Deep Mutational Scanning of Viral Glycoproteins and Their Host Receptors

Abstract

Deep mutational scanning or deep mutagenesis is a powerful tool for understanding the sequence diversity available to viruses for adaptation in a laboratory setting. It generally involves tracking an in vitro selection of protein sequence variants with deep sequencing to map mutational effects based on changes in sequence abundance. Coupled with any of a number of selection strategies, deep mutagenesis can explore the mutational diversity available to viral glycoproteins, which mediate critical roles in cell entry and are exposed to the humoral arm of the host immune response. Mutational landscapes of viral glycoproteins for host cell attachment and membrane fusion reveal extensive epistasis and potential escape mutations to neutralizing antibodies or other therapeutics, as well as aiding in the design of optimized immunogens for eliciting broadly protective immunity. While less explored, deep mutational scans of host receptors further assist in understanding virus-host protein interactions. Critical residues on the host receptors for engaging with viral spikes are readily identified and may help with structural modeling. Furthermore, mutations may be found for engineering soluble decoy receptors as neutralizing agents that specifically bind viral targets with tight affinity and limited potential for viral escape. By untangling the complexities of how sequence contributes to viral glycoprotein and host receptor interactions, deep mutational scanning is impacting ideas and strategies at multiple levels for combatting circulating and emergent virus strains.

Keywords: deep mutational scan; entry receptor; mutational landscape; selection; viral escape; viral fusion protein; virus spike.

FIGURE 1

Examples of selection strategies used in deep mutational scans of viral spike proteins. (A) Infectious virus variants (green) are enriched after passaging through a permissive cell line expressing host target receptors (blue). (B) Syncytia are enriched upon fusion of cells expressing variants of viral fusion protein (pale yellow cells) and cells expressing the entry receptor (pale blue cells) in the presence of two different antibiotics. (C) Soluble extracellular domains of the viral spike are displayed on the yeast cell wall via Aga1p–Aga2p (dark red). After fluorescence activated cell sorting (FACS), these viral proteins (green) are enriched if they possess high binding affinity and/or expression to fluorescent partners, such as soluble receptors (blue). (D) After FACS, mammalian cells expressing viral spike proteins (green) are enriched if they are highly expressed and bind with high affinity to fluorescently labeled antibodies (pink) or soluble receptors.

FIGURE 2

Deep mutagenesis of entry receptors identifies critical binding residues for viral fusion proteins. (A) Conservation from a deep mutational scan of the human immunodeficiency virus 1 (HIV-1) co-receptor CCR5 for interacting with CD4-bound Env is mapped to the structure (PDB 6MEO). For clarity, only residues of Env (peach ribbon) and CD4 (green ribbon) within proximity of CCR5 are shown. Critical CCR5 residues are blue, while residues that are under selection to change are yellow. The asterisk denotes CCR5 sulfotyrosine-14. (B) Deep mutagenesis data of human ACE2 (colored from blue for conserved to yellow for residues under selection to change) binding to the RBD of SARS-CoV-2 (peach ribbon) is mapped to structure (PDB 6M17). In the selection, residue conservation at the interface is bipartite, with one subsite on ACE2 (in dark blue) having very low mutational tolerance.