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

Summary Site-directed mutagenesis was used to study the biological significance of three N-linked glycans (linked to Asn 406, Asn 448, and Asn 463), situated in the CD4-binding region of gp120. Mutagenesis was carried out in a phage M13 system, and the mutatedenv genes were inserted into recombinant vaccinia virus (r-vaccinia virus). To evaluate if the level of expression affected the biological phenotype of mutant gp 120, we expressed the envelope glycoproteins using either a weak (7.5 K) or a strong (11 K) promoter of vaccinia virus. The expression of mutatedenv 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 asenv gene products lacking different permutations of one or two glycans were analyzed. All mutated gp 120 species had the expected electrophoretical mobility as anticipated from elimination of one, two, and three N-linked glycans, respectively. Moreover, all mutantenv 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 HIVenv gene products to function in CD4-binding and the subsequent fusion step.