima Nawaz1,3, Katija Jelicic1,

Donald Van Ryk1, Anthony Fauci1, and James Arthos1.

1National Institute of Allergy and Infectious Disease, National Institutes of Health, 2Johns Hopkins University, 3Sackler Institute of Biomedical Research, New York University

Mucosal HIV transmission is an inefficient process and is commonly the result of a single “founder” virus. This bottleneck-produced “founder” virus replicates in an activated CD4+ T cell and produces replicating virus.

Transmission of HIV across mucosal tissues involves a complex yet inefficient series of events that are not yet fully understood. The HIV-1 envelope protein (Env) of early-transmitting viruses has been found to present distinct transmission signatures. One such signature involves a reduced number of potential N-linked glycosylation sites, underscoring the importance of post-translational modifications in transmission fitness. Recently, an additional transmission signature has been identified in the signal peptide of Env. This signature involves the over-representation of basic residues at a specific position in the signal peptide. We investigated the potential impact of this signal peptide signature on gp120 glycosylation and antigenicity. Two recombinant Envs were constructed, one derived from a chronic isolate that lacks the signal peptide signature and a second from a transmitting isolate that includes the signal peptide signature. Chimeric Envs were also constructed in which the two signal peptides were swapped. All four gp120s were probed with glycan-, structure- and interaction-specific probes in a surface plasmon resonance binding assay. The signature found in the signal peptide of Env influences qualitative aspects of Env glycosylation that in turn affect the structure and antigenicity of Env in a major way. The signal peptide of a transmitting isolate increases Env’s affinity for DC-SIGN, a lectin receptor expressed on dendritic cells that is believed to be involved in mucosal transmission. Additionally, affinity for the monoclonal antibody 17b, which recognizes the CD4-induced (open) conformation of Env is also increased by the transmitting isolate’s signal peptide. We conclude that the HIV Env signal peptide is an important determinant of Env processing, folding and antigenicity. These observations may aid in the development of Env based HIV vaccines.

HIV-1 Env is heavily glycosylated with variable glycan forms (high-mannose, complex, and hybrid glycans). The number of potential N-linked glycosylation sites (PNGs) varies from 18 to 28 depending on the virus clade and maturation level.

Signature 1 of early transmitting viruses is demonstrated by transmitting HIV isolates bearing fewer PNGs resulting in hypo-glycosylation of viral Env.

Signature 2 is demonstrated by transmitting HIV isolate sequences bearing an over-represented basic residue at position 12 of the Env leader sequence.

Env containing a basic residue at position 12 of the leader sequence is associated with higher expression levels of HIV-1 Env.

Early (AA05) M.W. ~100 kD Early w. Chronic Leader M.W ~160 kD

Chronic (AC02) M.W. ~140 kD Chronic w. Early Leader M.W. ~120 kD

Figure 1. Addition of early transmitting leader peptide decreases the molecular weight of chronic gp120 as demonstrated by size-exclusion chromatography.

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Figure 2. Swapping Env leader peptide sequences between transmitting and chronic gp120s induces alternate glycosylation patterns as shown by Surface Plasmon Resonance (SPR) Assay (Biacore).

High-Mannose Oligosaccharide Narcissus Pseudonarcissus (Daffodil) Lectin

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Figure 3. Swapping Env leader peptides between transmitting and chronic gp120s induces alternate affinities for DC-SIGN, a C-type lectin expressed on dendritic cells. HIV Env binds to DC-SIGN and some reports suggest a relevant interaction between virus and DC-SIGN at the time of transmission.

AC02 AC02 (chronic) gp120

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AC02 AA05 (early) gp120

Figure 5. Swapping Env leader peptides changes the structure of mature gp120 as shown by alternate binding to mAb 17b.

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Figure 4. Removing two PNGs from a subtype A chronic gp120 changes the structure of mature gp120 as shown by alternate binding to mAb 17b, a conformational sensitive probe of gp120 structure.

mAb 17b Ug037 wildtype

mAb 17b Ug037 N144Q

gp120 was pre-bound with increasing concentrations of sCD4 (12.5-200nM) followed by 50nM 17b.

-  Two genetic signatures of transmitting HIV-1 impact N-linked glycosylation of viral Env.

-  Signature 2, an over-represented basic residue at position 12 of the Env leader sequence, biases Env to present high mannose glycans.

-  Signature 2 also alters the interaction between gp120 and DC-SIGN, which may play a key role in early transmission.

Asmal M, Hellmann I, Liu W, Keele BF, Perelson AS, et al. (2011) A Signature in HIV-1 Envelope Leader Peptide Associated with Transition from Acute to Chronic Infection Impacts Envelope Processing and Infectivity. PLoS ONE 6(8): e23673.

Binley J, Ban YA, Crooks ET, Eggink D, Osawa K, et al. (2010) Role of Complex Carbohydrates in Human Immunodeficiency Virus Type 1 Infection and Resistance to Antibody Neutralization. Journal of Virology 84(11): 5637-55. This work was supported by funding from the National Institute of Allergy and Infectious Disease (NIAID) Intramural Research Program at the National Institutes of Health (NIH).

Asmal et al. 2010

AC02 AC02 (Chronic) gp120

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Transmitting (early) Chronic (late)

MRVKGIRKNYQHLWRWGIMLLGMLMICSEEDKLWVTVY MRVKGIRKNYWWKWGTL--LLGMLMICSAEEQLWVTVY ********** * ******** * ******

transferred cotranslationally to the free amide of the aspara-gine in a sequon Asn-X-Thr/Ser, where X is not Pro (40).Terminal glucose and mannose moieties are then trimmed toyield Man5GlcNAc2 (Fig. 1B). Conversion to a hybrid glycan is

then initiated by N-acetylglucosamine transferase I (GnTI),which transfers a GlcNAc moiety to the D1 arm of theMan5GlcNAc2 substrate (19) (Fig. 1B). This hybrid glycoformis then a substrate for modification into complex glycans, in

FIG. 1. Glycan biosynthesis and distribution on gp120 and gp41. (A) Putative carbohydrate modifications are shown on gp120 and gp41secondary structures, based on various published works (26, 42, 63, 74, 119, 128). The gp120 outer domain is indicated, as are residues that formthe SOS gp120-gp41 disulfide bridge. The outer domain is divided into neutralizing and silent faces. Symbols distinguish complex, oligomannose,and unknown glycans. Generally, the complex glycans of the outer domain line the receptor binding sites of the neutralizing face, while theoligomannose glycans of the outer domain protect the silent domain (105). Asterisks denote sequons that are unlikely to be utilized, includingposition 139 (42), position 189 (26, 42), position 406 (42, 74), and position 637 (42). Glycans shown in gray indicate when sequon clustering maylead to some remaining unused, e.g., positions 156 and 160 (42, 119), positions 386, 392, and 397 (42), and positions 611 and 616 (42). There isalso uncertainty regarding some glycan identities: glycans at positions 188, 355, 397, and 448 are not classified as predominantly complex oroligomannose (26, 42, 63, 128). The number of mannose moieties on oligomannose glycans can vary, as can the number of antennae and sialic acidson complex glycans (77). The glycan at position 301 appears to be predominantly a tetra-antennary complex glycan, as is the glycan at position 88,while most other complex glycans are biantennary (26, 128). (B) Schematic of essential steps of glycan biosynthesis from the Man9GlcNAc2precursor to a mature multiantennary complex glycan. Mannosidase I progressively removes mannose moieties from the precursor, in a processthat can be inhibited by the drug kifunensine. GnTI then transfers a GlcNAc moiety to the D1 arm of the resulting Man5GlcNAc2 intermediate,creating a hybrid glycan. Mannose trimming of the D2 and D3 arms then allows additional GlcNAc moieties to be added by a series of GnT familyenzymes to form multiantennary complexes. This process can be inhibited by swainsonine. The antennae are ultimately capped and decorated bygalactose and sialic acid. Hybrid and complex glycans are usually fucosylated at the basal GlcNAc, rendering them resistant to endo H digestion.However, NgF is able to remove all types of glycan.

5638 BINLEY ET AL. J. VIROL.

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Differences in glycan processing and maturation can have a profound impact on protein folding, structuring and binding to target receptors.

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Figure 1. Size Exclusion Chromatography of Recombinant gp120s with Leader Sequence Variations. Caption.

Abstract

Introduction

Introduction (cont’d) Results (cont’d)

Results

Conclusions

References

AC02 AC02 (chronic) gp120

AA05 AC02 (chronic) gp120

AA05 AA05 (early) gp120

AC02 AA05 (early) gp120