HIV-1 encodes a viral protease that is essential for the maturation of infectious viral particles. While protease inhibitors are effective antiretroviral agents, recent studies have shown that prematurely activating – rather than inhibiting – protease function leads to the pyroptotic death of infected cells, with exciting implications for efforts to eradicate viral reservoirs. Despite 40 years of research into the kinetics of protease activation, it remains unclear exactly when protease becomes activated. Recent reports have estimated that protease activation occurs minutes to hours after viral release, suggesting that premature protease activation may be challenging to induce efficiently. Here, combining a powerful FRET-based assay to monitor viral protease activity with sensitive techniques including nanoscale flow cytometry and instant structured illumination microscopy, we demonstrate that the viral protease is activated within cells prior to the release of free virions. Using genetic mutants that lock protease into a ‘precursor’ conformation, we further show that both the precursor and mature protease have rapid activation kinetics and that the activity of the precursor protease is sufficient for viral fusion with target cells. Our finding that HIV-1 protease is activated within producer cells prior to release of free virions helps resolve a long-standing question of when protease is activated and suggests that only a modest acceleration of protease activation kinetics may be required to induce potent and specific elimination of HIV-infected cells.

Western blotting: 

Virus was mixed with 4x Laemmli buffer containing β-mercaptoethanol (BioRad #1610747) and boiled at 95℃for 10 minutes prior to loading on a precast SDS gel (BioRad #4561086DC). Cells were lysed with RIPA buffer (Thermo Fisher #89901) for one hour, vortexing every 10 minutes, prior to mixing with Laemmli buffer and loading on the gel. When staining for viral CA, cellular samples were boiled at 95℃ prior to loading, but when staining for the membrane protein Na/K ATPase, samples were instead incubated at 37℃ for 30 minutes to prevent membrane protein aggregation. Gels were run at 100V for approximately 1 hour before transferring to a nitrocellulose membrane using the Trans-Blot Turbo system (BioRad). Nitrocellulose membranes were stained using the iBind Flex (Thermo Fisher). CA protein was stained using an anti-CA monoclonal antibody obtained through the NIH HIV Reagent Program, Division of AIDS, NIAID, NIH (Anti-Human Immunodeficiency Virus 1 (HIV-1) CA Gag Monoclonal (#24-2), ARP-6457, contributed by Dr. Michael Malim). The Thermo Fisher GAPDH antibody (AM4300) and Abcam Na/K ATPase antibody (ab76020) were used to stain cytosolic and membrane fractions, respectively. All western blot samples were normalized for loading based on volume. Westerns were imaged using the iBright (Thermo Fisher). Densitometry analysis was performed using ImageJ.

Nanoscale flow cytometry:

Biosafety: All viruses were fixed with 4% paraformaldehyde prior to analysis to lower the risk of analyzing infectious HIV-1 particles. In addition, the FACSAria instrument used for analysis was equipped with a negative-pressure BioBubble (Propel Labs) containing a 0.3 µm HEPA filtration system at 200 cfm airflow. The system integrity is regularly maintained by the Case Western cytometry core using a “Glo-germ” protocol. All equipment and procedures comply with the proposed NIH/International Society for the Advancement of Cytometry standards and are approved by the Case Western Reserve University Environmental Health Safety office.

Analysis: Analysis was performed using a FACSAria II SORP sorter (Becton Dickinson). Thresholding was set using the 150mW 532 nm laser, which detects mKOκ positivity using the 575/26 filter. Processing signal was based on mUKG positivity that was detected with a 100mW 488 nm laser and 515/20 filter. Unprocessed FRET signal was similarly detected with the same 488 nm laser but with a 575/40 filter. Sheath fluid was filtered with a 0.2 µm pore and viruses were diluted to collect approximately 6,000 events per second or less to avoid coincidental swarm detection, 

Microscopy:

Virus production: HEK293T cells were plated in glass bottom 35 mm µ-dishes (Ibidi 81158) and transfected using PEI 1 day later with NL4-3 and VIPER-Vpr. Plates were treated with either DMSO or 5 µM darunavir at the time of transfection. 16 hours post-transfection, plates were washed with pre-warmed PBS and fixed with 4% paraformaldehyde for 20 minutes at room temperature prior to washing and storing in PBS at 4°C. 
iSIM imaging: Imaging was conducted on a commercial VT-iSIM (VisiTech International) module integrated with a Nikon Ti base52. Cells were imaged using a 100x 1.49 numerical aperture (NA) oil objective and sCMOS camera (ORCA-Fusion BT, HAMAMATSU). mUKG fluorescence was measured using a 488-nm laser and 525/50 filter with a 300 ms exposure. mKOk fluorescence was measured using a 561-nm laser, 605/52 filter, and 200 ms exposure. FRET was also measured with a 488-nm laser, 605/52 filter and 200 ms exposure. Images were deconvolved using the Nikon NIS-Elements Imaging software (Version 5.30). 
Analysis of images: Analysis was performed using ImageJ. While a z-stack was collected for each image, only the first slice was used for analysis to avoid potential complications from photobleaching. Regions of interest (ROIs) were defined around individual puncta manually, and the same ROIs were used to quantify fluorescence intensity in both the mUKG and mKOκ layers using the ImageJ ROI manager.

The data is divided based on the corresponding figure in the research article "The HIV-1 viral protease is activated during assembly and budding prior to particle release". The final values plotted in each figure and the corresponding statistical analysis can be found in the article in the supplementary file S2.