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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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TAR miRNA altered ERCC1 protein expression without altering mRNA levels. (A) 293T cells were transfected with psiCheckERCC-737 and either pPolIII TAR or pPolIIIScr. Renilla and firefly luciferase expression was measured after 72 hours. Data shown represent the normalized ratio of Renilla luciferase to firefly luciferase for two replicates. 293T cells were transfected with either pPolIII TAR or pPolIIIScr. Forty-eight hours after transfection the cells were harvested and protein and RNA extracts were prepared. (B) Protein extracts were Western blotted for ERCC1 and β-actin expression. (C) RNA extracts were used to generated cDNA, and ERCC1 and IER3 mRNA levels were determined by PCR.
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Figure 10: TAR miRNA altered ERCC1 protein expression without altering mRNA levels. (A) 293T cells were transfected with psiCheckERCC-737 and either pPolIII TAR or pPolIIIScr. Renilla and firefly luciferase expression was measured after 72 hours. Data shown represent the normalized ratio of Renilla luciferase to firefly luciferase for two replicates. 293T cells were transfected with either pPolIII TAR or pPolIIIScr. Forty-eight hours after transfection the cells were harvested and protein and RNA extracts were prepared. (B) Protein extracts were Western blotted for ERCC1 and β-actin expression. (C) RNA extracts were used to generated cDNA, and ERCC1 and IER3 mRNA levels were determined by PCR.

Mentions: Our results indicated that the TAR 5' miRNA repressed ERCC1 expression and cellular apoptosis. Presumably this effect is through the miRNA pathway; perhaps through the silencing of translation by recruitment of the mRNA to the P-body. To confirm this assumption, we sought to examine the level of mRNA expression of ERCC1 and IER3 in the presence and absence of the TAR miRNA. To further control for a miRNA effect, we sought to utilize a control that still produces a mature miRNA, but has a mutated seed sequence. We used a pair of Pol III expression vectors that express either a WT TAR or a TAR element with a scrambled sequence in the stem corresponding to positions 6–16 of TAR and the complementary based on the 3' side of the stem (Generous gift of Dr. Rossi, City of Hope, CA). Both the pPol III-TAR and pPol III-Scr vectors produce a mature miRNA (Rossi and Castanotto, unpublished data). To verify that Pol III-TAR produces a miRNA that can affect ERCC1, we again performed the luciferase reporter assay using the psiCheck and psiCheckERCC-737 vectors (Fig. 10A). 293T were transfected with psiCheck or psiCheckERCC-737 and pPol III-Scr or pPol III-TAR vectors, and luciferase expression was measured 72 hours later. Renilla expression was normalized to an internal control firefly luciferase. The data indicated that pPol III-TAR vector suppressed the expression of the luciferase mRNA containing the miRNA target sequence. This effect was not seen with a control vector (data not shown).


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)

TAR miRNA altered ERCC1 protein expression without altering mRNA levels. (A) 293T cells were transfected with psiCheckERCC-737 and either pPolIII TAR or pPolIIIScr. Renilla and firefly luciferase expression was measured after 72 hours. Data shown represent the normalized ratio of Renilla luciferase to firefly luciferase for two replicates. 293T cells were transfected with either pPolIII TAR or pPolIIIScr. Forty-eight hours after transfection the cells were harvested and protein and RNA extracts were prepared. (B) Protein extracts were Western blotted for ERCC1 and β-actin expression. (C) RNA extracts were used to generated cDNA, and ERCC1 and IER3 mRNA levels were determined by PCR.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 10: TAR miRNA altered ERCC1 protein expression without altering mRNA levels. (A) 293T cells were transfected with psiCheckERCC-737 and either pPolIII TAR or pPolIIIScr. Renilla and firefly luciferase expression was measured after 72 hours. Data shown represent the normalized ratio of Renilla luciferase to firefly luciferase for two replicates. 293T cells were transfected with either pPolIII TAR or pPolIIIScr. Forty-eight hours after transfection the cells were harvested and protein and RNA extracts were prepared. (B) Protein extracts were Western blotted for ERCC1 and β-actin expression. (C) RNA extracts were used to generated cDNA, and ERCC1 and IER3 mRNA levels were determined by PCR.
Mentions: Our results indicated that the TAR 5' miRNA repressed ERCC1 expression and cellular apoptosis. Presumably this effect is through the miRNA pathway; perhaps through the silencing of translation by recruitment of the mRNA to the P-body. To confirm this assumption, we sought to examine the level of mRNA expression of ERCC1 and IER3 in the presence and absence of the TAR miRNA. To further control for a miRNA effect, we sought to utilize a control that still produces a mature miRNA, but has a mutated seed sequence. We used a pair of Pol III expression vectors that express either a WT TAR or a TAR element with a scrambled sequence in the stem corresponding to positions 6–16 of TAR and the complementary based on the 3' side of the stem (Generous gift of Dr. Rossi, City of Hope, CA). Both the pPol III-TAR and pPol III-Scr vectors produce a mature miRNA (Rossi and Castanotto, unpublished data). To verify that Pol III-TAR produces a miRNA that can affect ERCC1, we again performed the luciferase reporter assay using the psiCheck and psiCheckERCC-737 vectors (Fig. 10A). 293T were transfected with psiCheck or psiCheckERCC-737 and pPol III-Scr or pPol III-TAR vectors, and luciferase expression was measured 72 hours later. Renilla expression was normalized to an internal control firefly luciferase. The data indicated that pPol III-TAR vector suppressed the expression of the luciferase mRNA containing the miRNA target sequence. This effect was not seen with a control vector (data not shown).

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