Project Grant F32AI203577

ENV GLYCOPROTEIN INCORPORATION AT HIV-1 ASSEMBLY SITES: STERIC REGULATION DRIVEN BY THE LONG CYTOPLASMIC TAIL - PROJECT SUMMARY. TO PRODUCE INFECTIOUS VIRAL PARTICLES, THE HIV-1 GLYCOPROTEIN ENVELOPE (ENV) NEEDS TO BE INCORPORATED INTO VIRAL BUDS COMPOSED OF THE STRUCTURAL PROTEIN GAG AT THE PLASMA MEMBRANE OF CELLS. ENV HAS A LONG (~ 150 AMINO ACID) CYTOPLASMIC TAIL (CT) THAT FORMS AN ALPHA HELICAL BASEPLATE STRUCTURE THAT ADDS BULK TO THE STRUCTURE OF ENV. IMPORTANTLY, THE ENV-CT IS REQUIRED FOR INFECTIOUS SPREAD IN MOST CELL TYPES AND HAS KNOWN INVOLVEMENT IN THE INCORPORATION OF ENV AT ASSEMBLY SITES. WHILE BIOCHEMICAL AND BIOPHYSICAL EXPERIMENTS HAVE PROVIDED INSIGHT, THE MECHANISM BY WHICH ENV INCORPORATION OCCURS IS NOT WELL UNDERSTOOD AND REMAINS A KNOWLEDGE GAP IN OUR UNDERSTANDING OF HIV-1 REPLICATION BIOLOGY. IN PARTICULAR, IT HAS NOT BEEN WELL EXPLORED WHETHER THE BULKY NATURE OF THE ENV-CT STERICALLY CONTROLS ENV INCORPORATION MECHANISMS. HERE, WE USE SINGLE-MOLECULE SUPERRESOLUTION FLUORESCENCE MICROSCOPY (SRM) APPROACHES TO INVESTIGATE THE ROLE STERIC PROPERTIES OF THE LONG AND BULKY ENV- CT PLAY IN ENV INCORPORATION AT ASSEMBLY SITES. EXPERIMENTS COMBINING PHOTOACTIVATED LOCALIZATION MICROSCOPY (PALM) OF GAG ASSEMBLIES WITH SIMULTANEOUS SINGLE-MOLECULE TRACKING OF ENV ENABLE US TO INVESTIGATE THE MOBILITY OF ENV WITH CT-TRUNCATIONS IN AND AROUND GAG ASSEMBLY SITES. INTERFEROMETRIC PHOTO-ACTIVATED LOCALIZATION MICROSCOPY (IPALM) ENABLES US TO CHARACTERIZE THE SUB-VIRAL LOCALIZATION OF TRUNCATED ENV VARIANTS TO GAIN INSIGHT INTO WHETHER ENV VARIANTS HAVE RESTRICTED ACCESS TO ASSEMBLY SITES (SPECIFIC AIM 1). IN SPECIFIC AIM 2, WE USE THE SRM APPROACHES ABOVE TO STUDY THE IMPACT OF A NATURALLY OCCURRING 7 AMINO ACID INSERTION IN THE ENV-CT OF HIV-1 CLADE C TO GAIN FURTHER INSIGHT INTO THE BALANCE BETWEEN STERIC EFFECTS AND ENV INCORPORATION INTO ASSEMBLIES. THROUGH EXPERIMENTS IN SPECIFIC AIMS 1 AND 2, WE TEST OUR CENTRAL HYPOTHESIS THAT THE BULKINESS OF THE ENV-CT PLAYS A COMPLEX ROLE IN REGULATING ENV INCORPORATION EVENTS. WE PROPOSE THAT WHILE THE BULKY NATURE OF THE ENV-CT CAN PROMOTE STERIC TRAPPING AND FACILITATE ENV INCORPORATION INTO ASSEMBLIES, ENV-CT STERIC EFFECTS CAN ALSO RESTRICT ACCESS TO ASSEMBLY SITES AND ON THE OTHER HAND, DETER INCORPORATION EVENTS. WE PREDICT THAT THE FULL-LENGTH CT PROVIDES A PROPER BALANCE OF STERIC EFFECTS THAT ENABLES LATTICE TRAPPING WHILE ALSO PARTIALLY RESTRICTING ENTRANCE TO ASSEMBLY SITES TO HELP KEEP ENV VIRUS COPY NUMBERS LOW. WE PROPOSE THAT THE IMPORTANCE OF THIS WORK IS TWO-FOLD. FIRST, SINCE LOW COPY NUMBERS ON ENV ARE THOUGHT TO PROMOTE IMMUNE EVASION, THIS WORK HAS IMPORTANT IMPLICATIONS FOR VIRAL PERSISTENCE. SECOND, HIV-1 IS HIGHLY MUTAGENIC, MAKING DRUG RESISTANCE A CONCERN AND THE DEVELOPMENT OF NEW ANTIRETROVIRALS CRUCIAL. THE INCORPORATION OF ENV AT ASSEMBLY SITES IS CURRENTLY AN UNDRUGGED PROCESS THAT HAS POTENTIAL AS AN ANTIVIRAL TARGET AND THE RESULTS OF THE PROPOSED WORK WILL HELP LAY THE SCIENTIFIC GROUNDWORK TO MAKE THIS POSSIBLE.