The current COVID-19 crisis starkly illustrates the need to develop new modalities for the therapeutic treatment of pathogenic single-stranded RNA (ssRNA) viruses, including against novel viruses that have yet to emerge. Human oxysterol-binding protein (OSBP) has recently been determined to be a critical mediator in the replication of a broad spectrum of ssRNA viral human pathogens, including the enteroviruses, rhinovirus, hepatitis C, Zika virus, Dengue fever viruses, and coronaviruses. OSBP is an ER-located, non-enzymatic protein reported to function as an important lipid sensor and lipid transporter in eukaryotic cells. Published research, including our own recent publications, has established the antiviral activity of structurally-diverse OSBP- targeting small molecules against multiple RNA pathogenic viruses. These discoveries present the opportunity for a paradigm shift in antiviral drug development: potentially drug targeting a human host protein, OSBP, that is required for viral proliferation of a broad-spectrum of RNA viruses, as opposed to targeting viral proteins present in individual viruses. We have discovered that transient, low dose treatment with the OSBP-targeting compound OSW-1-compound induces a longterm, multigenerational repression of OSBP, and the cells with repressed OSBP show a pronounced inhibition of ssRNA viral replication. Our preliminary results show that the OSW-1- compound has prophylactic antiviral activity at low nanomolar concentrations against several ssRNA viruses, including against one coronavirus tested. The longterm repression of OSBP, triggered by OSW-1, has no effect on cellular division, viability, or morphology. The purpose of this proposal is to understand the cellular role of OSBP in innate antiviral response. Our preliminary results show that OSBP: 1) regulates mTORC1 activity, 2) induces autophagy; 3) slows global protein translation; and 4) activates alternative splicing nonsense- mediated decay (AS-NMD) process, which is an RNA regulatory process. All of these OSBP- involved cellular processes would limit ssRNA viral replication individually, but there is little insight into the organization of these systems to establish a coordinated antiviral response.Our overall hypothesis is that OSBP serves in a major regulatory role to coordinate a multifaceted innate antiviral response to ssRNA infection. We propose a complete model of how OSBP senses early- stage viral infection and then triggers a multisystem response to block viral replication in cells, including through modulating mTOR1C activity and the AS-NMD system.