Identification of three N-linked glycans in the V4–V5 region of HIV-1 gp 120, dispensable for CD4-binding and fusion activity of gp 120

Abstract

Site-directed mutagenesis was used to study the biological significance of three N-linked glycans (linked to Asn406, Asn448, and Asn463), situated in the CD4-binding region of gp120. Mutagenesis was carried out in a phage M13 system, and the mutated env genes were inserted into recombinant vaccinia virus (r-vaccinia virus). To evaluate if the level of expression affected the biological phenotype of mutant gp120, we expressed the envelope glycoproteins using either a weak (7.5 K) or a strong (11 K) promoter of vaccinia virus. The expression of mutated env proteins was analyzed after infecting CD4-expressing HeLa cells with the r-vaccinia virus, by monitoring the ability of the infected cells to generate CD4-dependent syncytia. Env gene products lacking all three glycans as well as env gene products lacking different permutations of one or two glycans were analyzed. All mutated gp120 species had the expected electrophoretical mobility as anticipated from elimination of one, two, and three N-linked glycans, respectively. Moreover, all mutant env gene products demonstrated the same capacity to induce formation of syncytia, irrespective of using the weak or strong promoter for expression. These data indicate that the three N-linked glycans studied are dispensable for HIV env gene products to function in CD4-binding and the subsequent fusion step.

Consistent Patterns of Change during the Divergence of Human Immunodeficiency Virus Type 1 Envelope from That of the Inoculated Virus in Simian/Human Immunodeficiency Virus-Infected Macaques

“…It has been shown that the relative positions and number of PNG sites can vary without sacrificing infectivity (28,45); however, these sites do have functional roles. Several of the convergent PNG changes occur within hot spots proximal to the CD4bs, a conformational determinant of Env that is conserved among the primate lentivirus family.…”

Role of Human Immunodeficiency Virus Type 1 Envelope Structure in the Induction of Broadly Neutralizing Antibodies

“…The hypervariable V4 loop is highly heterogenous due to several insertions and deletions and, as a consequence, the increase of potential N-glycosylation sites (PNG) (2,41). In contrast, V5 is less glycosylated than V4 (21) and plays a role in CD4 binding and neutralizing antibody responses (51,71), as shown with VRC01 (2, 41). These host-derived carbohydrates protect the protein surface underlying the gp120 by masking them.…”

Marked Depletion of Glycosylation Sites in HIV-1 gp120 under Selection Pressure by the Mannose-Specific Plant Lectins ofHippeastrumHybrid andGalanthus nivalis

“…However, given the increased infectivity of our mutant virus strains, this phenomenon can not be ascribed to amino acid mutations 88 or 276. Hemming et al (1994) performed site-directed mutagenesis on the C-terminal Asn406, Asn448, and Asn463 that are located in the CD4 binding area of gp120. When abolished all together, the absence of these three glycosylation sites had no effect on syncytia induction and they are thus dispensable for an efficient CD4 binding and subsequent viral fusion.…”