Identification of Two N-Linked Glycosylation Sites within the Core of the Simian Immunodeficiency Virus Glycoprotein Whose Removal Enhances Sensitivity to Soluble CD4

Abstract

Using PCR mutagenesis to disrupt the NXT/S N-linked glycosylation motif of the Env protein, we created 27 mutants lacking 1 to 5 of 14 N-linked glycosylation sites within regions of gp120 lying outside of variable loops 1 to 4 within simian immunodeficiency virus strain 239 (SIV239). Of 18 mutants missing N-linked glycosylation sites predicted to lie within 10 Å of CD4 contact sites, the infectivity of 12 was sufficient to measure sensitivity to neutralization by soluble CD4 (sCD4), pooled immune sera from SIV239-infected rhesus macaques, and monoclonal antibodies known to neutralize certain derivatives of SIV239. Three of these 12 mutants (g3, lacking the 3rd glycan at position 79; g11, lacking the 11th glycan at position 212; and g3,11, lacking both the 3rd and 11th glycans) were approximately five times more sensitive to neutralization by sCD4 than wild-type (WT) SIV239. However, these same mutants were no more sensitive to neutralization than WT by pooled immune sera. The other 9 of 12 replication-competent mutants in this group were no more sensitive to neutralization than the WT by any of the neutralizing reagents. Six of the nine mutants that did not replicate appreciably had three or more glycosylation sites eliminated; the other three replication-deficient strains involved mutation of site 15. Our results suggest that elimination of glycan attachment sites 3 and 11 enhanced the exposure of contact residues for CD4. Thus, glycans at positions 3 and 11 of SIV239 gp120 may be particularly important for shielding the CD4-binding site from antibody recognition.

Removal of a Single N-Linked Glycan in Human Immunodeficiency Virus Type 1 gp120 Results in an Enhanced Ability To Induce Neutralizing Antibody Responses

“…As was observed by Kolchinsky et al (63) with a CCR5-using strain ADA, removal of this V2 loop proximal glycan in the dualtropic 89.6 strain enables the mutant envelope to mediate CD4-independent infection. Similar observations have been made by Koch et al (62) in HIV and Pikora et al (81) in SIV, indicating that this highly conserved glycan may be important for virus survival by controlling access here further demonstrate that this mutation at this glycan site increases exposure not only for the coreceptor binding site but also for epitopes recognized by several key neutralizing monoclonal antibody, including those that target the CD4-binding site, the CD4-induced epitope, the tip of the V3 loop and, to a lesser extent, the mannan-dependent epitope defined by 2G12 and the MPER epitope defined by 4E10. Elucidation of the basis of these observations will likely require better understanding of the structure and function of the envelope glycoproteins.…”

Conserved Role of an N-Linked Glycan on the Surface Antigen of Human Immunodeficiency Virus Type 1 Modulating Virus Sensitivity to Broadly Neutralizing Antibodies against the Receptor and Coreceptor Binding Sites

“…Previous work examined the role of the N7 glycan in only a limited number of isolates (21)(22)(23). Because the sequon for the N7 glycan is present in more than 95% of available HIV and SIV sequences (19,23,24), we examined if the N7 glycan plays a conserved role in diverse isolates of HIV-1. To test this, we modified the N7 glycan site on the Env from a panel of HIV-1 strains representing different clades, tissue origins, coreceptor usages, and neutralization sensitivities (Table 1), including two viruses, QA255 and JR-FL (25,29), that lack the N7 glycan site in the Env of WT viruses.…”

CD4 + T Cells Support Production of Simian Immunodeficiency Virus Env Antibodies That Enforce CD4-Dependent Entry and Shape Tropism In Vivo

“…2C and D). Q was chosen as a conservative change compared to N that would maintain the charge but abrogate glycosylation, as described previously (39). D470Q in control Envs did not substantially increase CD4-independent entry ( Fig.…”