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.