title: Mutational analysis of HIV-1 gp160-mediated receptor interference: intracellular complex formation.
abstract: Formation of CD4-gp160 intracellular complexes represents an important mechanism leading to the induction of receptor interference. Previous studies have demonstrated that cells coexpressing gp160 and CD4 formed complexes of CD4 and gp160 which became blocked within the endoplasmic reticulum (ER), preventing CD4 from reaching the cell surface. In this report we have investigated the domains and residues of CD4 and gp160 involved in intracellular interaction. Accordingly, we have introduced mutations in both CD4 and gp160 at sites previously shown to disrupt CD4-gp120 interactions at the cell surface. Using a T7-vaccinia virus transient expression system, we expressed these gp160 and CD4 mutants in HeLa cells and analyzed their effects on intracellular complex formation and CD4 surface modulation. We observed that a number of gp160 mutants which failed to interact with CD4 at the cell surface also failed to bind and trap CD4 within the ER as expected. However, mutations at a critical residue, W427, did not abrogate intracellular CD4 binding. These gp160 mutants continued to interact with intracellular CD4 and inhibit CD4 transport to the cell surface, although gp120 produced from these mutants did not bind CD4 at the cell surface as expected. A number CD4 mutants also continued to form intracellular complexes with gp160, resulting in the loss of CD4 surface expression. Again, these CD4 mutants did not bind to gp120 at the cell surface, consistent with earlier reports. These results demonstrate that intracellular interactions between gp160 and CD4 in the ER may utilize different contact sites compared to those used during CD4 and gp120 binding at the cell surface. The data provide further evidence that the environment in which CD4 and the HIV-1 envelope glycoprotein interact can have a significant effect on their interaction.
authors.affiliation: Department of Microbiology and Immunology, Jonsson Comprehensive Cancer Center, UCLA School of Medicine 90024-1747, USA.
authors.forename: R A
authors.initials: RA
authors.lastname: Martin
authors.forename: D P
authors.initials: DP
authors.lastname: Nayak
mesh_terms: D000818:Animals; D001483:Base Sequence; D015704:CD4 Antigens / Q000235:genetics / Q000378:metabolism*; D004279:DNA, Viral; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D015686:Gene Products, env / Q000235:genetics / Q000378:metabolism*; D015699:HIV Envelope Protein gp120 / Q000378:metabolism; D019302:HIV Envelope Protein gp160; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D012995:Solubility
chemical_list: D015704:CD4 Antigens; D004279:DNA, Viral; D015686:Gene Products, env; D015699:HIV Envelope Protein gp120; D019302:HIV Envelope Protein gp160; D011498:Protein Precursors