Single-Domain Antibodies as Tools to Perturb and Study RNA Viruses

In this thesis, I describe the generation and characterization of alpaca-derived, antiviral, single-domain antibody fragments (VHHs). The antiviral targets of the described VHHs are the nuclear proteins of influenza A virus (IAV) and vesicular stomatitis virus (VSV). The described VHHs protect cells from viral infection when expressed in the cytosol. IAV is a segmented, negative-strand RNA virus and is the current focus of antiviral strategies because it causes reoccurring epidemics and pandemics in the human population. A broadly active, antiviral measure with high efficacy is therefore urgently needed. In contrast, VSV is a non-segmented, negative-strand RNA virus that is typically asymptomatic in humans, but is extensively studied as a prototypic non-segmented RNA virus. I describe a novel lentivirus based VHH screening approach that selects VHH based on phenotype, e.g. survival of viral infection. To better understand the antiviral mechanisms that underlie VHH-mediated viral inhibition, I present a structure of a IAV-specific VHH in complex with its viral antigen, the IAV nucleoprotein, and two structures of VSV-specific VHHs in complex with their viral antigen, the VSV nucleoprotein N. The structurally defined binding sites explain the antiviral mechanism of the VHHs and give clues about the functional relevance of the targeted structures. Data presented herein for both IAV and VSV highlight the advantages of VHHs as neutralizing agents and as tools to study viral structure and replication. In conclusion I demonstrate a robust cycle that: first, enables the production and selection of VHHs that impede viral growth; second, that the epitope recognized can be accurately defined in structural terms; and third, that the combination of both can explain the functional relevance of the targeted viral structure.