Deletion of the Highly Conserved N-Glycan at Asn260 of HIV-1 gp120 Affects Folding and Lysosomal Degradation of gp120, and Results in Loss of Viral Infectivity

Abstract

N-linked glycans covering the surface of the HIV-1 glycoprotein gp120 are of major importance for the correct folding of this glycoprotein. Of the, on average, 24 N-linked glycans present on gp120, the glycan at Asn260 was reported to be essential for the correct expression of gp120 and gp41 in the virus particle and deletion of the N260 glycan in gp120 heavily compromised virus infectivity. We show here that gp160 containing the N260Q mutation reaches the Golgi apparatus during biosynthesis. Using pulse-chase experiments with [35S] methionine/cysteine, we show that oxidative folding was slightly delayed in case of mutant N260Q gp160 and that CD4 binding was markedly compromised compared to wild-type gp160. In the search of compensatory mutations, we found a mutation in the V1/V2 loop of gp120 (S128N) that could partially restore the infectivity of mutant N260Q gp120 virus. However, the mutation S128N did not enhance any of the above-mentioned processes so its underlying compensatory mechanism must be a conformational effect that does not affect CD4 binding per se. Finally, we show that mutant N260Q gp160 was cleaved to gp120 and gp41 to a much lower extent than wild-type gp160, and that it was subject of lysosomal degradation to a higher extent than wild-type gp160 showing a prominent role of this process in the breakdown of N260-glycan-deleted gp160, which could not be counteracted by the S128N mutation. Moreover, at least part of the wild-type or mutant gp160 that is normally targeted for lysosomal degradation reached a conformation that enabled CD4 binding.

Global site-specific N-glycosylation analysis of HIV envelope glycoprotein

“…Glycosite N276 on the edge of the CD4-binding site2456, and the highly conserved glycan N262 needed for folding of gp120 (ref. 59), were both found to be exclusively high-mannose glycans. The epitope of bNAb PGT151 is comprised of the gp120-gp41 interface of one protomer and glycans N611 and N637 of the adjacent protomer that were predicted to be complex-type glycans1260, consistent with results here.…”

Engineering of a Lectibody Targeting High-Mannose-Type Glycans of the HIV Envelope

“…35,36 However, deglycosylation of Env glycoproteins may result in the unmasking of critical epitopes, making the virus more sensitive to host immunoglobulins, 12,35,37 and AvFc may still be able to recognize and kill infected cells expressing partially deglycosylated Env, since complete deglycosylation of Env is unlikely due to the critical roles of at least some N-glycans in Env assembly and viral infectivity. [38][39][40] Additionally, the ability of AvFc to neutralize not only group M viruses but also group O, HIV-2, and SIV strains may be an advantage over (or a complement to) contemporary bNAbs, which may not recognize and neutralize beyond group M viruses. Because HMGs are widely found on the glycoproteins of enveloped viruses, 12 there is a possibility that AvFc exhibits neutralization activity against other viruses as well.…”

Molecular Architecture of the Cleavage-Dependent Mannose Patch on a Soluble HIV-1 Envelope Glycoprotein Trimer

“…One notable glycan is the N262 glycan, which is known to play a critical role in gp120 folding (62,63). While this site is exclusively dominated by oligomannose-type glycans on SOSIP.664, it has a mixed composition (ϳ50% complex glycans) on the pseudotrimer, although Man 9 GlcNAc 2 still remains the most prominent oligomannose-type structure.…”