Generation and Characterization of a Chimeric Rabbit/Human Fab for Co-Crystallization of HIV-1 Rev

Abstract

Rev is a key regulatory protein of HIV-1. Its function is to bind to viral transcripts and effect export from the nucleus of unspliced mRNA thereby allowing the synthesis of structural proteins. Despite its evident importance, the structure of Rev has remained unknown, primarily because Rev's proclivity for polymerization and aggregation is an impediment to crystallization. Monoclonal antibody antigen-binding domains (Fabs) have proven useful for the co-crystallization of other refractory proteins. In the present study, a chimeric rabbit/human anti-Rev Fab was selected by phage display, expressed in a bacterial secretion system, and purified from the media. The Fab readily solubilized polymeric Rev. The resulting Fab/Rev complex was purified by metal ion affinity chromatography and characterized by analytical ultracentrifugation which demonstrated monodispersity and indicated a 1:1 molar stoichiometry. The Fab binds with very high affinity, as determined by surface plasmon resonance, to a conformational epitope in the N-terminal half of Rev. The complex forms crystals suitable for structure determination. The ability to serve as a crystallization aid is a new application of broad utility for chimeric rabbit/human Fab. The corresponding single chain antibody (scFv) was also prepared, offering the potential of intracellular antibody therapeutics against HIV-1.

A Cell-penetrating Antibody Fragment against HIV-1 Rev Has High Antiviral Activity

“…Two truncated forms of the protein, Rev and Rev 1-69 , were prepared by standard procedures and purified in essentially the same way as Rev . The selection, expression, and purification of a Rev aminoterminal domain-specific chimeric rabbit/human antibody fragment (Fab) using phage display have also been described in detail, as have the expression and purification of the corresponding single chain variable domain fragment (scFv) (17). In the present study, two additional antibody fragments were prepared.…”

“…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).…”

“…This method differed from that which led to FabRev1 where the carboxyl-terminal biotinylated Rev was immobilized on strepavidin-coated microtiter plates (17). The single chain form of this Fab (scFvRev2) bound to Rev with a mean equilibrium dissociation constant of 1.9 ϫ 10 Ϫ8 M, characterized by both a high association rate and a high dissociation rate (Fig.…”

“…We have recently used a chimeric human/rabbit Fab generated by phage display as a crystallization chaperone to solve the structure of Rev (17). We found that the oligomerization domain of Rev, constituting approximately one-half of the molecule, consists of two coplanar ␣-helices arranged like a hairpin, and that these dimerize in a chevron-like arrangement with the open ends of the hairpins located toward the middle of the dimer.…”

Structural and Functional Characterization of RNA-binding Site of Rev and Rev-Response-Element RNA Complex via All Atom Molecular Dynamics Simulations

“…Then the export of RNA is carried out by Crm1 nuclear export pathway [17]. First Rev oligomerizes onto the RRE to form a ribonucleoprotein (RNP) complex of 200-300 kDa containing 8-10 Rev molecules [18][19], that binds Crm-1 (exportin-1), GTP-bound Ran, and other host cell proteins via the Rev nuclear export sequence (NES), to promote nuclear export [20]. Structure of REV contain 116 a.a in a single chain [21].…”