Imaging protease activation and maturation of single HIV-1 particles - HIV-1 is released from infected cells as an immature particle consisting of Gag and Gag-Pol polyproteins. At some point during/after virus budding from the plasma membrane, the viral protease (PR) autoactivates by cleaving itself off Gag-Pol and proceeds to cleave Gag and Gag-Pol at multiple sites and in defined order. This sequence of well-orchestrated cleavage events culminates in the formation of a mature cone-shaped viral core containing two copies of genomic RNA, nucleocapsid and viral enzymes, and surrounded by a lipid membrane. Although the timely PR activation is critical for the formation of mature infectious HIV-1, the mechanism and timing of PR autoactivation remain poorly defined. Recent studies revealed that inhibition of neutral sphingomyelinase 2 (nSMase2), which converts sphingomyelin to ceramide, blocks HIV-1 maturation, implicating lipids in regulation of PR autoactivation. We implemented and validated HIV-1 labeling and single virus imaging approaches to visualize single virus assembly, PR autoactivation and substrate cleavage by total internal reflection fluorescence microscopy. These approaches rely on FRET probes in which fluorescence donor and acceptor are linked by distinct PR cleavage sites from Gag and Gag-Pol. We also established a correlative light-electron microscopy (CLEM) workflow to link FRET substrate processing by PR to formation of mature virions. Finally, we have developed a panel of innovative assays to characterize the properties of viral membrane. Together, these technical advances put us in a position to elucidate the timing and regulation of PR autoactivation leading to HIV-1 maturation. We propose three Specific Aims. Specific Aim 1 will measure the timing of PR activation relative to HIV-1 scission from the plasma membrane. Using bifunctional FRET probes in which a fluorescence donor also serves as a pH-sensor, we will visualize both single virus scission from a cell, by probing the connectivity with the cytoplasm, and PR activation (loss of FRET signal). Specific Aim 2 will assess the relative rate and extent of PR substrate cleavage in single HIV-1 and correlate these with formation of mature virions. Specific Aim 3 will define the role of lipids in HIV-1 maturation by investigating the effects of nSMase2 inhibition on the properties of viral membrane and on Gag-Pol dimerization, which is believed to initiate PR autoactivation. Collectively, the proposed studies will define the timing of PR autoactivation, elucidate the relative rates of processing of distinct Gag and Gag-Pol cleavage sites, link surrogate substrate cleavage to HIV-1 maturation, and provide critical insights into regulation of PR activation by lipids. Knowledge of the mechanism and regulation of PR autoactivation can inform new therapeutic strategies.
