The HIV-1 Viral Protease Is Activated during Assembly and Budding Prior to Particle Release

Abstract

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 is challenging to induce efficiently. Here, monitoring 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 is required to induce potent and specific elimination of HIV-infected cells. IMPORTANCE HIV-1 protease inhibitors have been a mainstay of antiretroviral therapy for more than 2 decades. Although antiretroviral therapy is effective at controlling HIV-1 replication, persistent reservoirs of latently infected cells quickly reestablish replication if therapy is halted. A promising new strategy to eradicate the latent reservoir involves prematurely activating the viral protease, which leads to the pyroptotic killing of infected cells. Here, we use highly sensitive techniques to examine the kinetics of protease activation during and shortly after particle formation. We found that protease is fully activated before virus is released from the cell membrane, which is hours earlier than recent estimates. Our findings help resolve a long-standing debate as to when the viral protease is initially activated during viral assembly and confirm that prematurely activating HIV-1 protease is a viable strategy to eradicate infected cells following latency reversal.

Keywords: HIV-1 protease; instant structured illumination microscopy; mature protease; nanoscale flow cytometry; precursor protease; protease activation.

FIG 1

Protease maturation is necessary for HIV-1 replication. (A) Schematic representation of protease maturation in viral particles. In the immature viral particle, GagPol aggregates for protease to initially dimerize. This is followed by precursor protease activation, which results in limited processing. Mature protease activation occurs once the precursor protease autoprocesses the p6*-protease cleavage site, after which the remaining cleavage sites are processed and the mature particle forms. Abbreviations: MA, matrix; CA, capsid; SP1, spacer peptide 1; NC, nucleocapsid; SP2, spacer peptide 2; TF, transframe protein; PR, protease; RT, reverse transcriptase; IN, integrase; Env, envelope. (B) Table of mutations at the TF-p6* and p6*-protease cleavage sites that prevent precursor protease autoprocessing. The mutated amino acids are highlighted in red, with arrows marking the protease scission point. (C) Western blot analysis of virus with mature or precursor protease activity. Viruses with only precursor protease activity were compared to wild-type (WT) and D25N (PR D25N) mutant virus. p6*-PR denotes virus with a mutated p6*-protease cleavage site, and TF-PR denotes virus with both TF-p6* and p6*-protease cleavage sites mutated. Western blots were probed with either anti-capsid or anti-protease antibodies. (D) Infectivity of virus with only precursor protease activity. JLTRG-R5 cells were infected with or without spinoculation. Both fusion and productive infection were quantified in 3 biological replicates. Data are means with SEM (standard error of the mean).

FIG 2

Infected cells show Gag processing in both the cytosolic and membrane fractions. (A) Western blot analysis of sucrose gradient fractionation. Ten fractions were probed with either a capsid-specific antibody or with both GAPDH- and Na/K ATPase-specific antibodies. Infected cells were treated with either DMSO, darunavir (DAR), or a combination of bictegravir and efavirenz (BE). Fractions 3 and 10 were representative of the membrane and cytosolic fractions, respectively, and used in panel B. (B) Western blot analysis of fractions 3 and 10. The analysis in panel A was repeated but with only fractions 3 and 10 to facilitate side-by-side comparison of capsid expression and processing.

FIG 3

VIPER allows protease activity to be assessed in individual and intact HIV-1 particles. (A) Schematic representation of VIPER (viral protease reporter). VIPER consists of an mUKG-mKOκ FRET pair attached by a protease cleavage sequence. The level of FRET changes depending on if protease is active, allowing protease activity to be assessed based on mUKG fluorescence. (B) Schematic representation of VIPER-Vpr and iVIPER. VIPER is specifically incorporated into viral particles as either a polyprotein with Vpr (red star symbol), referred to as VIPER-Vpr, or by cloning it into the viral genome between matrix and capsid, referred to as iVIPER. (C) Representative nanoscale flow cytometry data of HIV-1 NL4-3 VIPER-Vpr processing. The supernatant of cells transfected with VIPER-Vpr alone was used as a negative control. The processing gates were drawn based on FRET and mUKG fluorescence, and an approximate 5% background processed signal was allowed when protease activity was inhibited by the protease inhibitor saquinavir (SQV).

FIG 4

VIPER-Vpr does not interfere with normal budding kinetics of wild-type NL4-3 virus. (A) Western blot analysis of budding kinetics and processing in virus containing VIPER-Vpr or iVIPER. HEK293T cells were transfected with either NL4-3 alone, with both NL4-3 and VIPER-Vpr, or with NL4-3 iVIPER. Cells and supernatant were collected every 2 h beginning 4 h posttransfection and analyzed by probing with a capsid-specific antibody. (B) Densitometry analysis of wild-type NL4-3 alone and NL4-3 VIPER-Vpr anti-capsid Western blots from panel A. (C) Nanoscale flow cytometry analysis of budding kinetics and processing of virus containing VIPER-Vpr or iVIPER. The same NL4-3 VIPER-Vpr and iVIPER viruses used for panel A were analyzed using nanoscale flow cytometry with 2 technical replicates. Relative viral particle counts were calculated based on the number of events detected in 20 s by the cytometer. Data are the means with SEM.

FIG 5

VIPER-Vpr validates the effects of increased GagPol expression and impaired ESCRT recruitment on protease activation. (A) Anti-capsid Western blot analysis of NL4-3 virus made with various ratios of Gag to GagPol. The Gag-to-GagPol ratio was modified by cotransfection of two plasmids expressing Gag and GagPol individually, keeping the total amount of DNA used the same. Twenty-one ratios between Gag and GagPol, from 20:0 to 0:20, were analyzed. The wild-type NL4-3 virus is included for reference. (B) Nanoscale flow cytometry analysis of NL4-3 VIPER-Vpr virus with various ratios of Gag and GagPol. The same ratios of Gag to GagPol used in panel A were repeated, except the virus was cotransfected with VIPER-Vpr. Two biological replicates were analyzed. Relative viral particle counts were normalized to the 20:0 Gag/GagPol ratio condition. Data are means with SEM. (C) Anti-capsid Western blot of NL4-3 virus with impaired ESCRT recruitment. Wild-type virus is compared to virus with mutations in the PTAP and/or YP domains of p6 responsible for recruiting ESCRT proteins. (D) Nanoscale flow cytometry analysis of NL4-3 VIPER-Vpr virus with impaired ESCRT recruitment. Viruses used in panel C were made with VIPER-Vpr, and particle production was monitored every 2 h from 10 to 20 h posttransfection of HEK293T cells. Two biological replicates were analyzed. Data are means with SEM.

FIG 6

Protease activity can be detected in newly released viruses using nanoscale flow cytometry. (A) Schematic representation of a washing experiment to analyze newly produced virions using nanoscale flow cytometry. HEK293T cells were cotransfected with NL4-3 and VIPER-Vpr and washed to remove residual virus, and new virions were collected over the following 8 min. The color of the cellular supernatant is reflective of the relative concentration of viral particles at each step of the experiment, with a darker color being the most concentrated. (B) Relative particle concentration and processing of NL4-3 VIPER-Vpr virus during washes using nanoscale flow cytometry. VIPER-Vpr was cotransfected with either wild-type NL4-3 or with a precursor protease-restricted NL4-3 containing a mutated p6*-protease cleavage site. The relative viral particle count and processing was monitored over 10 successive washes. Three biological replicates were analyzed. Data are means with SEM. (C) Relative particle concentration and processing of NL4-3 VIPER-Vpr virus after washing using nanoscale flow cytometry. Particle concentration and processing were monitored from 15 s up to 8 min after the washing performed for panel B. Three biological replicates were analyzed. Data are means with SEM.

FIG 7

Processing activity of the precursor and mature protease can be distinguished using VIPER-Vpr. (A) Table of protease cleavage sequences cloned into VIPER-Vpr. The natural location of the cleavage site in wild-type HIV-1 is listed where applicable, and arrows indicate the scission point. Abbreviations are listed in the legend for Fig. 1A. (B) Processing of a panel of VIPER-Vpr reporters in NL4-3 by nanoscale flow cytometry. Each VIPER-Vpr reporter was cotransfected with either wild-type NL4-3 or a precursor protease-restricted NL4-3 (with the p6*-protease cleavage site mutated). Processing of each individual VIPER-Vpr reporter was normalized based on results with the wild-type virus. Numbers indicating the protease cleavage sequence in VIPER-Vpr correspond to those listed in Fig. 7A. Three to four biological replicates were analyzed. Data are means with SEM. (C) Western blot analysis of NL4-3 VIPER-Vpr with either the MA-CA or SP2-p6 cleavage sequences. Both VIPER-Vpr reporters were made with either the wild-type or precursor protease-restricted NL4-3 viruses. Western blots were probed with either capsid- or mKOκ-specific antibodies to assess processing of Gag or VIPER-Vpr, respectively.

FIG 8

Both precursor and mature protease activity is detectable in newly released viruses. (A) Results of the nanoscale flow cytometry washing experiment using NL4-3 SP2-p6 VIPER-Vpr. Particle concentration and processing was monitored from 15 s up to 8 min after washing cells 10 times. Three biological replicates were analyzed. Data are means with SEM. (B) Results of the nanoscale flow cytometry washing experiment using NL4-3 SP2-p6 VIPER-Vpr in SupT1-R5 cells. Particle concentration and processing was monitored from 1 to 16 min postwashing 10 times. Three biological replicates were analyzed. Data are means with SEM. (C) Comparison of premature protease activation in wild-type NL4-3 and the CH106.c-NL4-3 hybrid. Virus was made with VIPER-Vpr to assess particle concentration using nanoscale flow cytometry. The ratio of viral particle counts when made with darunavir (DAR) compared to DMSO was assessed using seven biological replicates. Data are means with SEM. (D) Results of the nanoscale flow cytometry washing experiment using the CH106.c-NL4-3 hybrid virus. The washing experiment was repeated using the CH106.c-NL4-3 hybrid and either the MA-CA or SP2-p6 VIPER-Vpr. Processing and particle concentration were monitored for 15 s to 8 min after washing cells 10 times. Four biological replicates were analyzed. The bars are means with SEM.

FIG 9

Protease activation occurs both at the cell membrane and within the cytosol. (A) Representative image of VIPER-Vpr expression in viral puncta at the HEK293T cell membrane. HEK293T cells producing NL4-3 VIPER-Vpr were fixed and imaged using instant structured illumination microscopy. The image on the left contains a cell expressing NL4-3 VIPER-Vpr, imaged for mUKG fluorescence. The area inside the red rectangle is expanded in the panels on the right, showing both mUKG and mKOκ fluorescence with individual viral puncta segmented in yellow boxes. (B) Comparison of mUKG and mKOκ fluorescence intensity in individual, membrane-bound puncta. HEK293T cells producing NL4-3 VIPER-Vpr virus in the presence of either DMSO or the protease inhibitor darunavir were compared for fluorescence intensity of both mUKG and mKOκ. Sixty-one puncta from 9 cells were used for the DMSO-treated analysis, and 46 puncta from 8 cells were used for the darunavir-treated condition. A simple linear regression was performed for each condition and plotted. The Spearman two-tailed correlation values were 0.8229 and 0.9225 for the DMSO-treated and darunavir-treated conditions, respectively. (C) The ratio of mUKG to mKOκ fluorescence in the segmented membrane-bound viral puncta illustrated in panel B and within the cytosol. Data are means with SEM.