Analysis of SAMHD1 Restriction by Flow Cytometry in Human Myeloid U937 Cells

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.