Project Grant R01AI173044

MOLECULAR MECHANISMS GOVERNING CHIKUNGUNYA VIRUS BINDING, TROPISM, AND PATHOGENESIS - CHIKUNGUNYA VIRUS (CHIKV) IS AN ARTHRITOGENIC ALPHAVIRUS THAT CAUSES A PAINFUL MUSCULOSKELETAL INFLAMMATORY DISEASE, WHICH CAN PERSIST FOR MONTHS TO YEARS. CHRONIC CHIKV ARTHRITIS IS DEBILITATING AND PRODUCES SUBSTANTIAL SOCIAL AND ECONOMIC CONSEQUENCES. DESPITE THE BURDEN POSED BY ACUTE AND CHRONIC CHIKV DISEASE, LICENSED VACCINES AND ANTIVIRAL THERAPEUTICS ARE NOT AVAILABLE. VIRUS-HOST INTERACTIONS AT THE CELL-SURFACE DICTATE SPECIES AND TISSUE TROPISM AND ARE OFTEN DETERMINANTS OF VIRULENCE. WE DISCOVERED THAT MULTIPLE PATHOGENIC STRAINS OF CHIKV BIND TO HEPARAN SULFATE (HS), A TYPE OF GLYCOSAMINOGLYCAN (GAG) THAT IS HIGHLY VARIABLE IN SUBUNITS AND MODIFICATIONS, TO MEDIATE ADHESION TO CELLS. EFFICIENT BINDING AND INFECTION OF BIOLOGICALLY RELEVANT CELL TYPES BY CHIKV DEPENDS ON CELL-SURFACE HS, BUT LITTLE IS KNOWN ABOUT THE VIRAL AND HOST DETERMINANTS THAT MEDIATE THESE INITIAL CONTACTS OR HOW CHIKV-HS INTERACTIONS INFLUENCE VIRULENCE AND DISEASE OUTCOMES. WE HYPOTHESIZE THAT CHIKV HAS EVOLVED AN OPTIMUM AFFINITY FOR UNIQUE HS STRUCTURES, INCLUDING N-SULFATION, VIA SPECIFIC RESIDUES OF CHIKV E2 TO MEDIATE EFFICIENT CHIKV ATTACHMENT TO TARGET CELLS, WHICH INFLUENCES VIRAL DISEASE OUTCOMES. THREE INTEGRATED BUT INDEPENDENT SPECIFIC AIMS ARE PROPOSED. IN SPECIFIC AIM 1, WE WILL TEST THE HYPOTHESIS THAT CHIKV BINDS TO SPECIFIC MODIFICATIONS OF HS CHAINS TO ATTACH TO TARGET CELLS. A COMBINATION OF BIOCHEMICAL, BIOPHYSICAL, GENETIC, AND VIROLOGICAL TECHNIQUES WILL BE USED TO DEFINE SPECIFIC HS STRUCTURES REQUIRED FOR BINDING AND INFECTION OF TARGET CELLS, WHICH WILL BE GENETICALLY VALIDATED IN TARGET CELLS. BINDING PARAMETERS WILL BE BIOPHYSICALLY CHARAC- TERIZED. IN SPECIFIC AIM 2, WE WILL TEST THE HYPOTHESIS THAT MULTIPLE HS BINDING SITES ON CHIKV VIRIONS ARE MEDI- ATED BY SPECIFIC BASIC RESIDUES IN E2. THE STRUCTURAL FEATURES AND SPECIFIC RESIDES OF E2 THAT FACILITATE CHIKV-HS INTERACTIONS WILL BE DEFINED BY CRYO-EM, AND TARGETED MUTAGENESIS OF HS-BINDING SITES WILL BE USED TO ENGINEER MUTANTS WITH A LOSS OF FUNCTION IN HS BINDING. VIRAL MUTANTS WILL BE CHARACTERIZED FOR E2 FUNCTIONS IN VITRO AND USED TO DETERMINE THE REQUIREMENT OF VIRAL ENGAGEMENT WITH HS FOR BINDING AND INFECTION OF TARGET CELLS. IN SPECIFIC AIM 3, WE WILL TEST THE HYPOTHESIS THAT DECREASED ATTACHMENT TO HS WILL RESULT IN ATTENUATION IN A MOUSE MODEL OF DISEASE. WE WILL FIRST ELUCIDATE THE DEPENDENCE OF CHIKV BINDING TO HS ON TARGET CELLS USING MICE HARBORING CONDITIONAL KNOCKOUT OF THE HS-MODIFYING GENE NDST1 IN ENDOTHELIAL AND MUSCLE CELLS. MICE WILL ALSO BE INFECTED WITH A PANEL OF VIRUSES THAT DISPLAY DECREASED CAPACITY IN BINDING TO HS OR ENTRY RECEPTOR MXRA8 TO ASSESS THE SPECIFIC FUNCTION OF VIRUS ENGAGEMENT WITH HS IN DISSEMINATION, CELL TROPISM, IMMUNE RESPONSES, AND TISSUE. STUDIES IN THIS APPLICATION WILL DEFINE STRUCTURAL AND BIOPHYSICAL PARAMETERS OF CHIKV-HS BINDING, ELUCI- DATE THE SIGNIFICANCE OF HS-DEPENDENT ATTACHMENT TO TARGET CELLS, AND UNRAVEL FUNCTIONS OF VIRUS-HS BINDING IN CHIKV PATHOGENESIS. AS MANY PATHOGENIC VIRUSES USE GAGS AS ATTACHMENT FACTORS, OUR WORK WILL CONTRIBUTE TO BROAD CONCEPTUAL ADVANCES OF VIRUS-GAG INTERACTIONS. NEW KNOWLEDGE ABOUT CHIKV-HS INTERACTIONS MAY INFORM THE RATIONAL DESIGN OF SPECIFIC ANTIVIRALS TARGETING GAGS TO BLOCK VIRUS INFECTION AND DIMINISH DISEASE.