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HIV-1 TAR miRNA protects against apoptosis by altering cellular gene expression.

Klase Z, Winograd R, Davis J, Carpio L, Hildreth R, Heydarian M, Fu S, McCaffrey T, Meiri E, Ayash-Rashkovsky M, Gilad S, Bentwich Z, Kashanchi F - Retrovirology (2009)

Bottom Line: Specifically, the microRNA down-regulates ERCC1 and IER3, protecting the cell from apoptosis.Comparison to our cloned sequence reveals possible target sites for the TAR miRNA as well.The TAR microRNA is expressed in all stages of the viral life cycle, can be detected in latently infected cells, and represents a mechanism wherein the virus extends the life of the infected cell for the purpose of increasing viral replication.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Department of Microbiology, Immunology and Tropical Medicine program, The George Washington University School of Medicine, Washington, District of Columbia 20037, USA. bcmzak@gwumc.edu

ABSTRACT

Background: RNA interference is a gene regulatory mechanism that employs small RNA molecules such as microRNA. Previous work has shown that HIV-1 produces TAR viral microRNA. Here we describe the effects of the HIV-1 TAR derived microRNA on cellular gene expression.

Results: Using a variation of standard techniques we have cloned and sequenced both the 5' and 3' arms of the TAR miRNA. We show that expression of the TAR microRNA protects infected cells from apoptosis and acts by down-regulating cellular genes involved in apoptosis. Specifically, the microRNA down-regulates ERCC1 and IER3, protecting the cell from apoptosis. Comparison to our cloned sequence reveals possible target sites for the TAR miRNA as well.

Conclusion: The TAR microRNA is expressed in all stages of the viral life cycle, can be detected in latently infected cells, and represents a mechanism wherein the virus extends the life of the infected cell for the purpose of increasing viral replication.

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Related in: MedlinePlus

Transfection of TAR miRNA into 293T cells has an anti-apoptotic effect. (A) 293T cells were transfected with TAR-D control or TAR-WT RNA. Twenty-four hours post transfection the media was replaced with DMEM with 0.1% FBS. Cells were sampled at Zero and 48 hours post serum starvation, stained for cell cycle analysis using propidium iodide (PI), and analyzed by flow cytometry. (B) HeLaT4 cells were transfected TAR-D or TAR-WT RNA. Twenty-four hours post transfection the media was replaced with DMEM with 0.1% FBS. Cells were sampled at 96 hours, and apoptosis was determined via AnnexinV and PI co-staining. Data are representative of three experiments.
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Figure 2: Transfection of TAR miRNA into 293T cells has an anti-apoptotic effect. (A) 293T cells were transfected with TAR-D control or TAR-WT RNA. Twenty-four hours post transfection the media was replaced with DMEM with 0.1% FBS. Cells were sampled at Zero and 48 hours post serum starvation, stained for cell cycle analysis using propidium iodide (PI), and analyzed by flow cytometry. (B) HeLaT4 cells were transfected TAR-D or TAR-WT RNA. Twenty-four hours post transfection the media was replaced with DMEM with 0.1% FBS. Cells were sampled at 96 hours, and apoptosis was determined via AnnexinV and PI co-staining. Data are representative of three experiments.

Mentions: The flow cytometric breakdown indicated that the TAR miRNA had an effect on cell-cycle and survival when under stress. Serum starvation of TAR-D transfected cells led to an arrest in the G1 phase of the cell cycle by 24 hours (86.8% as compared to 46.7% in the cells with full serum). By 48 hours, nearly all the cells were in a sub-G1 peak indicative of possible apoptosis. Whereas the cells without the miRNA showed high levels of apoptosis after 48 hours of serum starvation (70%), the 293T cells with the TAR miRNA showed alterations in cell cycle but were not nearly as apoptotic (no significant change in apoptosis after 48 hours) (Fig. 2A). Interestingly, the TAR-WT containing cells not only survived, but continued to progress through the cell cycle as evidenced by the presence of cells in the S and G2/M phases. Although at 24 hours of serum starvation TAR-WT containing cells did start to accumulate in the G1 phase (66.6% as compared to 44.3%) this did not lead to cell death, and at 48 hours cells were observable in all phases of the cell cycle. The increase in cells in the S-phase as compared to cells with serum suggests that the cells are replicating more slowly. Indeed, TAR-WT transfected cells appear to have a greater portion of cells in the S phase than the control cells even in full serum (compare untreated TAR-D to TAR-WT). The induction of apoptosis was verified using Annexin V staining (2B). HeLa cells were transfected with TAR-D or TAR-WT RNA and serum starved for 96 hours. The increase in Annexin V positive, PI negative cells after serum starvation of the TAR-D transfected cells indicates apoptosis (2.0% to 9.9%). Apoptosis in serum starved, TAR-WT treated HeLa cells was not as high (4.5%). These results suggest that the TAR miRNA is able to decrease levels of apoptosis in stressed cells. To investigate this phenotype in another stress-context, we again used 293T cells transfected with either the TAR-D or TAR-WT, but this time we treated the cells with the DNA crosslinking agent Mitomycin C. Upon analysis by flow cytometry, we observed the same trend as when the cells were deprived of serum; the cells containing the miRNA were more resistant to apoptosis (No increase in the level of apoptosis) compared to the control transfection (TAR-D containing cells experienced over 40 fold increase in apoptosis) (Data not shown). These data indicate that the TAR miRNA has the ability to protect cells from stress-induced cell death.


HIV-1 TAR miRNA protects against apoptosis by altering cellular gene expression.

Klase Z, Winograd R, Davis J, Carpio L, Hildreth R, Heydarian M, Fu S, McCaffrey T, Meiri E, Ayash-Rashkovsky M, Gilad S, Bentwich Z, Kashanchi F - Retrovirology (2009)

Transfection of TAR miRNA into 293T cells has an anti-apoptotic effect. (A) 293T cells were transfected with TAR-D control or TAR-WT RNA. Twenty-four hours post transfection the media was replaced with DMEM with 0.1% FBS. Cells were sampled at Zero and 48 hours post serum starvation, stained for cell cycle analysis using propidium iodide (PI), and analyzed by flow cytometry. (B) HeLaT4 cells were transfected TAR-D or TAR-WT RNA. Twenty-four hours post transfection the media was replaced with DMEM with 0.1% FBS. Cells were sampled at 96 hours, and apoptosis was determined via AnnexinV and PI co-staining. Data are representative of three experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2654423&req=5

Figure 2: Transfection of TAR miRNA into 293T cells has an anti-apoptotic effect. (A) 293T cells were transfected with TAR-D control or TAR-WT RNA. Twenty-four hours post transfection the media was replaced with DMEM with 0.1% FBS. Cells were sampled at Zero and 48 hours post serum starvation, stained for cell cycle analysis using propidium iodide (PI), and analyzed by flow cytometry. (B) HeLaT4 cells were transfected TAR-D or TAR-WT RNA. Twenty-four hours post transfection the media was replaced with DMEM with 0.1% FBS. Cells were sampled at 96 hours, and apoptosis was determined via AnnexinV and PI co-staining. Data are representative of three experiments.
Mentions: The flow cytometric breakdown indicated that the TAR miRNA had an effect on cell-cycle and survival when under stress. Serum starvation of TAR-D transfected cells led to an arrest in the G1 phase of the cell cycle by 24 hours (86.8% as compared to 46.7% in the cells with full serum). By 48 hours, nearly all the cells were in a sub-G1 peak indicative of possible apoptosis. Whereas the cells without the miRNA showed high levels of apoptosis after 48 hours of serum starvation (70%), the 293T cells with the TAR miRNA showed alterations in cell cycle but were not nearly as apoptotic (no significant change in apoptosis after 48 hours) (Fig. 2A). Interestingly, the TAR-WT containing cells not only survived, but continued to progress through the cell cycle as evidenced by the presence of cells in the S and G2/M phases. Although at 24 hours of serum starvation TAR-WT containing cells did start to accumulate in the G1 phase (66.6% as compared to 44.3%) this did not lead to cell death, and at 48 hours cells were observable in all phases of the cell cycle. The increase in cells in the S-phase as compared to cells with serum suggests that the cells are replicating more slowly. Indeed, TAR-WT transfected cells appear to have a greater portion of cells in the S phase than the control cells even in full serum (compare untreated TAR-D to TAR-WT). The induction of apoptosis was verified using Annexin V staining (2B). HeLa cells were transfected with TAR-D or TAR-WT RNA and serum starved for 96 hours. The increase in Annexin V positive, PI negative cells after serum starvation of the TAR-D transfected cells indicates apoptosis (2.0% to 9.9%). Apoptosis in serum starved, TAR-WT treated HeLa cells was not as high (4.5%). These results suggest that the TAR miRNA is able to decrease levels of apoptosis in stressed cells. To investigate this phenotype in another stress-context, we again used 293T cells transfected with either the TAR-D or TAR-WT, but this time we treated the cells with the DNA crosslinking agent Mitomycin C. Upon analysis by flow cytometry, we observed the same trend as when the cells were deprived of serum; the cells containing the miRNA were more resistant to apoptosis (No increase in the level of apoptosis) compared to the control transfection (TAR-D containing cells experienced over 40 fold increase in apoptosis) (Data not shown). These data indicate that the TAR miRNA has the ability to protect cells from stress-induced cell death.

Bottom Line: Specifically, the microRNA down-regulates ERCC1 and IER3, protecting the cell from apoptosis.Comparison to our cloned sequence reveals possible target sites for the TAR miRNA as well.The TAR microRNA is expressed in all stages of the viral life cycle, can be detected in latently infected cells, and represents a mechanism wherein the virus extends the life of the infected cell for the purpose of increasing viral replication.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Department of Microbiology, Immunology and Tropical Medicine program, The George Washington University School of Medicine, Washington, District of Columbia 20037, USA. bcmzak@gwumc.edu

ABSTRACT

Background: RNA interference is a gene regulatory mechanism that employs small RNA molecules such as microRNA. Previous work has shown that HIV-1 produces TAR viral microRNA. Here we describe the effects of the HIV-1 TAR derived microRNA on cellular gene expression.

Results: Using a variation of standard techniques we have cloned and sequenced both the 5' and 3' arms of the TAR miRNA. We show that expression of the TAR microRNA protects infected cells from apoptosis and acts by down-regulating cellular genes involved in apoptosis. Specifically, the microRNA down-regulates ERCC1 and IER3, protecting the cell from apoptosis. Comparison to our cloned sequence reveals possible target sites for the TAR miRNA as well.

Conclusion: The TAR microRNA is expressed in all stages of the viral life cycle, can be detected in latently infected cells, and represents a mechanism wherein the virus extends the life of the infected cell for the purpose of increasing viral replication.

Show MeSH
Related in: MedlinePlus