title: A cell-penetrating antibody fragment against HIV-1 Rev has high antiviral activity: characterization of the paratope.
abstract: The HIV-1 protein Rev oligomerizes on viral transcripts and directs their nuclear export. Previously, a Fab against Rev generated by phage display was used to crystallize and solve the structure of the Rev oligomerization domain. Here we have investigated the capability of this Fab to block Rev oligomerization and inhibit HIV-1 replication. The Fab itself did not have antiviral activity, but when a Tat-derived cell-penetrating peptide was appended, the resulting molecule (FabRev1-Tat) was strongly inhibitory of three different CCR5-tropic HIV-1 isolates (IC50 = 0.09-0.44 μg/ml), as assessed by suppression of reverse transcriptase activity in infected peripheral blood mononuclear cells, and had low cell toxicity (TC50 > 100 μg/ml). FabRev1-Tat was taken up by both peripheral blood mononuclear and HEK293T cells, appearing in both the cytoplasm and nucleus, as shown by immunofluorescence confocal laser scanning microscopy. Computational alanine scanning was used to identify key residues in the complementarity-determining regions to guide mutagenesis experiments. Residues in the light chain CDR3 (LCDR3) were assessed to be important. Residues in LCDR3 were mutated, and LCDR3-Tyr(92) was found to be critical for binding to Rev, as judged by surface plasmon resonance and electron microscopy. Peptides corresponding to all six CDR regions were synthesized and tested for Rev binding. None of the linear peptides had significant affinity for Rev, but four of the amide-cyclic forms did. Especially cyclic-LCDR3 (LGGYPAASYRTA) had high affinity for Rev and was able to effectively depolymerize Rev filaments, as shown by both surface plasmon resonance and electron microscopy.
authors.affiliation: From the Protein Expression Laboratory and.
authors.forename: Xiaolei
authors.initials: X
authors.lastname: Zhuang
authors.affiliation: From the Protein Expression Laboratory and.
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Stahl
authors.affiliation: From the Protein Expression Laboratory and.
authors.forename: Norman R
authors.initials: NR
authors.lastname: Watts
authors.affiliation: the Laboratory of Structural Biology Research, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: Michael A
authors.initials: MA
authors.lastname: DiMattia
authors.affiliation: the Laboratory of Structural Biology Research, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: Alasdair C
authors.initials: AC
authors.lastname: Steven
authors.affiliation: From the Protein Expression Laboratory and pelpw@helix.nih.gov.
authors.forename: Paul T
authors.initials: PT
authors.lastname: Wingfield
mesh_terms: D000595:Amino Acid Sequence; D019380:Anti-HIV Agents / Q000737:chemistry / Q000276:immunology / Q000494:pharmacology*; D001666:Binding Sites, Antibody / Q000235:genetics / Q000276:immunology; D057846:Cell-Penetrating Peptides / Q000235:genetics / Q000276:immunology / Q000494:pharmacology*; D022801:Complementarity Determining Regions; D057809:HEK293 Cells; D015497:HIV-1 / Q000187:drug effects* / Q000276:immunology* / Q000502:physiology; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000276:immunology / Q000494:pharmacology*; D007700:Kinetics; D046529:Microscopy, Electron, Transmission; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D015202:Protein Engineering; D055503:Protein Multimerization / Q000187:drug effects; D014779:Virus Replication / Q000187:drug effects / Q000276:immunology; D054321:rev Gene Products, Human Immunodeficiency Virus / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism
chemical_list: D019380:Anti-HIV Agents; D057846:Cell-Penetrating Peptides; D022801:Complementarity Determining Regions; D007140:Immunoglobulin Fab Fragments; D054321:rev Gene Products, Human Immunodeficiency Virus; C514860:rev protein, Human Immunodeficiency Virus-1
keywords: AIDS; Cell-penetrating peptide (CPP); Cyclic Peptide; Monoclonal Antibody; Phage Display