Project Grant R01AI169457

UNDERSTANDING THE FUNDAMENTAL ENTEROVIRUS CAPSID ASSEMBLY AND MATURATION PATHWAY. - PROJECT SUMMARY/ABSTRACT: ENTEROVIRUSES (EVS) COMPRISE A FAMILY OF POSITIVE SENSE SSRNA VIRUSES. THE MOST WELL-STUDIED IS POLIOVIRUS, YET NON-POLIO ENTEROVIRUSES (NPEVS) CAUSE SERIOUS DISEASE, ESPECIALLY IN THE VERY YOUNG. THIS INCLUDES HAND, FOOT AND MOUTH DISEASE, FLACCID MYELITIS AND ENCEPHALITIS. CURRENTLY, IT IS ESTIMATED THAT NPEVS ARE RESPONSIBLE FOR OVER 10 MILLION INFECTIONS AND TENS OF THOUSANDS OF HOSPITALIZATIONS IN THE US ALONE. FURTHERMORE, COINFECTIONS ARE COMMON AND THEREFORE IT IS LIKELY THAT NEW VIRUSES WILL ARISE AS A RESULT OF RECOMBINATION. UNDERSTANDING KEY EVENTS THAT ARE SHARED ACROSS NPEVS IS THEREFORE KEY TO BEING ABLE TO CONTROL CURRENT INFECTIONS AND RESPOND TO EMERGING DISEASE IN THE FUTURE. ONE OF THESE KEY EVENTS IS POLYPROTEIN PROCESSING. THE NPEV GENOME ENCODES A POLYPROTEIN WHICH IS PROTEOLYTICALLY CLEAVED INTO THE MATURE VIRAL PROTEINS REQUIRED FOR THE GENERATION OF PROGENY VIRUS. THE STRUCTURAL REGION OF THE POLYPROTEIN IS PROCESSED INTO VP0, VP3 AND VP1. THESE ASSEMBLE INTO A PROTOMER, FIVE OF WHICH ASSEMBLE INTO A PENTAMER. TWELVE PENTAMERS COALESCE AROUND THE VIRAL GENOME, GENERATING A STRUCTURE TERMED THE PROVIRION. THE ASSEMBLY OF THE PROVIRION INDUCES A SERIES OF CONFORMATIONAL CHANGES WHICH RESULT IN THE CLEAVAGE OF VP0 INTO VP4 AND VP2. THIS OCCURS RAPIDLY UPON GENOME ENCAPSIDATION, THUS THE PROVIRION IS SHORT-LIVED AND POORLY CHARACTERIZED. THE CLEAVAGE OF VP0 INTO VP4 AND VP2 OCCURS WITHIN THE ASSEMBLED PARTICLE IN AN RNA-DEPENDENT MANNER AND IS A PREREQUISITE FOR EV INFECTIVITY. UNDERSTANDING THE PROVIRION AND CHARACTERISING THE KEY CONFORMATIONAL CHANGES WHICH PRECEDE VP0 CLEAVAGE IS THE FOCUS OF THIS APPLICATION. WE PROPOSE THAT THE MECHANISM OF VP0 MATURATION CLEAVAGE IS CONSERVED ACROSS ALL EVS, AND THAT UNDERSTANDING THIS PROCESS WILL INFORM THE DEVELOPMENT OF FUTURE VACCINES AND ANTI-VIRAL THERAPIES. THE CURRENT MODEL OF VP0 CLEAVAGE AND EV MATURATION IS HEAVILY BASED ON DATA FROM POLIOVIRUS (PV), AND SUGGEST A CATALYTIC ROLE FOR RNA IN VP0 MATURATION. HOWEVER, WE HYPOTHESISE THAT GENOME PACKAGING INITIATES CONFORMATIONAL CHANGES WHICH FACILITATE THE FORMATION OF A PROTEINACEOUS CATALYTIC POCKET. WE SUGGEST THAT IDENTIFYING ALLOSTERIC AND CATALYTIC CHANGES WHICH STABILIZE PROVIRIONS WILL ALLOW US TO CAPTURE THE MOLECULAR DETAILS OF THE CATALYTIC SITE AT ATOMIC RESOLUTION. USING A SERIES OF COMPLEMENTARY TECHNIQUES AND A REITERATIVE APPROACH, WE PROPOSE TO TRAP ASSEMBLY INTERMEDIATES AND CHARACTERIZE THE CRITICAL RESIDUES AND CONFORMATIONAL CHANGES THAT MEDIATE VP0 CLEAVAGE WITHIN THE ASSEMBLED VIRUS PARTICLE. WE WILL INITIALLY USE EVA71 AS OUR PROTOTYPE NPEV AND PAN-EV PHENOTYPES WILL BE CONFIRMED BY VERIFYING FINDINGS IN ECHOVIRUS 7, POLIOVIRUS, AND EVD68. OUR PRELIMINARY DATA DEMONSTRATE THE FEASIBILITY OF GENERATING MUTATIONALLY-STABILIZED PROVIRIONS WITH YIELDS SUITABLE FOR STRUCTURAL STUDIES. ADDITIONAL DATA SUPPORT THE TRACTABILITY OF DEVELOPING INHIBITORS OF CAPSID ASSEMBLY. BY THESE APPROACHES WE WILL DEFINE THE CONSERVED MECHANISM OF VP0 CLEAVAGE WHICH WILL BE APPLICABLE TO TREATING BOTH CURRENT NPEV INFECTIONS AND THOSE YET TO ARISE.