Tools for Studying Respiratory Syncytial Virus Infection with Fluorescence Microscopy

Respiratory Syncytial Virus (RSV) poses a significant burden in the United States, particularly among young children, older adults, and those with underlying health conditions. Despite recent progress in vaccine availability, there are still aspects of RSV infection that we do not understand, including mechanisms of viral entry and assembly. To address this challenge, we develop tools that are compatible with existing, widely available fluorescence-based imaging systems. Specifically, we develop a fluorescent RSV system harboring a fluorescent reporter and tags on the surface proteins using site-specific labeling. This engineered viral strain maintains strong replication kinetics, labels with high efficiency, and allows us to visualize infected cells and the virions they produce. This enables real-time tracking of viral interactions during the infection process, which we harness to identify a role for virus morphology in complement activation. We further expand this work by utilizing genetic code expansion to develop a recoded strain of RSV, in which the multifunctional nucleoprotein is site-specifically modified with a noncanonical amino acid. We leverage this tool to visualize RSV assembly, capturing the transfer of nucleoprotein complexes from cytoplasmic condensates directly to budding viral filaments at the cell surface and to cytoplasmic compartments containing viral surface proteins. In order to leverage these tools in the context of differentiated human airway cells HAECs, we develop devices to culture, differentiate, and perform live, high-resolution imaging on air-liquid interface cultures compatible with confocal fluorescence microscopy. These devices differentiate HAECs with equal capacity to existing platforms. We image RSV attachment in live ciliated cells with 300 nm of resolution at up to 100fps. Lastly, we investigate the role of cilia in RSV binding and infection in differentiated HAECs. Notably, we find that RSV preferentially binds to ciliated cells and that genetic mutations resulting in ciliary dysmotility influence RSV binding and cell-type selectivity. Collectively, these tools establish a new methodology that could be extended to study other respiratory disease.