Glycans Flanking the Hypervariable Connecting Peptide between the A and B Strands of the V1/V2 Domain of HIV-1 gp120 Confer Resistance to Antibodies That Neutralize CRF01_AE Viruses

Abstract

Understanding the molecular determinants of sensitivity and resistance to neutralizing antibodies is critical for the development of vaccines designed to prevent HIV infection. In this study, we used a genetic approach to characterize naturally occurring polymorphisms in the HIV envelope protein that conferred neutralization sensitivity or resistance. Libraries of closely related envelope genes, derived from virus quasi-species, were constructed from individuals infected with CRF01_AE viruses. The libraries were screened with plasma containing broadly neutralizing antibodies, and neutralization sensitive and resistant variants were selected for sequence analysis. In vitro mutagenesis allowed us to identify single amino acid changes in three individuals that conferred resistance to neutralization by these antibodies. All three mutations created N-linked glycosylation sites (two at N136 and one at N149) proximal to the hypervariable connecting peptide between the C-terminus of the A strand and the N-terminus of the B strand in the four-stranded V1/V2 domain β-sheet structure. Although N136 has previously been implicated in the binding of broadly neutralizing monoclonal antibodies, this glycosylation site appears to inhibit the binding of neutralizing antibodies in plasma from HIV-1 infected subjects. Previous studies have reported that the length of the V1/V2 domain in transmitted founder viruses is shorter and possesses fewer glycosylation sites compared to viruses isolated from chronic infections. Our results suggest that vaccine immunogens based on recombinant envelope proteins from clade CRF01_AE viruses might be improved by inclusion of envelope proteins that lack these glycosylation sites. This strategy might improve the efficacy of the vaccines used in the partially successful RV144 HIV vaccine trial, where the two CRF01_AE immunogens (derived from the A244 and TH023 isolates) both possessed glycosylation sites at N136 and N149.

Short Communication: The Distribution of Potential N-Linked Glycosylation Sites in Gp120 Differs Among Major HIV-1 Subtypes Circulating in China

“…As removing N-glycans from the V2/V4 domains was shown to significantly improve the immunogenicities of HIV-1 Env proteins 15,16 and the neutralization sensitivities of HIV-1/SIV viruses, 17 it is understandable that several HIV-1 CRF01_AE isolates were found to show slightly higher neutralization sensitivity by polyclonal antibodies in plasma of HIV-1-infected subjects. 18 Moreover, according to previous studies, the short V1/V2 and fewer PNGSs in V1/V2/V4 [19][20][21][22] are the major characteristics of mucosally transmitted founder viruses, hence our data emphasize the possibility that the fewer PNGSs in the V2 and V4 loops may contribute to the advantage of CRF01_AE in sexual transmission. To support this hypothesis, we compared the sexually and intravenously transmitted CRF01_AE-CN Env sequences.…”

Novel Acylguanidine-Based Inhibitor of HIV-1

“…We observed that SM111 selected for mutations in Vpu's transmembrane domain, alone or in combination with a mutation at Env codon 136 that is predicted to abolish a common N-linked glycosylation site adjacent to the V1-V2 loop (47). We observed that SM111 reduced extracellular, but not intracellular, p24…”

“…5), compared to outgrowth of control HIV-1 NL4.3 at day 4 in the absence of SM111. Bulk (direct) Sanger sequencing of the culture supernatant indicated that strain C featured both a premature stop codon in place of tryptophan 22 (W22*) in Vpu's transmembrane domain and a highly dominant N136Y variant substitution in Env gp120 that is predicted to abolish a common N-linked glycosylation site (47). Neither wild-type Vpu nor wild-type Env sequences were detected.…”

Plasticity and Epitope Exposure of the HIV-1 Envelope Trimer

“…The wild-type JR-FL pseudovirus is highly resistant to neutralization in the TZM-bl assay by virtually all V2i, V2p, and V2q MAbs tested, which is why it was selected for analysis (Table 1). Since glycosylation of Env shields and stabilizes Env from Abs of many specificities (5,7,24,25), we first tested the effect on the neutralization sensitivity of JR-FL and its variants from which selected gp120 glycans had been deleted ( Fig. 1 to 3).…”