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Updated: Oct 31, 2025
Analysis of SAMHD1 Restriction by Flow Cytometry in Human Myeloid U937 Cells
Published on: June 13, 2021
Analysis of SAMHD1 Restriction by Flow Cytometry in Human Myeloid U937 Cells
Paula Ordonez1, Kate N Bishop2, Jonathan P Stoye3
1Retrovirus-Host Interactions Laboratory, The Francis Crick Institute.
Abstract:
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.
Insights
Sterile alpha-motif/histidine-aspartate domain-containing protein 1 (SAMHD1) significantly restricts HIV-1 replication in myeloid cells. This study developed a robust assay to quantify SAMHD1
Area of Science:
- Virology
- Cell Biology
- Molecular Biology
Background:
- Sterile alpha-motif/histidine-aspartate domain-containing protein 1 (SAMHD1) is a key innate immune factor that restricts retroviral replication.
- Quiescent myeloid cells are a critical target for HIV-1, and SAMHD1's role in these cells is of significant interest.
- U937 cells offer a tractable system for studying SAMHD1 due to low endogenous expression, enabling precise analysis of its function.
Purpose of the Study:
- To establish and validate a quantitative assay for measuring SAMHD1's antiviral activity against HIV-1 in myeloid cells.
- To characterize the restriction efficiency of SAMHD1 using a novel two-color flow cytometry-based assay.
- To demonstrate the assay's utility in evaluating the impact of genetic modifications on SAMHD1's restriction capabilities.
Main Methods:
- Development of a two-color restriction assay using Murine Leukaemia Virus-like particles to express SAMHD1 and YFP in U937 cells.
- Induction of a quiescent phenotype in U937 cells via phorbol myristate acetate treatment.
- Infection with HIV-1 virus-like particles encoding a fluorescent reporter, followed by flow cytometry analysis to determine the restriction ratio.
Main Results:
- SAMHD1 expression resulted in a five-fold reduction in HIV-1 infection, yielding a restriction ratio of 0.2.
- The assay successfully identified loss-of-function mutations, such as the catalytic site substitution HD206-7AA, which showed a restriction phenotype of 1.
- The assay demonstrated flexibility in assessing different reporter viruses and can be adapted to study modulators of SAMHD1 activity.
Conclusions:
- The developed two-color assay provides a sensitive and quantitative method for evaluating SAMHD1's antiviral activity against HIV-1.
- The assay is effective in characterizing the functional impact of SAMHD1 mutations on viral restriction.
- This platform facilitates further investigation into the interplay between SAMHD1, cellular metabolic states, and viral restriction mechanisms.
