Glycosylation of the core of the HIV-1 envelope subunit protein gp120 is not required for native trimer formation or viral infectivity

Abstract

The gp120 subunit of the HIV-1 envelope (Env) protein is heavily glycosylated at ∼25 glycosylation sites, of which ∼7-8 are located in the V1/V2 and V3 variable loops and the others in the remaining core gp120 region. Glycans partially shield Env from recognition by the host immune system and also are believed to be indispensable for proper folding of gp120 and for viral infectivity. Previous attempts to alter glycosylation sites in Env typically involved mutating the glycosylated asparagine residues to structurally similar glutamines or alanines. Here, we confirmed that such mutations at multiple glycosylation sites greatly diminish viral infectivity and result in significantly reduced binding to both neutralizing and non-neutralizing antibodies. Therefore, using an alternative approach, we combined evolutionary information with structure-guided design and yeast surface display to produce properly cleaved HIV-1 Env variants that lack all 15 core gp120 glycans, yet retain conformational integrity and multiple-cycle viral infectivity and bind to several broadly neutralizing antibodies (bNAbs), including trimer-specific antibodies and a germline-reverted version of the bNAb VRC01. Our observations demonstrate that core gp120 glycans are not essential for folding, and hence their likely primary role is enabling immune evasion. We also show that our glycan removal approach is not strain restricted. Glycan-deficient Env derivatives can be used as priming immunogens because they should engage and activate a more divergent set of germlines than fully glycosylated Env. In conclusion, these results clarify the role of core gp120 glycosylation and illustrate a general method for designing glycan-free folded protein derivatives.

Bacterially expressed HIV-1 gp120 outer-domain fragment immunogens with improved stability and affinity for CD4-binding site neutralizing antibodies

“…3C). Native proteins run on a C5 analytical reverse-phase column elute as a single peak, thereby showing that they exist as a homogeneous species in solution and not as a mixture of different disulfide-bonded isomers (18). The denatured, reduced proteins eluted at a different acetonitrile concentration than the native protein, re-confirming that native proteins are well-folded and oxidized (Fig.…”

“…As all the above designs (⌬BS-OD EC , OD EC Consensus, OD EC CF, and OD EC SS) showed some improvement in binding with VRC01 bNAb, we decided to combine these mutations with the glycosylation-site mutations identified earlier by our group because these mutations also improve binding for CD4bs ligands (18). The first combined construct (OD EC C1) had mutations at all the 14 outer-domain glycosylation sites, cavityfilling mutations, an additional disulfide and was devoid of the bridging sheet region.…”

“…a GL-VRC01 refers to germline reverted VRC01 (18); S.D. is standard deviation for the data from two independent experiments; NB means no detectable binding.…”

“…Introducing various stabilizing mutations individually in the OD EC background did not result in achieving gp120-like affinity for bNAbs, so we decided to combine the various stabilizing mutations (described above) with the glycosylation site mutations earlier identified by our group (18). The first combined construct (OD EC C1) was made by deleting the bridging sheet region coupled with the introduction of an additional disulfide (294 -448) and cavity-filling mutations in the ⌬G14-OD EC background (all 14 glycosylation sites present in the outer domain were mutated) ( Table 1, no.…”

“…In another combined construct, the additional disulfide and cavity-filling mutations were introduced in the bridging sheet-deleted OD EC Consensus protein background. Four glycosylation sites close to the CD4bs (18) were also mutated in this construct to generate the OD EC C2 construct ( Table 1, no. 8).…”