Jonathan Owen, Emory University

Jonathan Einterz Owen1, Cheyanne Lynn Bemis1, Qingyi Wang1, Ambarish C. Varadan1, Jacob W. Vander Velden2,3, Laura Andačić4, Olus Uyar5, Mansi Gupta1, Christopher D. Scharer1, Laurent Chatel-Chaix5, Pietro Scaturro4,6, Mehul S. Suthar2,3, Christopher J. Neufeldt1 1Department of Microbiology and Immunology, School of Medicine, Emory University, Atlanta, GA, USA 2Center for Childhood Infections and Vaccines, Children's Healthcare of Atlanta, Division of Infectious Diseases, Department of Pediatrics, Emory University School of Medicine, Atlanta, GA, USA 3Emory Vaccine Center, Emory University, Atlanta, GA, USA 4Systems Arbovirology, Leibniz Institute for Experimental Virology, Hamburg, Germany 5Centre Armand-Frappier Santé Biotechnologie, Institut National de la Recherche Scientifique, Laval, QC, Canada 6German Center for Infection Research, Hamburg-Borstel-Lübeck-Riems, Germany

Expressing & Multiplying

Flaviviruses (genus Orthoflavivirus), including dengue virus (DENV) and Zika virus (ZIKV), are arthropod-borne pathogens which cause diseases of global importance. All known flaviviruses replicate in association with the host endoplasmic reticulum (ER), where genome replication is confined within virus-induced ER invaginations called viral replication organelles (vROs). These structures function to shield viral RNA from cellular innate immune recognition and facilitate efficient genome amplification. Despite the central role in flavivirus infection, the mechanisms of vRO biogenesis remain undefined – particularly the membrane rearrangements required for their formation. We report a conserved role for a cellular ER remodelling protein, atlastin-2 (ATL2), in the organization of vROs within flavivirus-infected cells. Using confocal and electron microscopy, as well as electron tomography, we show that ATL2 depletion alters vRO size and spatial distribution in both DENV- and ZIKV-infected cells. These changes corresponded with a decrease in virus production and increased induction of innate immune responses. Mutational analysis showed that a tethering-competent but fusion-defective ATL2 mutant was sufficient to rescue DENV and ZIKV replication in ATL2-knockout cells – demonstrating one of the first recorded biological functions for atlastin-mediated membrane tethering. Finally, inhibition of ATL2 activity using synthetic peptides significantly reduced DENV replication in both immortalized and human primary cells. Taken together, these results show that ATL2-mediated homotypic ER membrane tethering plays a critical and conserved role in flavivirus infection, functioning to organize membranes for vRO biogenesis and limit cellular immune activation. Importantly, we provide evidence that this function can be targeted to inhibit viral replication.