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Yeast As a Chassis for Developing Functional Assays to Study Human P53
Published on: August 4, 2019
Inhibition of HIV early replication by the p53 and its downstream gene p21
Binshan Shi1, Hamayun J Sharifi2, Sara DiGrigoli2
1Department of Basic and Clinical Sciences, Albany College of Pharmacy and Health Sciences, 106 New Scotland Ave, Albany, NY, 12208, USA. Binshan.shi@acphs.edu.
Background:
The tumor suppressor gene p53 has been found to suppress HIV infection by various mechanisms, but the inhibition of HIV at an early stage of replication by host cell p53 and its downstream gene p21 has not been well studied.
Method:
VSV-G pseudotyped HIV-1 or HIV-2 viruses with GFP or luciferase reporter gene were used to infect HCT116 p53+/+ cells, HCT116 p53-/- cells and hMDMs. The infections were detected by flow cytometry or measured by luciferase assay. Reverse transcription products were quantified by a TaqMan real time PCR. siRNA knockdown experiments were applied to study potential roles of p53 and p21 genes in their restriction to HIV infection. Western blot experiments were used to analyze changes in gene expression.
Results:
The infection of HIV-1 was inhibited in HCT116 p53+/+ cells in comparison to HCT116 p53-/- cells. The fold of inhibition was largely increased when cell cycle switched from cycling to non-cycling status. Further analysis showed that both p53 and p21 expressions were upregulated in non-cycling HCT116 p53+/+ cells and HIV-1 reverse transcription was subsequently inhibited. siRNA knockdown of either p53 or p21 rescued HIV-1 reverse transcription from the inhibition in non-cycling HCT116 p53+/+ cells. It was identified that the observed restrictions by p53 and p21 were associated with the suppression of RNR2 expression and phosphorylation of SAMHD1. These observations were confirmed by using siRNA knockdown experiments. In addition, p53 also inhibited HIV-2 infection in HCT116 p53+/+ cells and siRNA knockdown of p21 increased HIV-2 infection in hMDMs. Finally the expressions of p53 and p21 were found to be induced in hMDMs shortly after HIV-1 infection.
Conclusions:
The p53 and its downstream gene p21 interfere with HIV early stage of replication in non-cycling cells and hMDMs.
Insights
The tumor suppressor p53 and its gene p21 inhibit early HIV replication in non-cycling cells and human myeloid cells. This study reveals a novel mechanism of HIV restriction by host cell factors.
Area of Science:
- Virology
- Molecular Biology
- Immunology
Background:
- The tumor suppressor gene p53 is known to inhibit HIV infection through various mechanisms.
- However, the specific role of host cell p53 and its downstream gene p21 in restricting early-stage HIV replication remains understudied.
Purpose of the Study:
- To investigate the inhibition of early-stage HIV-1 and HIV-2 replication by host cell p53 and p21.
- To elucidate the molecular mechanisms underlying this restriction, particularly in non-cycling cells and human myeloid cells (hMDMs).
Main Methods:
- Utilized VSV-G pseudotyped HIV-1/HIV-2 viruses with reporter genes to infect HCT116 p53(+/+) and p53(-/-) cells, and hMDMs.
- Quantified viral reverse transcription using real-time PCR and assessed infection via flow cytometry or luciferase assays.
- Employed siRNA knockdown of p53 and p21, alongside Western blot analysis, to determine gene function and expression changes.
Main Results:
- HIV-1 infection was significantly inhibited in HCT116 p53(+/+) cells compared to p53(-/-) cells, with enhanced inhibition in non-cycling cells.
- Upregulation of p53 and p21 in non-cycling cells correlated with inhibited HIV-1 reverse transcription, which was rescued by siRNA knockdown of p53 or p21.
- p53 and p21 mediated restriction through suppression of RNR2 and phosphorylation of SAMHD1, and also inhibited HIV-2 infection.
Conclusions:
- Host cell p53 and its downstream gene p21 play a crucial role in interfering with the early stages of HIV replication.
- This restriction is particularly effective in non-cycling cells and human myeloid cells (hMDMs).
- The findings identify a novel host-intrinsic defense mechanism against HIV infection involving the p53/p21 pathway.
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