Limits...
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.

Show MeSH

Related in: MedlinePlus

Anti-apoptotic effect validated through the repression of ERCC1. (A) HeLaT4 were transfected with TAR-D, TAR-WT, siEGFP, or siERCC1 RNA. Twenty-four hours after transfection media were replaced with low serum (0.1%) media, and the cells were cultured for 96 hours. Apoptosis was measured at 96 hours after serum starvation using FACs analysis. Data are representative of two experiments. (B) 293T cells were transfected with siRNA against ERCC1 (lanes 5–8) or mock (lanes 1–4) for twenty-four hours, and then the media was replaced with low serum media. Cells were harvested for Western blot analysis at 0, 4, 8 and 24 hours after serum starvation and Western blotted for phosphor-p53 Ser15, Mdm2, p21 and β-actin. Pictured Western blots utilized 20 micrograms of total protein per lane.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2654423&req=5

Figure 8: Anti-apoptotic effect validated through the repression of ERCC1. (A) HeLaT4 were transfected with TAR-D, TAR-WT, siEGFP, or siERCC1 RNA. Twenty-four hours after transfection media were replaced with low serum (0.1%) media, and the cells were cultured for 96 hours. Apoptosis was measured at 96 hours after serum starvation using FACs analysis. Data are representative of two experiments. (B) 293T cells were transfected with siRNA against ERCC1 (lanes 5–8) or mock (lanes 1–4) for twenty-four hours, and then the media was replaced with low serum media. Cells were harvested for Western blot analysis at 0, 4, 8 and 24 hours after serum starvation and Western blotted for phosphor-p53 Ser15, Mdm2, p21 and β-actin. Pictured Western blots utilized 20 micrograms of total protein per lane.

Mentions: ERCC1 was upregulated by viral infection in the absence of Dicer by 18 fold (Fig. 5B). This is in keeping with published reports that viral infections, including HIV-1, up-regulate the expression of DNA repair proteins. ERCC1 is involved in the recognition and repair of DNA damage. Indeed, previously published reports indicate that increased levels of ERCC1 correlate with resistance to DNA damage induced apoptosis [30-32]. Our findings suggest a novel role for ERCC1 in inducing apoptosis in response to serum starvation. To confirm the role of ERCC1 in protection from serum starvation induced apoptosis, siRNA was utilized. 293T cells were transfected with TAR-D, TAR-WT or siRNA against EGFP or ERCC1. Cells were serum starved for 48 hours, and the level of apoptosis was determined at 96 hours post serum starvation (Fig. 8A). Control transfection of TAR-D showed that 9.9% of the cells were apoptotic. siRNA against ERCC1 prevented the induction of apoptosis at 48 hours, comparable to the transfection with wild type TAR RNA. Transfection of 293T cells and cell cycle analysis confirmed these results (data not shown). These results suggested that in the setting of 293T cells, repression of ERCC1 expression inhibited apoptosis triggered by serum starvation. IER3 has previously been shown to be involved also in serum starvation induced apoptosis [26-29]. Together these data suggested that the TAR miRNA prevented apoptosis by down-regulating both ERCC1 and IER3.


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)

Anti-apoptotic effect validated through the repression of ERCC1. (A) HeLaT4 were transfected with TAR-D, TAR-WT, siEGFP, or siERCC1 RNA. Twenty-four hours after transfection media were replaced with low serum (0.1%) media, and the cells were cultured for 96 hours. Apoptosis was measured at 96 hours after serum starvation using FACs analysis. Data are representative of two experiments. (B) 293T cells were transfected with siRNA against ERCC1 (lanes 5–8) or mock (lanes 1–4) for twenty-four hours, and then the media was replaced with low serum media. Cells were harvested for Western blot analysis at 0, 4, 8 and 24 hours after serum starvation and Western blotted for phosphor-p53 Ser15, Mdm2, p21 and β-actin. Pictured Western blots utilized 20 micrograms of total protein per lane.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 8: Anti-apoptotic effect validated through the repression of ERCC1. (A) HeLaT4 were transfected with TAR-D, TAR-WT, siEGFP, or siERCC1 RNA. Twenty-four hours after transfection media were replaced with low serum (0.1%) media, and the cells were cultured for 96 hours. Apoptosis was measured at 96 hours after serum starvation using FACs analysis. Data are representative of two experiments. (B) 293T cells were transfected with siRNA against ERCC1 (lanes 5–8) or mock (lanes 1–4) for twenty-four hours, and then the media was replaced with low serum media. Cells were harvested for Western blot analysis at 0, 4, 8 and 24 hours after serum starvation and Western blotted for phosphor-p53 Ser15, Mdm2, p21 and β-actin. Pictured Western blots utilized 20 micrograms of total protein per lane.
Mentions: ERCC1 was upregulated by viral infection in the absence of Dicer by 18 fold (Fig. 5B). This is in keeping with published reports that viral infections, including HIV-1, up-regulate the expression of DNA repair proteins. ERCC1 is involved in the recognition and repair of DNA damage. Indeed, previously published reports indicate that increased levels of ERCC1 correlate with resistance to DNA damage induced apoptosis [30-32]. Our findings suggest a novel role for ERCC1 in inducing apoptosis in response to serum starvation. To confirm the role of ERCC1 in protection from serum starvation induced apoptosis, siRNA was utilized. 293T cells were transfected with TAR-D, TAR-WT or siRNA against EGFP or ERCC1. Cells were serum starved for 48 hours, and the level of apoptosis was determined at 96 hours post serum starvation (Fig. 8A). Control transfection of TAR-D showed that 9.9% of the cells were apoptotic. siRNA against ERCC1 prevented the induction of apoptosis at 48 hours, comparable to the transfection with wild type TAR RNA. Transfection of 293T cells and cell cycle analysis confirmed these results (data not shown). These results suggested that in the setting of 293T cells, repression of ERCC1 expression inhibited apoptosis triggered by serum starvation. IER3 has previously been shown to be involved also in serum starvation induced apoptosis [26-29]. Together these data suggested that the TAR miRNA prevented apoptosis by down-regulating both ERCC1 and IER3.

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