Summary In the absence of selective delivery, many promising drugs do not reach the targeted cells, but rather cause toxic side effects. Many viruses, on the other hand, have mastered the art of identifying microenvironmental clues and selectively find and infect a specific cell. For instance, they depend on “local” proteases, sometimes two, to activate their fusion proteins. To develop better targeting devices, we aim to dissect molecular properties and functions embedded in viral surface proteins, specifically focusing on receptor interactions, stability, and fusion triggering (Aim I) for which we will employ surface display and infectivity assays. We will focus on the viral surface machinery of the paramyxoviruses (PMVs), specifically Parainfluenza virus 5 (PIV5). Most PMVs have a division of labor keeping host receptor binding and cell entry apart as two separate functions encoded into two molecules: one tetrameric protein responsible for molecular recognition and a trimeric fusion protein responsible for the merging of host and viral membranes. This compartmentalization makes the PMVs an excellent model system for repurposing as the fusion protein can remain untouched, while the recognition process can be re-engineered. We will take advantage of deep mutational scanning which allows us to evaluate all possible amino acid substitutions for any given genetic selection. By acquiring differential fitness landscapes for each of the aforementioned molecular properties, we will be able to address interesting questions about the biology of viruses, such as mutational tolerance in context of their protein chemistry. Importantly, fitness landscapes will have an immediate impact on engineering of delivery devices as they will provide rough blueprints of the molecular architecture of these complex machineries. We will use obtained sequence-function-structure maps for the development of a new, adaptable targeted delivery platform that will integrate viral surface machinery with antibody fragments (Aim II). The key point will be to develop an adapter molecule that integrates the antibody fragment while maintaining all regulatory function that the viral recognition machinery normally exhibits, which involves control of conformational changes. Previous efforts have not succeeded in developing an efficient, general delivery system. Here, we will obtain and leverage an invaluable database of virus protein structures together with our newly obtained sequence-function knowledge, which we combine with new technology – protein design – to advance this seemingly simple but ambitious engineering project. We aim to provide a generally applicable platform for a new targeting machinery that incorporates these molecular mechanisms while also taking advantage of the vast amount of identified and engineered antibodies. Through combining parts of the viral infection machinery with antibody fragments and adapter proteins, we anticipate that we will be able to significantly advance the development of drug and gene delivery systems and thereby also provide new and much needed precision targeting technology for genome engineering.

Relevance Through exhaustive mutagenesis study, we will dissect parts of a viral host recognition machinery and leverage obtained data to develop a general gene and drug delivery platform using protein design. We will mobilize previously discovered antibodies to be integrated into the delivery platform. We will fine-tune this new methodology through a new genetic selection for cell entry that can also be used to evaluate emerging viruses.