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MiR-23a facilitates the replication of HSV-1 through the suppression of interferon regulatory factor 1.

Ru J, Sun H, Fan H, Wang C, Li Y, Liu M, Tang H - PLoS ONE (2014)

Bottom Line: Suppression of IRF1 expression reduced RSAD2 gene expression, augmenting HSV-1 replication.Notably, IRF1 contributes to innate antiviral immunity by binding to IRF-response elements to regulate the expression of interferon-stimulated genes (ISGs) and apoptosis, revealing a complex interaction between miR-23a and HSV-1.MiR-23a thus contributes to HSV-1 replication through the regulation of the IRF1-mediated antiviral signal pathway, which suggests that miR-23a may represent a promising target for antiviral treatments.

View Article: PubMed Central - PubMed

Affiliation: Tianjin Life Science Research Center and Department of Microbiology, School of Basic Medical Sciences, Tianjin Medical University, Tianjin 300070, China; Department of Pathophysiology, School of Basic Medical Sciences, Yunnan University of Traditional Chinese Medicine, Kunming 650500, China.

ABSTRACT
MicroRNAs (miRNAs) are small, non-coding RNAs that negatively regulate gene expression. It has been reported that miRNAs are involved in host-virus interaction, but evidence that cellular miRNAs promote virus replication has been limited. Here, we found that miR-23a promoted the replication of human herpes simplex virus type 1 (HSV-1) in HeLa cells, as demonstrated by a plaque-formation assay and quantitative real-time PCR. Furthermore, interferon regulatory factor 1 (IRF1), an innate antiviral molecule, is targeted by miR-23a to facilitate viral replication. MiR-23a binds to the 3'UTR of IRF1 and down-regulates its expression. Suppression of IRF1 expression reduced RSAD2 gene expression, augmenting HSV-1 replication. Ectopic expression of IRF1 abrogated the promotion of HSV-1 replication induced by miR-23a. Notably, IRF1 contributes to innate antiviral immunity by binding to IRF-response elements to regulate the expression of interferon-stimulated genes (ISGs) and apoptosis, revealing a complex interaction between miR-23a and HSV-1. MiR-23a thus contributes to HSV-1 replication through the regulation of the IRF1-mediated antiviral signal pathway, which suggests that miR-23a may represent a promising target for antiviral treatments.

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IRF1 suppresses the replication of HSV-1 partially by up-regulation of RSAD2.(A) HeLa cells were transfected with IRF1 and pcDNA3 or co-transfected with IRF1 and Pri-miR-23a and control vector, as indicated. Total RNA was extracted, and RSAD2 mRNA was quantified by quantitative real-time PCR. (B) HeLa cells were transfected with Myc-RSAD2. At 48-h post-transfection, quantitative real-time PCR was used to detect the level of RSAD2 mRNA, and at 72 h post-transfection, a Western blot was used to detect the expression level of RSAD2. (C) HeLa cells were transfected with Myc-RSAD2 or pcDNA3. Cells were infected with HSV-1 at 0.01 PFU/cell and stained with neutral red at 36 h post-infection. The mean radius of the cytopathic area was measured. The scale bar represents 100 µm. (D) HeLa cells were transfected with Myc-RSAD2 or pcDNA3. Viral yields were determined by standard plaque assays at 48 h post-infection with HSV-1. (E) Model of miR-23a regulation in HSV-1 replication. Increased levels of miR-23a in HeLa cells led to decrease levels of IRF1 mRNA and RSAD2 mRNA, with a consequent increase in HSV-1 replication. All data represent the mean value ± SD of at least three independent experiments. *: p<0.05; **: p<0.01; ns: No significant differences by Student's t test.
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pone-0114021-g006: IRF1 suppresses the replication of HSV-1 partially by up-regulation of RSAD2.(A) HeLa cells were transfected with IRF1 and pcDNA3 or co-transfected with IRF1 and Pri-miR-23a and control vector, as indicated. Total RNA was extracted, and RSAD2 mRNA was quantified by quantitative real-time PCR. (B) HeLa cells were transfected with Myc-RSAD2. At 48-h post-transfection, quantitative real-time PCR was used to detect the level of RSAD2 mRNA, and at 72 h post-transfection, a Western blot was used to detect the expression level of RSAD2. (C) HeLa cells were transfected with Myc-RSAD2 or pcDNA3. Cells were infected with HSV-1 at 0.01 PFU/cell and stained with neutral red at 36 h post-infection. The mean radius of the cytopathic area was measured. The scale bar represents 100 µm. (D) HeLa cells were transfected with Myc-RSAD2 or pcDNA3. Viral yields were determined by standard plaque assays at 48 h post-infection with HSV-1. (E) Model of miR-23a regulation in HSV-1 replication. Increased levels of miR-23a in HeLa cells led to decrease levels of IRF1 mRNA and RSAD2 mRNA, with a consequent increase in HSV-1 replication. All data represent the mean value ± SD of at least three independent experiments. *: p<0.05; **: p<0.01; ns: No significant differences by Student's t test.

Mentions: In a recent study, IRF1 suppressed VSV replication through radical S-adenosyl methionine domain containing 2 (RSAD2) induction, leading to the expression of viperin protein, which is involved in innate immune responses [35]. To determine whether IRF-1 suppresses HSV-1 replication via a similar pathway, we first determined RSAD2 mRNA levels in HeLa cells transiently transfected with IRF-1 expressing vector. Fig. 6A showed that IRF1 significantly enhanced RSAD2 expression at both mRNA and protein levels. In contrast, ectopic expression of miR-23a caused the amount of RSAD2 mRNA and protein to decrease by about 40% and 30%, respectively (Fig. 6A). Next, we first constructed a RSAD2 expression vector (Myc-RSAD2), and verified the efficiency of the vector by Western blot (Fig. 6B). Plaque-formation assay and viral-titer assay to further explore the role of RSAD2 in HSV-1 replication was positive. (Fig. 6C, D). Most likely, by targeting IRF1, miR-23a indirectly suppresses RSAD2 expression to facilitate HSV-1 replication (Fig. 6E).


MiR-23a facilitates the replication of HSV-1 through the suppression of interferon regulatory factor 1.

Ru J, Sun H, Fan H, Wang C, Li Y, Liu M, Tang H - PLoS ONE (2014)

IRF1 suppresses the replication of HSV-1 partially by up-regulation of RSAD2.(A) HeLa cells were transfected with IRF1 and pcDNA3 or co-transfected with IRF1 and Pri-miR-23a and control vector, as indicated. Total RNA was extracted, and RSAD2 mRNA was quantified by quantitative real-time PCR. (B) HeLa cells were transfected with Myc-RSAD2. At 48-h post-transfection, quantitative real-time PCR was used to detect the level of RSAD2 mRNA, and at 72 h post-transfection, a Western blot was used to detect the expression level of RSAD2. (C) HeLa cells were transfected with Myc-RSAD2 or pcDNA3. Cells were infected with HSV-1 at 0.01 PFU/cell and stained with neutral red at 36 h post-infection. The mean radius of the cytopathic area was measured. The scale bar represents 100 µm. (D) HeLa cells were transfected with Myc-RSAD2 or pcDNA3. Viral yields were determined by standard plaque assays at 48 h post-infection with HSV-1. (E) Model of miR-23a regulation in HSV-1 replication. Increased levels of miR-23a in HeLa cells led to decrease levels of IRF1 mRNA and RSAD2 mRNA, with a consequent increase in HSV-1 replication. All data represent the mean value ± SD of at least three independent experiments. *: p<0.05; **: p<0.01; ns: No significant differences by Student's t test.
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pone-0114021-g006: IRF1 suppresses the replication of HSV-1 partially by up-regulation of RSAD2.(A) HeLa cells were transfected with IRF1 and pcDNA3 or co-transfected with IRF1 and Pri-miR-23a and control vector, as indicated. Total RNA was extracted, and RSAD2 mRNA was quantified by quantitative real-time PCR. (B) HeLa cells were transfected with Myc-RSAD2. At 48-h post-transfection, quantitative real-time PCR was used to detect the level of RSAD2 mRNA, and at 72 h post-transfection, a Western blot was used to detect the expression level of RSAD2. (C) HeLa cells were transfected with Myc-RSAD2 or pcDNA3. Cells were infected with HSV-1 at 0.01 PFU/cell and stained with neutral red at 36 h post-infection. The mean radius of the cytopathic area was measured. The scale bar represents 100 µm. (D) HeLa cells were transfected with Myc-RSAD2 or pcDNA3. Viral yields were determined by standard plaque assays at 48 h post-infection with HSV-1. (E) Model of miR-23a regulation in HSV-1 replication. Increased levels of miR-23a in HeLa cells led to decrease levels of IRF1 mRNA and RSAD2 mRNA, with a consequent increase in HSV-1 replication. All data represent the mean value ± SD of at least three independent experiments. *: p<0.05; **: p<0.01; ns: No significant differences by Student's t test.
Mentions: In a recent study, IRF1 suppressed VSV replication through radical S-adenosyl methionine domain containing 2 (RSAD2) induction, leading to the expression of viperin protein, which is involved in innate immune responses [35]. To determine whether IRF-1 suppresses HSV-1 replication via a similar pathway, we first determined RSAD2 mRNA levels in HeLa cells transiently transfected with IRF-1 expressing vector. Fig. 6A showed that IRF1 significantly enhanced RSAD2 expression at both mRNA and protein levels. In contrast, ectopic expression of miR-23a caused the amount of RSAD2 mRNA and protein to decrease by about 40% and 30%, respectively (Fig. 6A). Next, we first constructed a RSAD2 expression vector (Myc-RSAD2), and verified the efficiency of the vector by Western blot (Fig. 6B). Plaque-formation assay and viral-titer assay to further explore the role of RSAD2 in HSV-1 replication was positive. (Fig. 6C, D). Most likely, by targeting IRF1, miR-23a indirectly suppresses RSAD2 expression to facilitate HSV-1 replication (Fig. 6E).

Bottom Line: Suppression of IRF1 expression reduced RSAD2 gene expression, augmenting HSV-1 replication.Notably, IRF1 contributes to innate antiviral immunity by binding to IRF-response elements to regulate the expression of interferon-stimulated genes (ISGs) and apoptosis, revealing a complex interaction between miR-23a and HSV-1.MiR-23a thus contributes to HSV-1 replication through the regulation of the IRF1-mediated antiviral signal pathway, which suggests that miR-23a may represent a promising target for antiviral treatments.

View Article: PubMed Central - PubMed

Affiliation: Tianjin Life Science Research Center and Department of Microbiology, School of Basic Medical Sciences, Tianjin Medical University, Tianjin 300070, China; Department of Pathophysiology, School of Basic Medical Sciences, Yunnan University of Traditional Chinese Medicine, Kunming 650500, China.

ABSTRACT
MicroRNAs (miRNAs) are small, non-coding RNAs that negatively regulate gene expression. It has been reported that miRNAs are involved in host-virus interaction, but evidence that cellular miRNAs promote virus replication has been limited. Here, we found that miR-23a promoted the replication of human herpes simplex virus type 1 (HSV-1) in HeLa cells, as demonstrated by a plaque-formation assay and quantitative real-time PCR. Furthermore, interferon regulatory factor 1 (IRF1), an innate antiviral molecule, is targeted by miR-23a to facilitate viral replication. MiR-23a binds to the 3'UTR of IRF1 and down-regulates its expression. Suppression of IRF1 expression reduced RSAD2 gene expression, augmenting HSV-1 replication. Ectopic expression of IRF1 abrogated the promotion of HSV-1 replication induced by miR-23a. Notably, IRF1 contributes to innate antiviral immunity by binding to IRF-response elements to regulate the expression of interferon-stimulated genes (ISGs) and apoptosis, revealing a complex interaction between miR-23a and HSV-1. MiR-23a thus contributes to HSV-1 replication through the regulation of the IRF1-mediated antiviral signal pathway, which suggests that miR-23a may represent a promising target for antiviral treatments.

Show MeSH
Related in: MedlinePlus