The N-linked Glycan g15 within the V3 Loop of the HIV-1 External Glycoprotein gp120 Affects Coreceptor Usage, Cellular Tropism, and Neutralization

Abstract

We have studied infectivity and neutralization of X4, R5, and R5X4 tropic HIV-1 mutants, which are lacking N-linked glycosylation sites for glycans g13, g14, g15, and g17 in the V3 loop region of gp120. X4-tropic NL4-3 mutants lacking combinations of g14/15 or g15/17 showed markedly higher infectivity in CXCR4-specific infection. The role of g15 in CCR5-specific infection was investigated using viruses with high (NL-918, R5-monotropic), medium (NL-991, R5-monotropic), and low (NL-952, R5X4-dualtropic) CCR5-specific infectivity. For NL-991, a reduction of infectivity on GHOST-CCR5 cells was observed for a mutant lacking g15. For NL-952 mutants all lacking g15, a complete loss of CCR5-specificity was observed and NL-952 was shifted from R5X4 to X4 tropism. For all mutants of NL4-3, NL-991, and NL-952, where the lack of g15 markedly influenced infectivity or coreceptor usage, neutralization was enhanced. In contrast, NL-918 mutants with or without g15 showed no difference in neutralization and no difference in GHOST-CCR5 infection rates. Thus, for viruses with a low or medium CCR5-specificity the role of g15 for changing CCR5-usage and sensitivity to neutralization was more significant than for viruses with high infection rates on GHOST-CCR5 cells. Our data demonstrate that V3 glycans play an important role in the usage of CXCR4 and CCR5. The lack of g15 was relevant for a more efficient use of CXCR4, whereas interaction with CCR5 was facilitated in the presence of g15. This study also demonstrates that glycan g15 is involved in blocking of neutralizing antibodies and shifting HIV tropism from R5X4 to X4.

Emergence of CXCR4-Using Human Immunodeficiency Virus Type 1 (HIV-1) Variants in a Minority of HIV-1-Infected Patients following Treatment with the CCR5 Antagonist Maraviroc Is from a Pretreatment CXCR4-Using Virus Reservoir

“…Genotypic prediction of viral tropism based on the PSSM method of Jensen et al (21) correlated with the phenotypic tropism assigned to the virus for all clones from patient A and 11/12 unique V3 loop sequences of Env clones from patient C. However, in patient B, the PSSM scores failed to predict CXCR4 usage in all but three sequenced clones assigned a CXCR4-tropic phenotype. Instead, the loss of the g15 N-linked glycosylation site (amino acids 6 to 8), reported to enable more efficient use of CXCR4 (34) and observed in all 28 CXCR4-using clones, appeared to be the most reliable genotypic predictor of CXCR4 usage in this patient.…”

“…V3 loop sequences of clones were also analyzed to predict CXCR4 coreceptor usage on the basis of the PSSM (http://ubik.microbiol.washington.edu/computing /pssm/) (21), the loss of the g15 N-linked glycosylation site (amino acids 6 to 8) (34), and the presence of basic amino acid residues at positions 11 and/or 25 (8,14,20). The results of these genotypic analyses were compared to the coreceptor tropism assigned to the individual Env clones from the phenotypic assay described above.…”

Characterization of glycopeptides from HIV-ISF2 gp120 by liquid chromatography mass spectrometry

“…One of these, the V3 loop, elicits neutralizing anti-HIV antibodies and has been a target for HIV vaccine development [1]. It has been shown that carbohydrates within and around the V3 loop shield binding sites for the chemokine receptors, and loss of these carbohydrates increases infectivity [2][3][4]. The Nlinked glycans on this loop have been reported to strongly influence coreceptor usage, with CCR5 being used during the early stages of disease and CXCR4 being used as the disease progresses [3,4].…”

Development of a Novel Codon-Specific Polymerase Chain Reaction for the Detection of CXCR4-Utilizing HIV Type 1 Subtype B

“…However, it would be relatively easy to design CS-PCR for any target codon of interest -for example, monitoring the loss of N-linked glycosylation sites associated with coreceptor utilization. 22,23 An additional constraint is that CS-PCR was specifically designed for subtype B. Additional primers would be needed to test other subtypes.…”