Formation and restriction of coronavirus double-membrane vesicles - Coronaviruses induce the formation of double-membrane vesicles (DMVs) derived from the endoplasmic reticulum (ER), highly conserved structures in positive-sense RNA viruses which serve as the hub for viral RNA synthesis. Yet a detailed mechanistic understanding of their formation, innate immune processes which target them, and whether viruses have evolved to evade such targeting is unknown. During my postdoctoral training, I have identified and validated ring finger protein 213 (RNF213) as an interferon-gamma induced gene (ISG) which restricts the human coronavirus OC43. I developed novel reporter viruses to define the role of RNF213 in infection and discovered that it inhibits primary translation, but not entry, of OC43. RNF213 co-localized with and co-immunoprecipitated OC43 nonstructural protein 3 (NSP3), the main structural component of the coronavirus DMV. However, RNF213 had no impact on SARS-CoV-2 and did not localize to SARS-CoV-2 DMVs. My work has identified not only an ISG that restricts coronavirus DMVs, but also raises new questions about differences between DMV formation in coronaviruses. In the mentored phase of this K99 Pathway to Independence award, I will obtain a complete mechanistic understanding of the restriction of coronaviruses by RNF213. In Aim 1, I will define how RNF213 binds directly or indirectly to OC43 NSP3, the domains required, and how SARS-CoV-2 evades restriction. In Aim 2, I will define cellular co-factors required for the localization and activity of RNF213 against OC43, including which of the domains of RNF213 are required for restriction. I will also determine the role of cellular RNF213-interacting proteins I have recently identified. Achieving these aims will train me in virology, molecular biology, and microscopy methods to study coronavirus DMVs. This will prepare me for Aim 3, in which I will define the requirements for DMVs formation in positive-sense RNA viruses. I will determine how viral proteins are inserted into the ER to form DMVs, what cellular co-factors are required for the process of DMV formation, and whether these mechanisms vary between different coronaviruses. In addition to building my scientific knowledge, my training plan will provide me with the tools necessary to advance my career development and navigate the transition to independence. The training phase will take place in the laboratory of Dr. Paul Bieniasz at The Rockefeller University, one of the world’s experts in studying how ISGs restrict viruses. I will execute a comprehensive training plan cementing not only my scientific skills, but also workshops and seminars on laboratory management, conducting a job search, research ethics, and completing the transition to independence. I will benefit from a high knowledgeable advisory committee, including Drs. Charles Rice, Seth Darst, and Elizabeth Campbell, who will both provide essential scientific feedback and career advice. The training provided by this award will be essential to achieving my goal to become an independent investigator, and the completion of this work will provide novel and significant insights into the mechanisms of DMV formation, and antiviral mechanisms to restrict them.
