title: Conserved residues in the coiled-coil pocket of human immunodeficiency virus type 1 gp41 are essential for viral replication and interhelical interaction.
abstract: The human immunodeficiency virus type 1 (HIV-1) gp41 plays an important role in mediating the fusion of HIV with host cells. During the fusion process, three N-terminal helices and three C-terminal helices pack in an anti-parallel direction to form a six-helix bundle. X-ray crystallographic analysis of the gp41 core demonstrated that within each coiled-coil interface, there is a deep and large pocket, formed by a cluster of residues in the N-helix coiled-coil. In this report, we systematically analyzed the role of seven conserved residues that are either lining or packing this pocket on the infectivity and interhelical interaction using novel approaches. Our results show that residues L568, V570, W571, and K574 of the N-helix that are lining the side chain and right wall of the pocket are important for establishing a productive infection. Mutations V570A and W571A completely abolished replication, while replication of the L568A and K574A mutants was significantly attenuated relative to wild type. Similarly, residues W628, W631, and I635 of the C-helix that insert into the pocket are essential for infectivity. The impaired infectivity of these seven mutants is in part attributed to the loss in binding affinity of the interhelical interaction. Molecular modeling of the crystal structure of the coiled-coil further shows that alanine substitution of those residues disrupts the hydrophobic interaction between the N- and C-helix. These results suggest that the conserved residues in the coiled-coil domain play a key role in HIV infection and this coiled-coil pocket is a good target for development of inhibitors against HIV. In addition, our data indicate that the novel fluorescence polarization assay described in this study could be valuable in screening for inhibitors that block the interhelical interaction and HIV entry.
authors.affiliation: Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, IL 60064, USA. Hongmei.Mo@abbott.com
authors.forename: Hongmei
authors.initials: H
authors.lastname: Mo
authors.forename: Alex K
authors.initials: AK
authors.lastname: Konstantinidis
authors.forename: Kent D
authors.initials: KD
authors.lastname: Stewart
authors.forename: Tatyana
authors.initials: T
authors.lastname: Dekhtyar
authors.forename: Teresa
authors.initials: T
authors.lastname: Ng
authors.forename: Kerry
authors.initials: K
authors.lastname: Swift
authors.forename: Edmund D
authors.initials: ED
authors.lastname: Matayoshi
authors.forename: Warren
authors.initials: W
authors.lastname: Kati
authors.forename: William
authors.initials: W
authors.lastname: Kohlbrenner
authors.forename: Akhteruzzaman
authors.initials: A
authors.lastname: Molla
mesh_terms: D000595:Amino Acid Sequence; D014408:Biomarkers, Tumor; D002096:C-Peptide / Q000737:chemistry / Q000235:genetics; D002461:Cell Line, Transformed; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics; D005454:Fluorescence Polarization / Q000379:methods; D015700:HIV Envelope Protein gp41 / Q000737:chemistry / Q000235:genetics*; D015497:HIV-1 / Q000235:genetics* / Q000472:pathogenicity; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010751:Phosphopyruvate Hydratase / Q000737:chemistry / Q000235:genetics; D017354:Point Mutation; D013329:Structure-Activity Relationship; D025521:Tumor Suppressor Proteins / Q000737:chemistry / Q000235:genetics; D014779:Virus Replication / Q000235:genetics*
chemical_list: D014408:Biomarkers, Tumor; D002096:C-Peptide; D004268:DNA-Binding Proteins; D015700:HIV Envelope Protein gp41; D025521:Tumor Suppressor Proteins; C095157:ENO1 protein, human; D010751:Phosphopyruvate Hydratase