Abstract
Viral infection and pathogenesis is mediated by host protein—viral protein complexes that are important targets for therapeutic intervention as they are potentially less prone to development of drug resistance. We have identified human, recombinant antibodies (Fabs) from a phage display library that bind to three HIV-host complexes. We used these Fabs to 1) stabilize the complexes for structural studies; and 2) facilitate characterization of the function of these complexes. Specifically, we generated recombinant Fabs to Vif-CBF-β-ELOB-ELOC (VCBC); ESCRT-I complex and AP2-complex. For each complex we measured binding affinities with KD values of Fabs ranging from 12–419 nM and performed negative stain electron microscopy (nsEM) to obtain low-resolution structures of the HIV-Fab complexes. Select Fabs were converted to scFvs to allow them to fold intracellularly and perturb HIV-host protein complex assembly without affecting other pathways. To identify these recombinant Fabs, we developed a rapid screening pipeline that uses quantitative ELISAs and nsEM to establish whether the Fabs have overlapping or independent epitopes. This pipeline approach is generally applicable to other particularly challenging antigens that are refractory to immunization strategies for antibody generation including multi-protein complexes providing specific, reproducible, and renewable antibody reagents for research and clinical applications. The curated antibodies described here are available to the scientific community for further structural and functional studies on these critical HIV host-factor proteins.
Highlights
HIV, like other viruses, is an obligate intracellular parasite and requires host cellular machinery at all stages of its life cycle to replicate and counteract the effect of the host immune system
X-ray and cryogenic electron microscopy (EM) structural studies of difficult to study proteins and protein complexes we have developed conformation-specific recombinant antibodies for protein and complex stabilization, capture, and labelling [1,2,3,4]
Specific and potent recombinant antibody fragments that bind to distinct conformational states of HIV-host complexes are good reagents for structural and functional characterization of these interactions and can lead to new strategies for therapeutic intervention
Summary
HIV, like other viruses, is an obligate intracellular parasite and requires host cellular machinery at all stages of its life cycle to replicate and counteract the effect of the host immune system. Viruses manipulate the cellular mechanisms of host organisms via pathogenhost interactions to take advantage of the capabilities of host cells, leading to viral replication. Understanding the structure and function of human host factors and host-virus interactions is essential for a complete knowledge of the viral infection and the identification of new targets for therapeutic intervention. Fabs (fragments antigen binding) have been used to facilitate crystal packing of soluble and integral membrane proteins by binding specific epitopes resulting in stabilization of desired conformations [5,6,7]. Non-antigenic sequences in a protein or the need for three-dimensional epitopes can make antigens “hybridoma-refractory” and challenging targets for monoclonal antibody identification since the complexes are unstable and do not maintain their three-dimensional structure during the immunization process
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
