The Sequence Basis for Selectivity of Ephrin-B2 for Eph Receptors and Pathogenic Henipavirus G Glycoproteins [EFNB2-D62Q DEEP MUTAGENESIS]
Ephrin-B2 (EFNB2) is a ligand for six Eph receptors in humans and also functions as a cell entry receptor for several henipaviruses, which are zoonotic viruses with pandemic potential. Two such viruses, Nipah (NiV) and Hendra (HeV), are highly pathogenic in humans yet do not have any widely approved virus-specific therapeutics or vaccines. Soluble Fc-fusions of EFNB2 (sEFNB2-Fc) are known to bind the attachment glycoprotein G of both viruses and inhibit viral infection of cells. However, the potential of sEFNB2-Fc as a neutralizing agent in vivo is hindered by its binding activity to native Eph receptors. Therefore, using deep mutagenesis, variants of EFNB2 at cell surface are identified with increased binding to the soluble head domain of NiV-G over soluble Fc-fusions of the extracellular domains of Eph receptors. Specificity mutations for NiV-G are found primarily at the binding interface, especially around the G-H binding loop. The mutational landscape offers a preliminary blueprint for engineering sEFNB2-Fc variants with high specificity and affinity for potent neutralization of henipaviruses. Overall design: The cDNA encoding full-length human EFNB2 isoform 1 (GenBank NP_004084.1) with an N-terminal c-myc tag was diversified by site-saturation mutagenesis at 137 positions (a.a. I31-G168); a glutamine substitution at position D62 was unchanged. This was accomplished by introducing degenerate NNK codons at the respective sites, which fully spanned the known receptor binding domain for Eph receptors and the attachment glycoprotein G of Nipah virus (NiV-G). The EFNB2 library was expressed in human Expi293F cells under conditions where most cells express no more than a single coding variant. The cell culture was then incubated with soluble Fc-fused extracellular domains of EphB3 and EphB4 (subcloned from plasmids from Sino Biological) and simultaneously stained with Alexa 647-conjugated anti-myc to measure surface EFNB2 expression. The cell culture was then incubated with the head domain of NiV-G (a.a. G183-T602; GenBank NP_112027.1). Soluble NiV-G head domain was fused at its C-terminus to sfGFP for fluorescence detection in an oxidizing extracellular environment. After washing, bound NiV-G-sfGFP was found to be proportional to EFNB2 levels. There were unambiguous populations of cells expressing EFNB2 variants with high and low NiV-G-sfGFP binding; these cell populations were collected by FACS and are called NiV-G-High and NiV-G-Low, respectively. Transcripts in the collected cell populations were Illumina sequenced and their frequencies compared to the naive plasmid library. The enrichment or depletion of all 2,740 single amino acid variants in the library was calculated, which acts as a proxy for relative “binding” to NiV-G. This “binding” signal is a product both of EFNB2/NiV-G affinity and surface display of properly folded EFNB2.