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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

HIV-1 miRNA down-regulated the expression of proteins related to apoptosis. (A) 293T cells were mock transfected (lane 1) or transfected with TAR-D mutant (lane 2) or TAR-WT RNA (lane 3). After 48 hours cell lysates were prepared and Western blotted for ERCC1, PIASγ, GIT2, IER3 or β-actin. (B) Cell lysates were prepared from HeLaT4, HLM-1, CEM, ACH2, U1 and U937 cell lines and Western blotted for ERCC1, PIASγ, GIT2, IER3 or β-actin. Densitometry was performed, and the expression levels were normalized to actin. The average expression level of each protein from three experiments was determined and displayed as the ratio of expression in the infected cells (HLM-1, ACH2 and U937) to their uninfected counterpart (HeLaT4, CEM, U1). (C) HLM-1 cells were transfected with mock (lane 1) or TAR 5' antagomir (lane 2). Cells were lysed after 96 hours and 20 micrograms of protein were used to Western blot for the expression of ERCC1. Coomassie staining of the ~25–50 kDa portion of the gel is included as a loading control.
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Figure 7: HIV-1 miRNA down-regulated the expression of proteins related to apoptosis. (A) 293T cells were mock transfected (lane 1) or transfected with TAR-D mutant (lane 2) or TAR-WT RNA (lane 3). After 48 hours cell lysates were prepared and Western blotted for ERCC1, PIASγ, GIT2, IER3 or β-actin. (B) Cell lysates were prepared from HeLaT4, HLM-1, CEM, ACH2, U1 and U937 cell lines and Western blotted for ERCC1, PIASγ, GIT2, IER3 or β-actin. Densitometry was performed, and the expression levels were normalized to actin. The average expression level of each protein from three experiments was determined and displayed as the ratio of expression in the infected cells (HLM-1, ACH2 and U937) to their uninfected counterpart (HeLaT4, CEM, U1). (C) HLM-1 cells were transfected with mock (lane 1) or TAR 5' antagomir (lane 2). Cells were lysed after 96 hours and 20 micrograms of protein were used to Western blot for the expression of ERCC1. Coomassie staining of the ~25–50 kDa portion of the gel is included as a loading control.

Mentions: In examining the potential list of HIV-1 miRNA regulated genes, we selected four genes with possible links to apoptosis and cell survival for further study; ERCC1, PIASγ, GIT2 and IER3. Excision repair cross complementing-group 1 (ERCC1) is involved in the detection and base excision repair of damaged nucleotides [22]. Protein inhibitor of activated STAT Y (PIASγ) is an inhibitor of STAT1 signaling, and is capable of modulating NFκB signaling, and also functions as a transcriptional co-repressor due to E3 Sumo ligase activity [23,24]. G protein-coupled receptor interacting protein (GIT2) is involved in G-protein signaling [25]. Intermediate early response 3 (IER3) is up-regulated after cellular insult and has been shown to be required for induction of apoptosis after serum starvation and DNA damage [26-29]. We tested the ability of the TAR miRNA to down-regulate these four genes using Western blotting (Fig. 7A). 293T cells were transfected with TAR-WT, TAR-D or were mock transfected. Forty-eight hours after transfection, cell extracts were prepared, and protein expression was examined by Western blotting. While there was no change in the expression level between mock and TAR-D transfections, ERCC1, PIASγ, GIT2 and IER3 were all down-regulated in the presence of TAR-WT when normalized to actin.


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)

HIV-1 miRNA down-regulated the expression of proteins related to apoptosis. (A) 293T cells were mock transfected (lane 1) or transfected with TAR-D mutant (lane 2) or TAR-WT RNA (lane 3). After 48 hours cell lysates were prepared and Western blotted for ERCC1, PIASγ, GIT2, IER3 or β-actin. (B) Cell lysates were prepared from HeLaT4, HLM-1, CEM, ACH2, U1 and U937 cell lines and Western blotted for ERCC1, PIASγ, GIT2, IER3 or β-actin. Densitometry was performed, and the expression levels were normalized to actin. The average expression level of each protein from three experiments was determined and displayed as the ratio of expression in the infected cells (HLM-1, ACH2 and U937) to their uninfected counterpart (HeLaT4, CEM, U1). (C) HLM-1 cells were transfected with mock (lane 1) or TAR 5' antagomir (lane 2). Cells were lysed after 96 hours and 20 micrograms of protein were used to Western blot for the expression of ERCC1. Coomassie staining of the ~25–50 kDa portion of the gel is included as a loading control.
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Related In: Results  -  Collection

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Figure 7: HIV-1 miRNA down-regulated the expression of proteins related to apoptosis. (A) 293T cells were mock transfected (lane 1) or transfected with TAR-D mutant (lane 2) or TAR-WT RNA (lane 3). After 48 hours cell lysates were prepared and Western blotted for ERCC1, PIASγ, GIT2, IER3 or β-actin. (B) Cell lysates were prepared from HeLaT4, HLM-1, CEM, ACH2, U1 and U937 cell lines and Western blotted for ERCC1, PIASγ, GIT2, IER3 or β-actin. Densitometry was performed, and the expression levels were normalized to actin. The average expression level of each protein from three experiments was determined and displayed as the ratio of expression in the infected cells (HLM-1, ACH2 and U937) to their uninfected counterpart (HeLaT4, CEM, U1). (C) HLM-1 cells were transfected with mock (lane 1) or TAR 5' antagomir (lane 2). Cells were lysed after 96 hours and 20 micrograms of protein were used to Western blot for the expression of ERCC1. Coomassie staining of the ~25–50 kDa portion of the gel is included as a loading control.
Mentions: In examining the potential list of HIV-1 miRNA regulated genes, we selected four genes with possible links to apoptosis and cell survival for further study; ERCC1, PIASγ, GIT2 and IER3. Excision repair cross complementing-group 1 (ERCC1) is involved in the detection and base excision repair of damaged nucleotides [22]. Protein inhibitor of activated STAT Y (PIASγ) is an inhibitor of STAT1 signaling, and is capable of modulating NFκB signaling, and also functions as a transcriptional co-repressor due to E3 Sumo ligase activity [23,24]. G protein-coupled receptor interacting protein (GIT2) is involved in G-protein signaling [25]. Intermediate early response 3 (IER3) is up-regulated after cellular insult and has been shown to be required for induction of apoptosis after serum starvation and DNA damage [26-29]. We tested the ability of the TAR miRNA to down-regulate these four genes using Western blotting (Fig. 7A). 293T cells were transfected with TAR-WT, TAR-D or were mock transfected. Forty-eight hours after transfection, cell extracts were prepared, and protein expression was examined by Western blotting. While there was no change in the expression level between mock and TAR-D transfections, ERCC1, PIASγ, GIT2 and IER3 were all down-regulated in the presence of TAR-WT when normalized to actin.

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