Rhinoviruses (RVs) play a major role in causing asthma exacerbations, which drive disease and economic burdens. Among the RVs the RV-C subgroup is especially problematic, due to its virulence. This proposal is focused on RV-C, and ameliorating its effects with anti-viral agents derived from pulmonary surfactant lipids and proteins. Two minor anionic phospholipids of pulmonary surfactant, palmitoyl-oleoyl- phosphatidylglycerol (POPG) and phosphatidylinositol (PI), antagonize respiratory viral infections and suppress inflammatory sequelae triggered by Toll-like receptor activation, by acting as decoy ligands. In addition, the pulmonary surfactant protein, SP-A, also disrupts RV-C infections, in part by direct interactions with virions. In this proposal we plan to investigate the mechanisms by which surfactant lipids and proteins inhibit RV infections and replication. In Aim 1, we will examine how POPG and PI and their structural analogs interfere with RV-C infection/replication using Air-Liquid Interface (ALI) cultures. Our current data support a mechanism in which the lipids act intracellularly to alter the infection/replication processes. Indeed, preliminary RNAseq data suggest that PI interferes with ciliagenesis, rendering host cells refractory to being infected; since infection requires mature, motile cilia. The same RNAseq data set also suggests that POPG acts at a different intracellular site from PI. Transgenic mouse studies are also proposed in Aim 1 and will investigate the efficacy of the anti-viral phospholipids in the context of the whole organism. In Aim 2, we will investigate how SP-A disrupts RV-C infection/replication using Air-Liquid-interface cultures ex vivo. We will also perform RNAseq analysis to probe the host cell and viral transcriptomes. We will use our collection of purified SP-A isoforms to determine the rank order potency of different SP-A structural variants both ex vivo and in vivo. The ex vivo SP- A studies will be expanded to studies in transgenic mice to understand the actions of SP-A in the context of an intact lung and whole animal. From the experiments described in this proposal we anticipate learning the mechanisms of action of surfactant lipids and proteins as anti-viral agents targeting RVs that aggravate asthma and cause exacerbations. We expect this information will identify novel reagents for controlling RV infections in humans, and lead to new data that will improve the actions of the surfactant constituents.