Chemokine receptors CXCR4 and CCR5 regulate WBC trafficking and are engaged by the HIV-1 envelope glycoprotein gp120 during infection. We combine a selection of human CXCR4 and CCR5 libraries comprising nearly all of ∼7000 single amino acid substitutions with deep sequencing to define sequence-activity landscapes for surface expression and ligand interactions. After consideration of sequence constraints for surface expression, known interaction sites with HIV-1-blocking Abs were appropriately identified as conserved residues following library sorting for Ab binding, validating the use of deep mutational scanning to map functional interaction sites in G protein-coupled receptors. Chemokine CXCL12 was found to interact with residues extending asymmetrically into the CXCR4 ligand-binding cavity, similar to the binding surface of CXCR4 recognized by an antagonistic viral chemokine previously observed crystallographically. CXCR4 mutations distal from the chemokine binding site were identified that enhance chemokine recognition. This included disruptive mutations in the G protein-coupling site that diminished calcium mobilization, as well as conservative mutations to a membrane-exposed site (CXCR4 residues H79 and W161) that increased ligand binding without loss of signaling. Compared with CXCR4-CXCL12 interactions, CCR5 residues conserved for gp120 (HIV-1 BaL strain) interactions map to a more expansive surface, mimicking how the cognate chemokine CCL5 makes contacts across the entire CCR5 binding cavity. Acidic substitutions in the CCR5 N terminus and extracellular loops enhanced gp120 binding. This study demonstrates how comprehensive mutational scanning can define functional interaction sites on receptors, and novel mutations that enhance receptor activities can be found simultaneously.

HIV-1 infection is initiated by viral Env engaging the host receptor CD4, triggering Env to transition from a “closed” to “open” conformation during the early events of virus-cell membrane fusion. To understand how Env sequence accommodates this conformational change, mutational landscapes decoupled from virus replication were determined for Env from BaL (clade B) and DU422 (clade C) isolates interacting with CD4 or antibody PG16 that preferentially recognizes closed trimers. Sequence features uniquely important to each bound state were identified, including glycosylation and binding sites. Notably, the Env apical domain and trimerization interface are under selective pressure for PG16 binding. Based on this key observation, mutations were found that increase presentation of quaternary epitopes associated with properly conformed trimers when Env is expressed at the plasma membrane. Many mutations reduce electrostatic repulsion at the Env apex and increase PG16 recognition of Env sequences from clades A and B. Other mutations increase hydrophobic packing at the gp120 inner-outer domain interface and were broadly applicable for engineering Env from diverse strains spanning tiers 1, 2, and 3 across clades A, B, C, and BC recombinants. Core mutations predicted to introduce steric strain in the open state show markedly reduced CD4 interactions. Finally, we demonstrate how our methodology can be adapted to interrogate interactions between membrane-associated Env and the matrix domain of Gag. These findings and methods may assist vaccine design. IMPORTANCE HIV-1 Env is dynamic and undergoes large conformational changes that drive fusion of virus and host cell membranes. Three Env proteins in a trimer contact each other at their apical tips to form a closed conformation that presents epitopes recognized by broadly neutralizing antibodies. The apical tips separate, among other changes, to form an open conformation that binds tightly to host receptors. Understanding how Env sequence facilitates these structural changes can inform the biophysical mechanism and aid immunogen design. Using deep mutational scans decoupled from virus replication, we report mutational landscapes for Env from two strains interacting with conformation-dependent binding proteins. Residues in the Env trimer interface and apical domains are preferentially conserved in the closed conformation, and conformational diversity is facilitated by electrostatic repulsion and an underpacked core between domains. Specific mutations are described that enhance presentation of the trimeric closed conformation across diverse HIV-1 strains.

A trimeric glycoprotein complex on the surface of human cytomegalovirus (HCMV) binds to platelet-derived growth factor (PDGF) receptor α (PDGFRα) to mediate host cell recognition and fusion of the viral and cellular membranes. Soluble PDGFRα potently neutralizes HCMV in tissue culture, and its potential use as an antiviral therapeutic has the benefit that any escape mutants will likely be attenuated. However, PDGFRα binds multiple PDGF ligands in the human body as part of developmental programs in embryogenesis and continuing through adulthood. Any therapies with soluble receptor therefore come with serious efficacy and safety concerns, especially for the treatment of congenital HCMV. Soluble virus receptors that are orthogonal to human biology might resolve these concerns. This engineering problem is solved by deep mutational scanning on the D2-D3 domains of PDGFRα to identify variants that maintain interactions with the HCMV glycoprotein trimer in the presence of competing PDGF ligands. Competition by PDGFs is conformation-dependent, whereas HCMV trimer binding is independent of proper D2-D3 conformation, and many mutations at the receptor-PDGF interface are suitable for functionally separating trimer from PDGF interactions. Purified soluble PDGFRα carrying a targeted mutation succeeded in displaying wild type affinity for HCMV trimer with a simultaneous loss of PDGF binding, and neutralizes trimer-only and trimer/pentamer-expressing HCMV strains infecting fibroblasts or epithelial cells. Overall, this work makes important progress in the realization of soluble HCMV receptors for clinical application.

Chemokine receptors are members of the rhodopsin-like class A GPCRs whose signaling through G proteins drives the directional movement of cells in response to a chemokine gradient. Chemokine receptors CXCR4 and CCR5 have been extensively studied due to their roles in white blood cell development and inflammation and their status as coreceptors for HIV-1 infection, among other functions. Both receptors form dimers or oligomers but the function/s of self-associations are unclear. While CXCR4 has been crystallized in a dimeric arrangement, available atomic resolution structures of CCR5 are monomeric. To investigate the dimerization interfaces of these chemokine receptors, we used a bimolecular fluorescence complementation (BiFC)-based screen and deep mutational scanning to find mutations that modify receptor self-association. Many disruptive mutations promoted self-associations nonspecifically, suggesting they aggregated in the membrane. A mutationally intolerant region was found on CXCR4 that matched the crystallographic dimer interface, supporting this dimeric arrangement in living cells. A mutationally intolerant region was also observed on the surface of CCR5 by transmembrane helices 3 and 4. Mutations from the deep mutational scan that reduce BiFC were validated and were localized in the transmembrane domains as well as the C-terminal cytoplasmic tails where they reduced lipid microdomain localization. The reduced self-association mutants of CXCR4 had increased binding to the ligand CXCL12 but diminished calcium signaling. There was no change in syncytia formation with cells expressing HIV-1 Env. The data highlight that multiple mechanisms are involved in self-association of chemokine receptor chains.