High-resolution mapping reveals the mechanism and contribution of genome insertions and deletions to RNA virus evolution.

RNA viruses rapidly adapt to selective conditions due to the high intrinsic mutation rates of their RNA-dependent RNA polymerases (RdRps). Insertions and deletions (indels) in viral genomes are major contributors to both deleterious mutational load and evolutionary novelty, but remain understudied. To characterize the mechanistic details of their formation and evolutionary dynamics during infection, we developed a hybrid experimental-bioinformatic approach. This approach, called MultiMatch, extracts insertions and deletions from ultradeep sequencing experiments, including those occurring at extremely low frequencies, allowing us to map their genomic distribution and quantify the rates at which they occur. Mapping indel mutations in adapting poliovirus and dengue virus populations, we determine the rates of indel generation and identify mechanistic and functional constraints shaping indel diversity. Using poliovirus RdRp variants of distinct fidelity and genome recombination rates, we demonstrate tradeoffs between fidelity and Indel generation. Additionally, we show that maintaining translation frame and viral RNA structures constrain the Indel landscape and that, due to these significant fitness effects, Indels exert a significant deleterious load on adapting viral populations. Conversely, we uncover positively selected Indels that modulate RNA structure, generate protein variants, and produce defective interfering genomes in viral populations. Together, our analyses establish the kinetic and mechanistic tradeoffs between misincorporation, recombination, and Indel rates and reveal functional principles defining the central role of Indels in virus evolution, emergence, and the regulation of viral infection.

Fragment-based computational design of antibodies targeting structured epitopes.

De novo design methods hold the promise of reducing the time and cost of antibody discovery while enabling the facile and precise targeting of predetermined epitopes. Here, we describe a fragment-based method for the combinatorial design of antibody binding loops and their grafting onto antibody scaffolds. We designed and tested six single-domain antibodies targeting different epitopes on three antigens, including the receptor-binding domain of the SARS-CoV-2 spike protein. Biophysical characterization showed that all designs are stable and bind their intended targets with affinities in the nanomolar range without in vitro affinity maturation. We further discuss how a high-resolution input antigen structure is not required, as similar predictions are obtained when the input is a crystal structure or a computer-generated model. This computational procedure, which readily runs on a laptop, provides a starting point for the rapid generation of lead antibodies binding to preselected epitopes.

π-Clamp-Mediated Homo- and Heterodimerization of Single-Domain Antibodies via Site-Specific Homobifunctional Conjugation.

Post-translational protein-protein conjugation produces bioconjugates that are unavailable via genetic fusion approaches. A method for preparing protein-protein conjugates using π-clamp-mediated cysteine arylation with pentafluorophenyl sulfonamide functional groups is described. Two computationally designed antibodies targeting the SARS-CoV-2 receptor binding domain were produced (KD = 146, 581 nM) with a π-clamp sequence near the C-terminus and dimerized using this method to provide a 10-60-fold increase in binding (KD = 8-15 nM). When two solvent-exposed cysteine residues were present on the second protein domain, the π-clamp cysteine residue was selectively modified over an Asp-Cys-Glu cysteine residue, allowing for subsequent small-molecule conjugation. With this strategy, we build molecule-protein-protein conjugates with complete chemical control over the sites of modification.