Project Grant F31AI203598

ENGINEERED CHANGES IN HIV-1 ENVELOPE THAT OVERCOME MACAQUE ENTRY BARRIER. - PROJECT SUMMARY/ABSTRACT INFECTION OF MACAQUES WITH CHIMERIC SIV/HIV-1 VIRUSES (SHIVS) PROVIDES A CRITICAL MODEL FOR VACCINE STUDIES BECAUSE SHIVS ENCODE HIV-1 ENVELOPE GLYCOPROTEIN (ENV) - THE SOLE TARGET OF HIV-1 NEUTRALIZING ANTIBODIES. BECAUSE THE PRIMARY GOAL OF VACCINES IS TO PREVENT NEW INFECTION, SHIVS BASED ON TRANSMITTED/FOUNDER ENV VARIANTS (T/F SHIVS) ARE DESIRED AS CHALLENGE VIRUSES, AS THEY MORE ACCURATELY REPRESENT THE VIRAL VARIANTS RESPONSIBLE FOR EARLY TRANSMISSION. HOWEVER, MOST T/F ENVS DO NOT USE MACAQUE CD4 (MCD4) EFFICIENTLY FOR VIRAL ENTRY, AND, THEREFORE, T/F SHIVS REPLICATE POORLY IN MACAQUES AND DO NOT ESTABLISH PERSISTENT INFECTION. SINCE MCD4-ENV INTERACTION IS A MAJOR DETERMINANT OF SHIV INFECTION IN MACAQUES, SUBSTANTIAL EFFORTS HAVE BEEN MADE TO ENGINEER T/F SHIVS THAT EFFICIENTLY UTILIZE MCD4 AND REPLICATE IN MACAQUES WITHOUT EXTENSIVE ADAPTATION. RECENT STUDIES EMPLOYING STRUCTURE-GUIDED DESIGN AND IN VIVO SELECTION OF ENV VARIANTS IN MACAQUES HAVE IDENTIFIED RESIDUES IN THE GP120 RECEPTOR-BINDING SUBUNIT OF ENV (SUCH AS SERINE 375 AND ALANINE 281) WHOSE MUTATIONS IMPROVE MCD4 USAGE AND SHIV REPLICATION. IDENTIFICATION OF ADDITIONAL ENV RESIDUES THAT ENHANCE MCD4 BINDING WILL HELP ADVANCE THE FIELD BY ENABLING A MORE PRECISE DEFINITION AND COMPARISON OF THE DETERMINANTS THAT GOVERN SHIV REPLICATION IN MACAQUES. HIV-1 ENV EXISTS IN A METASTABLE STATE AND TRANSITIONS FROM A PREFUSION `CLOSED' STATE TO AN INTERMEDIATE `OPEN-OCCLUDED' STATE THAT BINDS CD4 TO AN `OPEN' STATE THAT MEDIATES FUSION WITH THE HOST MEMBRANE. WE RECENTLY SHOWED THAT OPEN ENV CONFORMATIONS CAN OVERCOME THE MCD4 ENTRY BARRIER AND INCREASE SHIV REPLICATION. USING STRUCTURE-GUIDED DESIGN, WE HAVE NOW IDENTIFIED FIVE RESIDUES IN THE GP41 TRANSMEMBRANE FUSION SUBUNIT OF ENV WHOSE ALTERATIONS HAVE THE POTENTIAL TO RESHAPE ENV CONFORMATION IN A MANNER THAT PROMOTES TRANSITIONS TO DOWNSTREAM, OPEN ENV CONFORMATIONS. OUR GOAL IS TO INTRODUCE GP41 SUBSTITUTIONS THAT MINIMALLY AFFECT OVERALL ENV ARCHITECTURE-THEREBY PRESERVING ITS ANTIGENIC PROPERTIES-WHILE STILL PROMOTING ENHANCED MCD4 ENGAGEMENT, VIRAL FUSION, AND SHIV REPLICATION. OUR PRELIMINARY DATA SUGGEST THAT POINT MUTATION IN ONE OF THESE GP41 RESIDUES IS SUFFICIENT TO INCREASE SHIV FUSION IN MACAQUE LYMPHOCYTES. THIS F31 PROPOSAL ENGAGES IN FUNCTIONAL AND STRUCTURAL ANALYSES OF GP41 MUTATIONS TO DEFINE HOW ENGINEERED CHANGES IN ENV CAN HELP OVERCOME MACAQUE VIRAL ENTRY BARRIER. THIS RESEARCH PROPOSAL WILL: 1) INVESTIGATE THE EFFECT OF GP41 MUTATIONS ON MACAQUE CD4 USAGE AND SHIV REPLICATION; AND 2) STUDY THE STRUCTURAL CHANGES IN ENV INDUCED BY POINT MUTATIONS IN THE GP41 SUBUNIT OF ENV. THIS PROPOSAL EMPLOYS INNOVATIVE CRYO- ELECTRON MICROSCOPY TO DETERMINE THE FIRST STRUCTURE OF HIV-1 ENV IN COMPLEX WITH MACAQUE CD4, WHICH WILL HELP REVEAL THE STRUCTURAL VARIANCE BETWEEN HUMAN VERSUS MACAQUE CD4 ENGAGEMENT. THIS PROPOSAL IS SIGNIFICANT AS IT PROVIDES FUNDAMENTAL UNDERSTANDING OF HOW MODULATION OF KEY GP41 TRANSMEMBRANE RESIDUES REGULATE ENV STRUCTURE AND FUNCTION AND HELP OVERCOME MACAQUE VIRAL ENTRY BARRIER.