Project Grant R01AI181618

HIV-1 MEMBRANE FUSION AND INHIBITION - SUMMARY VIRUS ENTRY BEGINS WITH THE FIRST ENCOUNTER BETWEEN THE VIRUS AND THE CELL SURFACE AND ENDS WITH DELIVERY OF THE CONTENTS OF THE VIRUS INTO THE HOST CELL. HIV-1 MEMBRANE FUSION IS THE FIRST KEY DELIVERY STEP, MEDIATED BY THE VIRUS-ENCODED ENVELOPE GLYCOPROTEIN [ENV; TRIMERIC (GP160)3 CLEAVED TO (GP120/GP41)3], WHICH BELONGS TO THE GROUP OF CLASS I VIRAL FUSION PROTEINS INCLUDING INFLUENZA HEMAGGLUTININ, SARS-COV-2 SPIKE PROTEIN AND EBOLA GLYCOPROTEIN. A MATURE ENV SPIKE HAS THREE COPIES EACH OF NONCOVALENTLY-ASSOCIATED RECEPTOR-BINDING SUBUNIT GP120 AND FUSION SUBUNIT GP41. SEQUENTIAL BINDING OF GP120 TO THE PRIMARY RECEPTOR CD4 AND A CORECEPTOR (CHEMOKINE RECEPTOR CCR5 OR CXCR4) LEADS TO LARGE, IRREVERSIBLE STRUCTURAL REARRANGEMENTS IN GP41, WHICH DRIVE FUSION. THIS PICTURE, DERIVED LARGELY FROM STRUCTURAL STUDIES OF THE SOLUBLE FRAGMENTS AND FROM CELLULAR STUDIES WITH INHIBITORS AND ANTIBODIES, IS STILL INCOMPLETE BECAUSE IT LACKS EXTENSION TO A HIGH-RESOLUTION PICTURE OF THE COMPLETE ENV TRIMER IN THE CONTEXT OF A LIPID-BILAYER MEMBRANE, WHICH IS THE SUBSTRATE OF THE FUSION REACTION. WE HAVE DETERMINED BY NMR THE STRUCTURES OF THE HIV-1 ENV TRANSMEMBRANE DOMAIN (TMD), MEMBRANE PROXIMAL EXTERNAL REGION (MPER), AND CYTOPLASMIC TAIL (CT) IN BICELLES THAT MIMIC LIPID BILAYERS. THESE REGIONS ALL FORM WELL-ORDERED, TRIMERIC CLUSTERS IN A LIPID BILAYER. DISRUPTION OF ANY OF THEM CAN REDUCE MEMBRANE FUSION EFFICIENCY AND ALTER THE ANTIGENIC STRUCTURE OF THE ENTIRE ENV, SUGGESTING THAT THEY HAVE STRUCTURAL AND FUNCTIONAL ROLES IN FUSION AND IN TRIMER CONFORMATIONAL STABILIZATION. VERY RECENTLY, WE HAVE COMPLETED A HIGH-RESOLUTION STRUCTURE OF THE INTACT SARS-COV-2 POSTFUSION SPIKE IN MEMBRANE, SHOWING HOW THE FUNCTIONALLY CRITICAL MEMBRANE-INTERACTING REGIONS INTERACT WITH MEMBRANE AND WITH EACH OTHER. THESE FINDINGS ARE THE BASIS OF OUR OVERALL HYPOTHESIS THAT THAT STRUCTURES OF THE HIV-1 FUSION COMPLEX EITHER ALONE OR BOUND WITH FUSION INHIBITORS IN MEMBRANE WILL REVEAL NEW STRUCTURAL FEATURES OF THE MEMBRANE-INTERACTING REGIONS AND SUBSTANTIALLY ADVANCE OUR MECHANISTIC UNDERSTANDING OF THE VIRAL FUSION AND ITS INHIBITION, THEREBY INFORMING FUTURE DEVELOPMENT OF INTERVENTION STRATEGIES. WE WILL APPLY ADVANCED TECHNOLOGIES IN CRYOGENIC ELECTRON MICROSCOPY (CRYO-EM) AND TOMOGRAPHY (CRYO-ET) TO STUDY STRUCTURAL AND FUNCTIONAL PROPERTIES OF THE HIV-1 FUSION COMPLEX, AS RECONSTITUTED IN MEMBRANES AND ON THE SURFACE OF A VIRUS PARTICLE. WE WILL ALSO INVESTIGATE MOLECULAR MECHANISMS OF HIV-1 INHIBITION BY TWO DISTINCT TYPES OF FUSION INHIBITORS. OUR GOAL IS A "MOLECULAR MOVIE" OF HIV-1 FUSION, TO INFORM DEVELOPMENT OF NEW INTERVENTION STRATEGIES. WE PROPOSE THE FOLLOWING SPECIFIC AIMS TO ADDRESS OUR HYPOTHESIS: 1) WE WILL DETERMINE STRUCTURE OF THE HIV-1 FUSION COMPLEX CONTAINING INTACT ENV, CD4 AND CCR5 IN THE CONTEXT OF A LIPID BILAYER. 2) WE WILL INVESTIGATE MOLECULAR MECHANISM OF HIV-1 FUSION INHIBITION BY ANTI-CD4 ANTIBODY IBALIZUMAB. 3) WE WILL DISSECT MECHANISM OF ACTION OF SMALL-MOLECULE FUSION INHIBITORS TARGETING THE MPER OF HIV-1 ENV.