Described here is an established method to determine the extent of HIV-1 restriction by the cellular inhibitory protein SAMHD1. Human myeloid lineage U937 cells are transduced with a SAMHD1 expression vector co-expressing YFP, differentiated and then challenged with HIV-RFP. The level of restriction is determined by flow cytometry analysis.
Sterile α-motif/histidine-aspartate domain-containing protein 1 (SAMHD1) inhibits replication of HIV-1 in quiescent myeloid cells. U937 cells are widely used as a convenient cell system for analyzing SAMHD1 activity due to a low level of SAMHD1 RNA expression, leading to undetectable endogenous protein expression. Based on similar assays developed in the Stoye laboratory to characterize other retroviral restriction factors, the Bishop lab developed a two-color restriction assay to analyze SAMHD1 in U937 cells. Murine Leukaemia Virus-like particles expressing SAMHD1, alongside YFP expressed from an IRES, are used to transduce U937 cells. Cells are then treated with phorbol myristate acetate to induce differentiation to a quiescent phenotype. Following differentiation, cells are infected with HIV-1 virus-like particles expressing a fluorescent reporter. After 48 h, cells are harvested and analyzed by flow cytometry. The proportion of HIV-infected cells in the SAMHD1-expressing population is compared to that in internal control cells lacking SAMHD1. This comparison reveals a restriction ratio. SAMHD1 expression leads to a five-fold reduction in HIV infection, corresponding to a restriction ratio of 0.2. Our recent substitution of RFP for the original GFP as the reporter gene for HIV infection has facilitated flow cytometry analysis.
This assay has been successfully used to characterize the effect of amino acid substitutions on SAMHD1 restriction by transducing with viruses encoding altered SAMHD1 proteins, derived from site-directed mutagenesis of the expression vector. For example, the catalytic site substitutions HD206-7AA show a restriction phenotype of 1, indicating a loss of restriction activity. Equally, the susceptibility of different tester viruses can be determined. The assay can be further adapted to incorporate the effect of differentiation status, metabolic status, and SAMHD1 modifiers to better understand the relationship between SAMHD1, cell metabolic state, and viral restriction.
Sterile α-motif/histidine-aspartate domain-containing protein 1 (SAMHD1) impedes replication of Human Immunodeficiency Virus type 1 (HIV-1) in quiescent cells of the myeloid lineage. SAMHD1 blocks replication through its enzymatic activity as a dNTP triphosphohydrolase, which results in decreased levels of intracellular dNTPs. As a result, HIV-1 cannot perform the process of reverse transcription efficiently. Much progress has been made in the few years since this initial observation, particularly regarding the contribution of specific domains and amino acids to SAMHD1's antiviral activity. These insights have been made using biochemical assays as well as cell systems mimicking the physiologically relevant quiescent myeloid environment. U937 cells1 are widely used as a convenient myeloid cell system for analyzing SAMHD1 activity. This is due to a lack of endogenous SAMHD1 expression, thought to be due to low RNA levels compared to SAMHD1-expressing cells (an area of ongoing research). Here, the protocol describes the transient transduction of U937 cells with virus-like particles co-expressing SAMHD1 and a yellow fluorescent protein (YFP) reporter to examine the mechanism of SAMHD1 restriction of HIV-1. Such transient two-color flow cytometry assays to examine retroviral restrictions were first developed in the Stoye laboratory2 and have since been adapted to investigate other restriction factors, including the tripartite motif (TRIM) proteins3. Inspired by these assays, the Bishop lab developed a two-color assay to analyze SAMHD1 restriction in U937 cells.
The workflow for the experiment is shown in Figure 1. Murine Leukaemia Virus (MLV)-like particles containing a bi-cistronic message encoding SAMHD1 alongside YFP expressed from an IRES are used to transduce U937 cells. Cells are then treated with phorbol myristate acetate (PMA) to induce differentiation to a quiescent phenotype. Cells are next infected with HIV-1 virus-like particles expressing red fluorescent protein (RFP). After 48 h, the cells are harvested and analyzed by two-color flow cytometry. This assay is also used with YFP and green fluorescent protein (GFP), requiring less standard filter combinations for flow cytometry analysis. Use of HIV-RFP allows for more straightforward compensation and thus the assay is more easily accessible to less experienced flow cytometry users, and achievable with most cytometers.
During analysis, the proportion of HIV infected cells is compared between SAMHD1-expressing cells and those lacking SAMHD1 within the same well of cells. This affords an internal control in well/flow cytometry tube, which is a key feature. The comparison of infection levels in transduced and untransduced cells reveals a restriction ratio. A ratio of 1.0 indicates that the transduced factor has no effect on infectivity. Wild type SAMHD1 expression leads to a five-fold reduction in HIV infection in this assay, corresponding to a restriction ratio of 0.2. While this effect is modest in comparison to more classic restriction factors such as TRIM5, the effect is nonetheless reproducible and allows the classification of modified SAMHD1 expressors into those that restrict in an equivalent manner to the wild-type protein, those that fail to restrict, and those with an intermediate phenotype.
This assay has been used successfully to characterize SAMHD1 domain and amino acid mutants' restriction phenotypes by transducing with viruses encoding mutant SAMHD1 sequences. For example, the catalytic site substitutions HD206-7AA fail to restrict in this assay. Equally, the susceptibility of different tester viruses can be determined. For example, HIV-1 reverse transcriptase mutants are more susceptible to SAMHD1-mediated restriction (e.g., 151V4). This protocol outlines the details of virus-like particle (VLP) production, transduction of U937 with SAMHD1 expression vectors, infection with HIV carrying a fluorescent reporter and the subsequent analysis by flow cytometry. This article discusses what data to expect and how to avoid suboptimal results. Finally, alternative uses for the assay are outlined, in addition to examining different SAMHD1 variants to understand the interplay between SAMHD1, dNTP levels and viral infection more thoroughly. Given the role of SAMHD1 at the center of cell metabolism, with additional links to cancer, this continues to be an area of intense interest.
This protocol does not contain any studies involving animals or human participants performed by any of the authors. All the steps of this protocol were performed following the guidelines and codes of practice of the institutions where they were carried out.
1. Transfection of 293T cells for VLP production (both SAMHD1-expression vectors and tester HIV particles)
NOTE: The most significant contributors to the successful production of virus particles are the health of the producer 293T cells, confluency at transfection and time of harvest. Laboratories will typically have their preferred transfection reagents and protocol for retroviral particle production. The following protocol yields good quality infectious particles, but equivalent protocols would also be suitable for this application. To maintain good quality 293T cells, passage at least three times per week to maintain a constant growth rate. Cells should be seeded in the log phase of growth for efficient transfection.
2. Titering new VLP prior to use
3. Transduction of U937 with VLP expressing SAMHD1 and YFP
NOTE: Steps 3-6 constitute the restriction experiment. Days are numbered for ease of planning.
4. Differentiation of transduced U937
5. Infection of differentiated, SAMHD1-expressing U937 with HIV-RFP
6. Flow cytometry analysis
NOTE: It is good practice to include a live-dead stain in flow cytometry pipelines. However, if the U937 cells are of good quality, this step can be omitted without negative consequences for downstream analysis. Gentle pipetting of trypsinized cells should easily result in a single cell suspension.
7. Flow cytometry analysis
8. Data analysis
NOTE: The steps that follow correspond to analysis in FlowJo v10; however, equivalent steps can easily be performed in other analysis software.
The results of the analysis above should yield a restriction ratio of 0.25 or lower for the wild-type SAMHD1 positive control, and 1.0 for the negative control. If these two quality control checks are valid, then consider the statistical significance of the results. SAMHD1 variants that show no significant difference from wild type therefore carry substitutions which do not impact SAMHD1 restriction in this context. Those significantly different from wild type show impaired restriction. If these are not significantly different from the negative control, then they lack the ability to restrict in this context (Figure 4 left panels).
If the wild-type SAMHD1 restriction value is greater than 0.3, then results for tester viruses may be indicative but cannot be relied upon. Ineffective restriction by wild-type protein may result from using U937 cells too early post-recovery from reconstitution (within 2 weeks), or when they are too old (>2-3 months). These parameters may need to be empirically determined for a given cell stock. Typically, the lower the passage, the more reliably the cells differentiate and therefore provide the appropriate environment for SAMHD1 restriction. Inappropriate infection level with either SAMHD1-YFP or HIV-RFP may also result in difficulties with both compensation and downstream determination of restriction ratio. An example is illustrated in the right-hand panels of Figure 4.
If the negative control deviates from 1.0 (outside the range of 0.9-1.2), this may indicate a problem with the analysis, either the proportion of infected cells, gating strategy, or health of the cells are affecting the assay. Refer to NOTES above.
Figure 1: Schematic outlining protocol. VLP, virus-like particles, PMA, phorbol myristate acetate. Numbered stages correspond to stages in protocol. Figure produced using BioRender.com. Please click here to view a larger version of this figure.
Figure 2: Schematic of retroviral plasmids. (A) Packaging vectors (B) Transfer vectors (C) VSV-G envelope expressor. Key coding and regulatory elements are shown. For details refer to the Table of Materials. CMV IE: cytomegalovirus immediate-early promoter, BGH: bovine growth hormone, pA: polyA, RRE: Rev Response Element, CMV-LTR: composite CMV-HIV-1 LTR promoter, Psi: HIV-1 packaging signal, cPPT/CTS: central polypurine tract/central termination sequence, SV40: simian vacuolating virus 40. Figure produced using BioRender.com. Please click here to view a larger version of this figure.
Figure 3: Gating strategy for flow cytometry analysis. (A) Compensation controls: left panel shows FSC-A/SSC-A gating on in-tact cells for the untransduced, uninfected control. Central and right panels show screenshots of single-color compensation controls for YFP (mid) and RFP (right) with uncompensated data in black and compensated in blue. (B) Corresponding compensation matrix and plots for compensation controls above. (C) Gating strategy. Debris is eliminated through analysis of all cells by FSC-A/SSC-A (left panel). Doublets are excluded by gating on FSC height versus area (central panel). Right panel shows an example of HIV-1 restriction by wild-type SAMHD1. Axes show compensated blue and yellow laser fluorescence corresponding to YFP (SAMHD1) and RFP (HIV)-positive cells respectively. Quadrant gates are drawn though comparison of negative and single-color controls for RFP and YFP. Numbers indicate percentage of parent population. Compensation values for YFP with GFP will be much higher but possible to discriminate using appropriate filter sets. Please click here to view a larger version of this figure.
Figure 4: Expected results: ideal versus suboptimal data. (A) Representative YFP/RFP plots for optimal (left) and suboptimal (right) data. Numbers indicate percentage of parent population. In the right panel, HIV infection is too low creating difficulties with compensation and gating. Restriction ratio is higher than expected at 0.5. (B) Plots of restriction ratio for variants of SAMHD1 with respect to wild type (WT, red) or the negative control (HD206-7AA, black) generated using statistical software. Each point represents a replicate value. Mean and standard deviation are shown. Paired t-tests between each group were significant in all cases expect where shown. Left: Ideal data - WT shows the expected restriction of approximately 0.2, negative shows 1.0. The variant R372D (gray) is significantly different from WT but not significant from the negative control and thus has lost the ability to restrict. Right: Suboptimal data. Here, the negative behaves as expected but in all six replicates the WT only shows a restriction ratio of 0.5, due to low infection rate. The low variance within the groups means R143H shows an intermediate phenotype statistically different from WT and the negative, while G209S does not restrict; however, this should be repeated with fresh cells as the positive control has not given the expected restriction ratio. Please click here to view a larger version of this figure.
As discussed in the notes above, the critical aspects of the protocol center around retaining the correct cell phenotype for VLP production and for creating the appropriate environment for observing SAMHD1 restriction. Firstly, the accurate analysis of restriction by two-color flow cytometry relies on achieving appropriate proportions of transduced and infected cells so that there are enough cells in each area of the plot for analysis. As such the researcher should be aiming for an MOI of less than 1 (see Protocol). Transduction and infection rates that are either too high or too low lead to problems with analysis due to lack of uninfected or doubly infected cells. Equally, a similar transduction rate for SAMHD1 vectors to be compared is key to allowing the same compensation matrix and gating to be applied to the whole experiment, increasing confidence in the subsequent analysis.
Secondly, the age of the U937 cells used is a key factor in whether they differentiate successfully. Successful differentiation can be monitored through observation of adherence, a reduced acidification of the media over time following differentiation and adequate restriction by the wild type positive control in the assay. Differentiation of up to 5 days has been successful.
One of the key advantages to this assay is its flexibility. A variety of modified versions of SAMHD1 (domains, amino acids, species sequences) can be tested via simple site-directed mutagenesis of the vector or cloning. Equally, variants of the tester virus can be explored. The assay has been used previously to demonstrate that HIV-1 reverse transcriptase mutants with reduced capacity to bind dNTP are more sensitive to SAMHD1 restriction. The same principle can be used to test the restriction of other viruses by SAMHD1. Furthermore, varying differentiation conditions or artificial manipulation of intracellular dNTP concentrations can be used to further probe the interaction between intracellular conditions and SAMHD1 activity, an area of active research in the Bishop lab. The interaction of SAMHD1 with various nucleoside reverse transcriptase inhibitors has also been described, and the effect of SAMHD1 shown using this assay modified for use in primary cells12,13,14,15.
Adaptation of the protocol for use in primary cells overcomes the limitation posed by examining metabolic phenotypes in transformed cells. However, the existing format allows for a convenient and less technically challenging protocol for high throughput analysis, especially with use of a flow cytometer with a high-throughput sampler. When adapting the protocol, the susceptibility of the internally untransduced cells to bystander effects (e.g., immune signaling) should be considered.
As with many aspects of cell biology, it is essential that such assays are used as part of a holistic approach for understanding a given phenomenon. The parallel use of biochemical assays for SAMHD1 activity16, quantification of intracellular dNTP pools, and observation of SAMHD1 mutant phenotypes in humans, ex vivo and in animal models (being carried out by labs around the world, some described in this issue) are key for the evolving understanding of this complex protein.
This work was supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001042 and FC001162), the UK Medical Research Council (FC001042 and FC001162), and the Wellcome trust (FC001042 and FC001162) and by a Wellcome Trust Investigator Award to JPS (108012/Z/15/Z).Work at the University of Cambridge was co-funded by the NIHR Cambridge Biomedical Research Centre, The Evelyn Trust (16/21), The Rosetrees Trust (M590), and the UK Medical Research Council (MR/S009752/1). The latter UK funded award is part of the EDCTP2 programme supported by the European Union.
Name | Company | Catalog Number | Comments |
293T cells | ATCC | CRL-3216 | |
0.45 µm syringe filters | Sartorius | FCT122 | |
10 cm dishes | Corning | 430167 | virus preps can be scaled up through transfection in higher capacity plates/flasks - see transfection reagent guidance |
12 well plates | Greiner | 665180 | |
24 well plates | Greiner | 662160 | |
BD LSRFortessa cell analyzer | BD Bioscience | 649225 | alternative analysers can be used as long as they can read RFP and YFP fluorescence |
DMEM | Sigma | D6429 | ATCC recommends Dulbecco's Modified Eagle's Medium (DMEM) is modified to contain 4 mM L-glutamine, 4500 mg/L glucose, 1 mM sodium pyruvate, and 1500 mg/L sodium bicarbonate. |
DMSO | Fisher | BP-231-100 | |
fetal bovine serum | Gibco | 10500064 | |
Flowjo | BD Bioscience | - | alternative analysis software can be used |
light microscope | - | - | Any bright field light microscope |
p8.91 | - | - | HIV GagPol expressor (PMID: 8602510) |
paraformaldehyde (4 % in PBS) | Alfa Aesar | J61889 | |
PBS | Sigma | D8537 | |
Penicillin-Streptomycin (10,000 U/mL) | ThermoFisher | 15140122 | |
phorbol 12-myristate 13-acetate (PMA) | Sigma | P8139 | |
polybrene | Sigma | TR-1003 | |
pKB4 | - | - | Murine leukaemia Virus GagPol expressor (PMID: 23035841) |
pLGatewaySAMHD1eYFP | - | - | MLV packaging plasmid encoding wild-type human sequence. Details and cloning procedures Arnold et al 2015 (Ref 1). |
pLGatewaySAMHD1HD206-7AAeY FP | - | - | As above, encodes amino acid substitutions at crucial active sites residues |
Prism | Graphpad | - | https://www.graphpad.com/scientific-software/prism/ Other data representation software are also suitable |
pVSV-G | - | - | VSV-G expressor (https://www.addgene.org/138479/), alternative: pMD2.G (https://www.addgene.org/12259/) |
RPMI | ThermoFisher | 21875034 | |
screw-cap tubes | StarLab | E1415-2231 | |
SCRPSY | - | - | HIV packaging plasmid encoding RFP, Sam Wilson, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5026698/ |
stripettes 10 mL | Corning | 10084450 | |
stripettes 5 mL | Corning | 10420201 | |
TrypLE express | Gibco | 12604021 | Trypsin will also be suitable |
Turbofect | ThermoFisher | R0531 | alternative transfection reagents will also work well |
U937 cells | ATCC | CRL-1593.2 |
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