Project Grant R01AI185849

HOST FACTOR CONTRIBUTION TO POSITIVE-STRAND RNA VIRUS INDUCED MEMBRANE REORGANIZATION - POSITIVE-SENSE SINGLE-STRAND RNA VIRUSES [(+)RNA VIRUSES] ARE RESPONSIBLE FOR A SIGNIFICANT PORTION OF HUMAN INFECTIONS AND REPRESENT A HIGH RISK FOR NEW AND EMERGING HUMAN DISEASES. THIS GROUP OF VIRUSES INCLUDES FLAVIVIRUSES SUCH AS DENGUE VIRUS (DENV), ZIKA VIRUS (ZIKV), AND WEST NILE VIRUS (WNV), WHICH ARE COLLECTIVELY RESPONSIBLE FOR >400 MILLION HUMAN INFECTIONS EACH YEAR. CHANGES IN HUMAN POPULATION DISTRIBUTION AND CLIMATE CHANGE THAT DRIVE MOVEMENT OF INSECT POPULATIONS THAT MEDIATE SPREAD OF FLAVIVIRUSES INTO NEW ENVIRONMENTS INCREASE THE RISK OF WIDESPREAD INFECTIONS IN NAIVE POPULATIONS. THIS RISK IS EXEMPLIFIED BY THE 2016 OUTBREAK OF ZIKA VIRUS IN SOUTH AMERICA AND IS ENHANCED BY THE CURRENT LACK OF EFFECTIVE VACCINES OR ANTIVIRALS. STUDIES AIMED AT DETERMINING THE MECHANISMS OF VIRUS INFECTION AND INTERACTIONS BETWEEN VIRUSES AND HOST CELLS ARE VITAL TO UNDERSTANDING AND CONTROLLING THESE PATHOGENS. THE OBJECTIVES OF THIS STUDY ARE TO INTERROGATE THE MECHANISMS BY WHICH FLAVIVIRUSES UTILIZE HOST PROTEIN NETWORKS THAT FACILITATE VIRAL REPLICATION AND TO INVESTIGATE METHODS TO DISRUPT THESE PROCESSES. ALL STUDIED (+)RNA VIRUSES REPLICATE IN HOST MEMBRANES WITH THE MAJORITY REQUIRING THE ER FOR THE FORMATION OF THEIR VIRAL REPLICATION ORGANELLES (VROS) THAT HAVE A HIGH LEVEL OF MORPHOLOGICAL CONSERVATION BETWEEN DISTANTLY RELATED (+)RNA VIRUS SPECIES. DESPITE WIDESPREAD INFECTION, RELATIVELY LITTLE IS KNOWN ABOUT THE SPECIFIC CONTRIBUTION OF HOST PROTEINS TO THE FORMATION OR STRUCTURE OF (+)RNA VIRUS VROS. OUR PRELIMINARY EXPERIMENTS HAVE IDENTIFIED A NETWORK OF HOST MEMBRANE FACTORS THAT ARE CRITICAL TO DENV AND ZIKV REPLICATION, LIKELY HAVING A ROLE IN THE FORMATION OF VROS. IN OUR FIRST AIM, WE WILL DEFINE THE ROLE OF THESE HOST MEMBRANE PROTEINS IN THE FORMATION OF VROS FOR TWO CLOSELY RELATED FLAVIVIRUSES, DENV AND ZIKV. TO DETERMINE IF THESE HOST PROTEINS ARE DIRECTLY INVOLVED IN VRO BIOGENESIS, WE WILL EMPLOY UNIQUE VIRUS EXPRESSION AND INFECTION SYSTEMS, WHICH ALLOW PRECISE EVALUATION OF EACH STEP IN THE VIRUS REPLICATION CYCLE, IN COMBINATION WITH INTEGRATED MICROSCOPY AND BIOCHEMICAL APPROACHES. WE WILL ALSO EXPAND OUR ANALYSIS TO DETERMINE IF THE IDENTIFIED HOST FACTORS HAVE COMMON FUNCTIONS FOR OTHER (+)RNA VIRUSES. IN OUR SECOND AIM, WE WILL USE GENE MANIPULATION AND MUTAGENESIS TO MECHANISTICALLY INTERROGATE THE FUNCTION OF SPECIFIC HOST MEMBRANE FACTORS IN VIRUS REPLICATION AND VRO FORMATION. THIS WORK WILL SIGNIFICANTLY ADVANCE UNDERSTANDING OF THE MECHANISMS UNDERPINNING FLAVIVIRUS MANIPULATION OF ER MEMBRANES, FURNISHING NOVEL INSIGHTS INTO FUNDAMENTAL VIRAL PROCESSES LIKELY TO BE SHARED BY A BROAD RANGE OF (+)RNA VIRUSES.