Project Summary/Abstract Viruses in the genus Orthomyxoviridae and family Thogotovirus are globally widespread pathogens capable of causing significant and fatal human disease. These enveloped, segmented, negative-sense RNA viruses are mainly transmitted to hosts by a tick vector, though there is some evidence for transmission by mosquitoes and aerosols. Thogoto, Dhori, and Bourbon virus infections in humans have caused significant disease and, in some cases, death. There are no vaccines or therapeutics to prevent or alleviate thogotovirus infections, as most steps in the viral lifecycle of this entire family of viruses are poorly understood. Defining the lifecycle of thogotoviruses is the first step towards developing treatments to combat thogotovirus infections. Thogotoviruses use a single viral glycoprotein to mediate entry into host cells. While these glycoproteins have been structurally defined as class III viral fusogens and shown to undergo conformational changes in response to low pH, their role in entry is otherwise unknown. I have successfully replaced the single glycoprotein of eGFP-expressing vesicular stomatitis virus (VSV) with the glycoproteins of Bourbon, Dhori, and Thogoto virus in order to generate biosafety level 2, replication-competent, reporter viruses. With these novel tools, I have initially confirmed a role for pH in the Bourbon virus entry pathway and have conducted a genome-wide CRISPR-Cas9 screen, which has revealed the requirement for a host protein, glucosylceramide synthase (UGCG), in viral entry. The underlying hypothesis for my proposal is that virus-specific host factors dictate thogotovirus entry into cells. I will examine this hypothesis in two specific aims. In specific aim 1, I will 1) perform additional, more robust CRISPR-Cas9 screens, and 2) determine the role of identified host factors in thogotovirus entry. In specific aim 2, I will use genetic, chemical, and imaging approaches to define the role of UGCG in thogotovirus entry. Completion of these studies will reveal the entry pathways and host factors required for thogotovirus entry into cells and will define the mechanistic role of UGCG in the entry pathway of these viruses. Consequently, these studies will provide insight into the basic virology of an understudied family of human-infecting viruses.