title: Development of an in vitro assay system for screening of gp41 inhibitory compounds.
abstract: The transmembrane protein of HIV-1, gp41, mediates fusion between membranes of the virus and target cell. Strong interaction between the helical regions in the ectodomain of gp41 has been exploited to develop a method that can detect a potential inhibitor against gp41. The N-terminus coiled-coil or the C-terminus helical sequences within the ectodomain of gp41 were inserted into the C-terminus of thioredoxin (Trx) or glutathione S-transferase (GST) to generate the fusion proteins, Trx-N and GST-C, respectively. The inserted sequences of GST-C and Trx-N cause the two proteins to interact with each other and to form a complex. Furthermore, GST-C binds specifically to the surface-coated Trx-N, and the amount of attached GST-C is detected by an ELISA assay using anti-GST antibodies. Peptides derived from the helical regions of gp41 compete with GST-C for binding to Trx-N as well as prevent the gp41-mediated cell fusion. This in vitro assay system can be applied to screening compounds that have an inhibitory activity against gp41.
authors.affiliation: Structural Biology Center, Korea Institute of Science and Technology, Seoul.
authors.forename: J R
authors.initials: JR
authors.lastname: Ryu
authors.forename: J
authors.initials: J
authors.lastname: Lee
authors.forename: S
authors.initials: S
authors.lastname: Choo
authors.forename: S H
authors.initials: SH
authors.lastname: Yoon
authors.forename: E R
authors.initials: ER
authors.lastname: Woo
authors.forename: Y G
authors.initials: YG
authors.lastname: Yu
mesh_terms: D000595:Amino Acid Sequence; D019380:Anti-HIV Agents / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D001667:Binding, Competitive; D002459:Cell Fusion / Q000187:drug effects; D004797:Enzyme-Linked Immunosorbent Assay; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D015700:HIV Envelope Protein gp41 / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008561:Membrane Fusion / Q000187:drug effects; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013879:Thioredoxins / Q000235:genetics / Q000378:metabolism
chemical_list: D019380:Anti-HIV Agents; D015700:HIV Envelope Protein gp41; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D013879:Thioredoxins; D005982:Glutathione Transferase