We describe a cell-based assay to report on HIV-1 fusion via the expression of green fluorescent protein detectable by flow cytometry or fluorescence microscopy. It can be used to test inhibitors of viral entry (specifically at the fusion step) in cell-free and cell-to-cell infection systems.
This assay is designed to specifically report on HIV-1 fusion via the expression of green fluorescent protein (GFP) detectable by flow cytometry or fluorescence microscopy. An HIV-1 reporter virus (HIV-1 Gag-iCre) is generated by inserting Cre recombinase into the HIV-1 genome between the matrix and the capsid proteins of the Gag polyprotein. This results in a packaging of Cre recombinase into virus particles, which is then released into a target cell line stably expressing a Cre recombinase-activated red fluorescent protein (RFP) to GFP switch cassette. In the basal state, this cassette expresses RFP only. Following the delivery of Cre recombinase into the target cell, the RFP, flanked by loxP sites, excises, resulting in GFP expression. This assay can be used to test any inhibitors of viral entry (specifically at the fusion step) in cell-free and cell-to-cell infection systems and has been used to identify a class of purinergic receptor antagonists as novel inhibitors of HIV-1 viral membrane fusion.
The need for novel antiretroviral therapies has prompted the developement of high-throughput screens for inhibitors of HIV-1 entry. The Gag-iCre reporter assay is developed to identify inhibitors of viral entry at the fusion step in a cell-to-cell infection system by specifically measuring viral membrane fusion with the host cell membrane1. An assay was developed to screen for novel inhibitors that acted specifically at the early stages of HIV-1 infection up to the point of viral membrane fusion. One challenge to measuring cell-cell infection is that the initial inoculum contains infected donor cells and ininfected target cells, so measuring new infection is difficult to discriminate from the input cells. The ideal system would involve a heterologous gene marker that can be induced by the initiation of a target cell infection and is not present in the donor cell. A popular viral contents mixing assay based on a viral protein-enzyme fusion, BlaM-Vpr, can be effective, but has limitations for high throughput, cell-cell screening2. This assay involves a beta-lactamase (BlaM) reporter gene that is fused to HIV-1 Vpr, packaged into virions, and delivered into target cells upon viral membrane fusion. The substrate CCF2-AM is loaded into the cytoplasm of target cells and undergoes a fluorescence shift upon cleavage. The CCF2-AM substrate is expensive and can be prohibitive for high-throughput screens. In order to distinguish donor and target cells, it is necessary to dye-label the target populations. Finally, the protocol requires several wash and incubation steps which can be burdensome and costly when testing large numbers of compounds.
The Gag-iCre assay was developed as a solution to these issues, to develop a high-throughput screening for inhibitors of fusion in cell-to-cell transmission. This system does not require substrate to be loaded into cells. The assay can also be used for cell-free studies and is adaptable to other viral fusion studies using pseudo-typed HIV-1 Gag-iCre virus particles. HIV Env mediated cell-cell fusion assays can measure virus Env protein-mediated cell fusion, however these do not use actual virus particles as the HIV-1 Gag-iCre assay does3,4,5. This assay can be read with flow cytometry or fluorescence microscopy. It has been successfully used to screen the FDA library as well as a small library of purinergic inhibitors1,6. Other investigators have also identified purinergic inhibitors in HIV-1 fusion using an adapted and optimized high-throughput fusion assay that reports the transfer of virus-encapsulated beta-lactamase into the cytoplasm7,8.
The Gag-iCre virus was designed with a similar approach to the Gag-iGFP virus, inserting Cre recombinase between matrix and capsid, flanked by HIV-1 protease sites9. The Cre enzyme is made as a precursor inserted within the Gag polyprotein that matures when HIV-1 protease is activated within the nascent virus particle. The Cre delivery is, thus, dependent upon the delivery of the contents of a protease-activated HIV-1 particle into a target cell via a viral membrane fusion process. Two versions of the protocol are provided here. The first uses an HIV-1 infected population to infect target cells, to study direct cell-to-cell transmission of HIV. The second version uses a cell-free virus to study a cell-free viral infection. The cell-to-cell transmission assay takes 7 days to complete from the day the cells are thawed, or 5 days if the cells are already thawed and passaged. The cell-free infection assay can be performed in 5 days if the cells need to be thawed and 3 days if the cells are thawed and passaged. Instructions are also given to generate an RG (red-to-green) target cell line in the desired cell type (if a preexisting RG target cell line is not being used) using a plasmid created by the Clevers Lab10. It is recommended that appropriate biosafety precautions are taken with the virus and the virus-expressing cells in this assay. We conduct the infectious portion of this assay in a BSL2+ tissue culture facility. After the cells are fixed, they can be analyzed in standard flow cytometry and microscopy facilities.
Here we describe the application of this assay to screen for novel compounds that inhibit HIV-1 viral membrane fusion (Figure 1). Purinergic receptors are pro-inflammatory mediators. Our laboratory has demonstrated that non-selective inhibitors of purinergic receptors act as inhibitors of HIV-1 viral membrane fusion6. We report that the utilization of this assay in high throughput can identify novel HIV-1 viral membrane fusion inhibitors. We demonstrate that an inhibitor of the purinergic class of receptors represents a novel class of HIV-1 viral membrane fusion inhibitors.
1. Generation of Target Cell Lines
NOTE: This step is optional; if using an existing RG target cell line, start at step 2.
2. Cell-to-cell Virus Transmission
3. Alternative Protocol for a Cell-free Virus Infection
Uninfected RG Jurkat Cells exhibit a low level of background GFP signal (0.3%) with a very strong RFP signal (Figure 2A, uninfected column). The infection with Gag-iCre causes an increase in GFP signal (24.9%,) while the presence of the HIV-1 fusion inhibitor AMD3100 (20 µM) inhibits the development of this signal, bringing it down to uninfected background levels (0.34%). When an inhibitor of a post-fusion event such as the reverse transcription inhibitor AZT (2 µM) is used, the signal is not affected significantly (23.5%), indicating that the GFP signal produced by the assay is specific to HIV-1 fusion. Figure 2B indicates a dose-dependent inhibition of Gag-iCre fusion with PPADS (100 µM), a non-selective purinergic inhibitor.
Figure 3 indicates a comparison of cell-free HIV-1 viral membrane fusion using the Gag-iCre assay (Figure 3A) and the convention BlaM-Vpr assay (Figure 3B). A titration of Gag-iCre and BlaM-Vpr viruses was used to spinoculate—a 2 h long 1,200 x g spin of the 96-well infection plate at the beginning of the infection to increase its efficiency—RG Jurkat cells with Gag-iCre and Jurkat cells with the BlaM-Vpr virus as previously described2. At 3 ng of the virus, both assays offered a robust signal of 64.7% (Gag-iCre) and 47.2% (BlaM-Vpr) of the cells were fused. At 0.3 ng of the virus, the signals were 1.18% (Gag-iCre) and 2% (BlaM-Vpr). These data suggest that Gag-iCre can offer a comparable signal for viral fusion to the well-established BlaM-Vpr assay at a range of virus concentrations.
Figure 1: Gag-iCre assay overview. RG Jurkat cells are co-incubated with donor Jurkat cells transfected with HIV Gag-iCre. Upon viral membrane fusion, the Cre is released into the target cells, which can cleave the dsRed gene at the loxP sites to allow EGFP expression, resulting in the cells fluorescing green. Please click here to view a larger version of this figure.
Figure 2: Representative results from a cell-free and a cell-to-cell Gag-iCre infection. (A) Cell-free Gag-iCre fusion is inhibited by the fusion inhibitor AMD3100, but not by the reverse transcription inhibitor AZT. These panels show uninfected RG Jurkat cells, untreated RG Jurkat cells 48 h after the infection, and RG Jurkat cells 48 h after the infection but treated with AMD3100 or AZT, as indicated. The upper panels represent fluorescent micrographs on the GFP channel, the middle panels represent fluorescent micrographs on the RFP channel, while the lower panels represent fluorescence-activated cell sorting (FACS) data for samples showing GFP positive of a percentage indicative of fusion. Scale bar = 100 µm. This panel has been modified with permission from Esposito et al.1. (B) The HIV-1 viral membrane fusion following cell-to-cell infection is inhibited by PPADS. RG Jurkat cells were mixed with Jurkat cells transfected with HIV-1 Gag-iCre, and the inhibitors AMD3100 and PPADS were tested for their ability to block viral membrane fusion at 100 µM, as shown in representative fluorescence-activated cell sorter plots. This figure has been modified from Swartz et al.6. Please click here to view a larger version of this figure.
Figure 3: HIV Gag-iCre reporter signal is comparable to BlaM-Vpr. These panels show the results of a FACS analysis of (A) RG cells spinoculated with a Gag-iCre virus and (B) Jurkat cells spinoculated with NL43 BlaM-Vpr, 16 h after a 6 h infection at the indicated concentration of the virus. This panel has been modified with permission from Esposito et al.1. Please click here to view a larger version of this figure.
The Gag-iCre assay has proven to be very useful for screening drug candidates which may inhibit the fusion step of viral replication. When performing this assay, the most important steps to get a good signal are similar to most viral infection assays. The first critical step is producing high titers of good-quality virus. This step requires that the 293T cells are passaged frequently (at least 1x every 48 h) so they do not become overconfluent and clump together. Additionally, it may be worth experimenting with different transfection methods, as some researchers find that calcium-phosphate transfection gives better results, while others favor lipid-based reagents. Similarly, the other critical step in this protocol is to not let the target cells overgrow but to keep them in the exponential growth phase right before the infection, as the stationary phase reduces their ability to be infected.
Compared to the well-established BlaM-Vpr assay2, the Gag-iCre assay offers a substrate-free assay with no need to label target cells. This makes it well suited for certain high-throughput applications1. Additionally, the assay provides a permanent marker for viral entry even if the cell becomes latent, offering some potential to study latent infection. Future studies for latency may utilize this system in mice where RG cells which have been switched to green due to Gag-iCre exposure, yet are not productively infected, could be isolated to study the cell populations with a latent virus or nonproductive infection. In addition to latency, the assay has other potential applications. It has been successfully utilized for pseudo-typing with other viral envelope proteins such as Ebola and VSV (unpublished data), opening up the potential for this assay to identify fusion inhibitors for a variety of viruses in a safer manner than working with a live virus.
One potential limitation of the Gag-iCre assay is that it does take an additional day for the signal to develop, compared to BlaM-Vpr. In addition, the Gag-iCre virus in its current state only replicates for a single cycle, which works for drug screening under the conditions we have described but would not be suitable for long-term studies or growth curves. In the future, new versions of the assay will be made with different reporter signals, such as luciferase, allowing for a wider variety of uses. Further, the screening of larger compound libraries will likely yield novel inhibitors specific to the fusion step of viral replication.
The authors have nothing to disclose.
This research was supported by grants NIH/NIAID AI112423 and NIH/NIGMS GM113885 to Benjamin K. Chen and NIH/NIAID K08-AI120806 to Talia H. Swartz. We would like to thank the Icahn School of Medicine at Mount Sinai Dean's flow Cytometry CORE.
Dulbecco's Modified Eagle's Medium (DMEM) | Sigma Aldrich | D5546 | Media for 293 Cells |
RPMI-1640 | Sigma Aldrich | R0883 | Media for Jurkats |
Fetal Bovine Serum Albumin | Gibco | 16140-071 | Serum for 293 Cells |
Cosmic Calf Serum | Hyclone | SH30087.03 | Serum for Jurkat Cells |
Hyclone Pennecillin Streptomycin solution | GE Healthcare Life sciences | SV30010 | Penn/Strep used in both media |
T75 Flasks | Corning | 3073 | Used for Culture of Jurkat Cells |
10cm Tissue Culture Plates | Corning | 430167 | Used for Culture of 293 Cells |
96 Well Plates (tissue culture Treated) | Corning | 3595 | Used for fusion assay |
Polyjet Transfection Reagent | Signagen | SL100688 | Used to transfect 293 Cells |
Dulbecco's Phosphate Buffered Saline | Sigma Aldrich | D8537-100ML | Used in wash steps |
Hyclone Trypsin Protease | GE Healthcare Life sciences | SH30042.01 | For Trypsinization of 293 cells |
Amaxa Cell Line Nucleofector Kit V | Lonza | VACA-1003 | For Nucleofection of Jurkats |
Ficoll-Paque plus | GE Healthcare Life sciences | 17144002 | For Nucleofection of Jurkats |
Serological Pipettes | Fisher Brand | 13-678-11E | For all tissue culture |
Pipettor Tips | Denville Scientific | P3020-CPS | For all tissue culture and liquid handling steps |
Millex Syringe Filter (0.45 micron) | Millipore | SLHA033 | For filtration of virus |
BD Slip Tip Sterile syringes | BD Diagnostics | 309656 | For filtration of virus |
Amaxa Nucleofector | Lonza | 2b | for Nucleofection of Jurkats (various models available) |
BD Fortessa Flow Cytometer | BD Biosciences | for flow cytometry analyss of samples | |
Tissue Culture Hood | Various models | Fortessa 2 | |
pMSCV-loxp-dsRed-loxp-eGFP-Puro-W PRE | Addgene | 32702 | Koo BK et al. Controlled gene expression in primary Lgr5 organoid cultures. Nature Methods 9:81-83 (2011). |
pCL10a1 | Novus Bio | NBP2-29542 | Naviaux, RK, Costanzi, E, Haas, M and Verma, I. The pCL vector system: Rapid production of helper-free, high titer, recombinant retroviruses. Journal of Virology 70: 5701-5705 (1996) |
Gag-iCre | Benjamin Chen Lab | Esposito AM et al. A high throughput Cre-lox activated viral membrane fusion assay identifies pharmacological inhibitors of HIV entry. Virology 490:6-16 (2016). |