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.
Highlights
There is a need for alternative HIV-1 inhibitors
We further show that the light chain CDR3 (LCDR3) is a critical component of the Fab paratope, and that a cyclic form of LCDR3 effectively disrupts Rev polymers in vitro
Antibody Fragments Binding to the Amino-terminal Region of Rev—We have previously used phage display to generate a chimeric rabbit/human anti-Rev Fab [17], which binds to a conformational epitope involving the two ␣-helices in the aminoterminal domain of Rev (Fig. 1A) [18]
Summary
Results: An engineered antibody fragment (Fab) against the essential HIV-1 protein Rev significantly reduces reverse transcriptase activity in human cell culture, and synthetic antigen-binding peptides from the Fab block Rev polymerization. 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 g/ml), as assessed by suppression of reverse transcriptase activity in infected peripheral blood mononuclear cells, and had low cell toxicity (TC50 > 100 g/ml). Residues in LCDR3 were mutated, and LCDR3-Tyr was found to be critical for binding to Rev, as judged by surface plasmon resonance and electron microscopy. 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
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