TMEM41B: a pan-flavivirus and pan-coronavirus host factor with antiviral potential

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Project Summary Arthropod-borne flaviviruses and respiratory-transmitted coronaviruses have the potential to cause severe epidemics and pandemics. One strategy to prepare for and respond to viral outbreaks is to develop drugs that target host factors viruses require to complete their lifecycles. Through a series of CRISPR/Cas9 gene disruption screens, we identified transmembrane protein 41B (TMEM41B) and the closely related vacuole membrane protein 1 (VMP1) as critical pan-flavivirus and pan-coronavirus host factors. Both proteins are highly conserved lipid scramblases with roles in autophagy. Our current model is that viruses from both the Flavivirdae and Coronaviridae families hijack TMEM41B and VMP1 for their ability to remodel ER membranes and induce membrane curvature to establish membrane-protected viral RNA replication organelles. Our overall goal for this proposal is to understand how, on a mechanistic level, both proteins support flavivirus and coronavirus infection. Our previous work indicates that TMEM41B is required at a post-entry step at or prior to viral RNA replication. In Aim 1, we will interrogate early events of the virus lifecycle including primary translation, polyprotein processing, and replication organelle formation in WT, TMEM41B and VMP1 knockout (KO) cells to determine how far the flavivirus and coronavirus lifecycles progress in the absence of either protein. We previously showed that lack of TMEM41B and VMP1, induces a heightened innate immune response upon flavivirus infection. We hypothesize that both proteins are recruited to sites of viral RNA replication, and that in their absence, RNA replication initiates and viral double stranded RNA (dsRNA) is produced. However, without a proper replication organelle dsRNA is exposed and triggers an innate immune response. Alternatively, given TMEM41B’s and VMP1’s lipid scramblase activity and function in lipid homeostasis, their absence may induce ER stress, which triggers an unfolded protein response (UPR) that in synergy with dsRNA may cause a heightened innate immune response. In Aim 2, we will test virus infection in double KO cells that lack either protein in addition to genes that are essential for pathogen sensing, IFN signaling, and UPR activation. We will further conduct RNAseq experiments to investigate lack of TMEM41B in stem cells and stem cell-derived primary-like cells representing different tissue lineages in the absence and presence of viral replication. Lastly, in Aim 3, will use a panel of phenotypic and mechanistic assays to characterize naturally occurring SNPs in TMEM41B that we previously found to impact flavivirus replication, and several reported VMP1 loss-of- function mutants. We will further take a deep mutational scanning approach to comprehensively characterize TMEM41B and VMP1 and determine if any domains or amino acids are differentially required for their cellular and proviral functions. This functional characterization will identify mutants that can be studied in detail in mechanistic assays and may identify amino acids or interfaces in both proteins that can be targeted to prevent virus infection with minimal disruption to cellular biology.