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HIV-1 TAR miRNA Protects Against Apoptosis by Altering Cellular Gene Expression

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HIV-1 TAR miRNA Protects Against Apoptosis by Altering Cellular Gene Expression

Zachary Klase et al. Retrovirology.

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.

Figures

Figure 1
Figure 1
Determination of the sequence of that HIV-1 TAR miRNA. RNA from cMagi cells infected with HIVIIIB was used to construct miRNA libraries and used for cloning. (A) Cloned sequences of the TAR-5p and TAR-3p (5' and 3' arm) miRNA obtained as compared to predicted sequence registered with the Sanger miRNA database. (B) Diagram showing the TAR hairpin and the position of the mature miRNA within the TAR sequence. (C) Structure of the TAR-WT and truncated TAR-D mutant used for 293T transfections. (D) 293T cells were mock transfected (lane 2) or transfected with TAR-WT (lane 3) or TAR-D (lane 4) RNA. Forty-eight hours after transfection RNA was isolated and subjected to Northern blotting for TAR sequence. Numbers to the left indicate the size of the RNA ladder in nucleotides. Diagrams to the right show the positions of the wild-type TAR and the mature TAR miRNA.
Figure 2
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.
Figure 3
Figure 3
HIV-1 infected cell lines are resistant to apoptosis. HelaT4 (A, B) and HLM-1 (C, D) were cultured in the presence of 10% serum (A, C) or 0.1% serum (B, D) for 96 hours. Cells were then collected and apoptosis determined via AnnexinV and PI co-staining. Data are representative of three experiments.
Figure 4
Figure 4
Infected T-cell lines are resistant to apoptosis. CEM (A, B) and ACH2 (C, D) were cultured in the presence of 10% serum (A, C) or 0.1% serum (B, D) for 96 hours. Cells were then collected and apoptosis determined via AnnexinV and PI co-staining. Data are representative of three experiments.
Figure 5
Figure 5
Serum starvation induced cleavage of Caspase 3 in uninfected but not infected cells. (A) HeLaT4 (lanes 1 and 3) and HLM-1 (lanes 2 and 4) were Western blotted for Caspase 3 expression and cleavage at Zero (lanes 1 and 2) and 48 (lanes 3 and 4) hours after serum starvation. (B) CEM (lanes 1 and 3) and ACH2 (lanes 2 and 4) were Western blotted for Caspase 3 expression and cleavage at Zero (lanes 1 and 2) and 48 (lanes 3 and 4) hours after serum starvation. Densitometry was performed to determine the density of the cleaved 17 and 20 kDa Caspase 3 bands as compared to the 32 kDa inactive form.
Figure 6
Figure 6
Specifically blocking TAR miRNA sensitized cells to apoptosis. HLM-1 cells were transfected with antagomir complementary to the TAR 5' miRNA. Twenty-four hours post transfection the media was replaced with DMEM with 0.1% FBS. Cells were sampled at 96 hours and apoptosis determined via AnnexinV and PI co-staining. Percentages shown indicate the number of AnnexinV positive, PI negative and AnnexinV, PI double positive cells.
Figure 7
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.
Figure 8
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.
Figure 9
Figure 9
TAR 5' miRNA targeted the ERCC1 gene directly. (A) miRanda software was used to determine potential target sites in the ERCC1 and IER3 mRNA sequences for the HIV-1 TAR 5' miRNA. (B) 293T cells were transfected with TAR-D or TAR-WT RNA. Twenty-four hours after RNA transfection the cells were transfected with psiCheckERCC-737 reporter vector. Extracts were prepared and luciferase expression determined at 24, 48 and 72 hours after reporter transfection. Data shown represent the normalized expression of Renilla luciferase (with the target region) to firely luciferase. Data are representative of two replicates.
Figure 10
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.

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