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Isolation, Transfection, and Culture of Primary Human Monocytes
Published on: December 16, 2019
Stimulated trans-acting factor of 50 kDa (Staf50) inhibits HIV-1 replication in human monocyte-derived macrophages
Abdellatif Bouazzaoui1, Marina Kreutz, Veronika Eisert
1Georg-Speyer-Haus, Institute for Biomedical Research, 60596 Frankfurt/M., Germany.
Abstract:
In order to identify cellular genes which interfere with HIV-1 replication in monocyte-derived macrophages (MAC), cells were stimulated with interferon (IFN) or lipopolysaccharide (LPS) leading to a pronounced inhibition of HIV-1 infection in these cells, and the resulting gene expression was analyzed. Using the microarray technology we identified a gene named Stimulated Trans-Acting Factor of 50 kDa (Staf50), which is known to repress the activity of the HIV-1 LTR. Analysis of the Staf50 expression by real-time PCR showed an overexpression in IFNalpha (up to 20-fold) and LPS (up to 10-fold)-stimulated MAC as well as in infected cells (up to 3-fold). For stable overexpression, 293 T cells and primary macrophages were transduced with Staf50-IRES-GFP bicistronic pseudotype viruses. After transduction, 293 T CD4/CCR5 and MAC were infected with HIV-1, and virus replication was monitored by p24 ELISA. Overexpression of Staf50 inhibited the HIV-1 infection between 50% and 90% in 293 T CD4/CCR5 as well as in MAC. Our findings suggest that host genetic effects in combination with viral properties determine the susceptibility of an appropriate target cell for HIV-1 infection as well as the replication potential of the virus in the cell resulting in an overall productive infection.
Insights
Stimulated Trans-Acting Factor of 50 kDa (Staf50) gene overexpression inhibits HIV-1 replication in macrophages. This host factor plays a key role in controlling viral infection, suggesting potential therapeutic targets for HIV.
Area of Science:
- Virology
- Immunology
- Molecular Biology
Background:
- Monocyte-derived macrophages (MAC) are key targets for HIV-1 infection.
- Cellular genes can influence HIV-1 replication.
- Interferon (IFN) and lipopolysaccharide (LPS) stimulation inhibit HIV-1 in MAC.
Purpose of the Study:
- Identify cellular genes that interfere with HIV-1 replication in MAC.
- Investigate the role of Stimulated Trans-Acting Factor of 50 kDa (Staf50) in HIV-1 infection.
Main Methods:
- Microarray analysis to identify differentially expressed genes.
- Real-time PCR to quantify Staf50 expression.
- Viral transduction for stable Staf50 overexpression in 293 T and MAC cells.
- HIV-1 infection assays and p24 ELISA for viral replication monitoring.
Main Results:
- Staf50 was identified as a gene overexpressed in IFN- and LPS-stimulated MAC, and in HIV-1 infected cells.
- Stable overexpression of Staf50 significantly inhibited HIV-1 replication (50-90%) in both 293 T CD4/CCR5 cells and primary MAC.
- Staf50 represses the activity of the HIV-1 Long Terminal Repeat (LTR).
Conclusions:
- Host genetic factors, like Staf50, significantly impact HIV-1 replication.
- Staf50 overexpression demonstrates potent antiviral activity against HIV-1.
- Findings suggest Staf50 as a potential therapeutic target for controlling HIV-1 infection.
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