IDENTIFICATION OF 3 N-LINKED GLYCANS IN THE V4-V5 REGION OF HIV-1 GP-120, DISPENSABLE FOR CD4-BINDING AND FUSION ACTIVITY OF GP-120

Site-directed mutagenesis was used to study the biological significanc e of three N-linked glycans (linked to Asn406, Asn448, and Asn463), si tuated in the CD4-binding region of gp 120. Mutagenesis was carried ou t in a phage M13 system, and the mutated env genes were inserted into recombinant vaccinia virus (r-vaccinia virus). To evaluate if the leve l 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 mutated e nu proteins was analyzed after infecting CD4-expressing HeLa cells wit h the r-vaccinia virus, by monitoring the ability of the infected cell s to generate CD4-dependent syncytia. Env gene products lacking all th ree glycans as well as env gene products lacking different permutation s of one or two glycans were analyzed. All mutated gp 120 species had the expected electrophoretical mobility as anticipated from eliminatio n of one, two, and three N-linked glycans, respectively. Moreover, all mutant enu gene products demonstrated the same capacity to induce for mation of syncytia, irrespective of using the weak or strong promoter for expression. These data indicate that the three N-linked glycans st udied are dispensable for HIV env gene products to function in CD4-bin ding and the subsequent fusion step.