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Robust expression of vault RNAs induced by influenza A virus plays a critical role in suppression of PKR-mediated innate immunity.

Li F, Chen Y, Zhang Z, Ouyang J, Wang Y, Yan R, Huang S, Gao GF, Guo G, Chen JL - Nucleic Acids Res. (2015)

Bottom Line: Importantly, silencing vtRNA in A549 cells significantly inhibited IAV replication, whereas overexpression of vtRNAs markedly promoted the viral replication.The vtRNA knockdown animals exhibited significantly enhanced resistance to IAV infection, as evidenced by attenuated acute lung injury and spleen atrophy and consequently increased survival rates.In addition, increased expression of vtRNAs was required for efficient suppression of PKR by NS1 during IAV infection.

View Article: PubMed Central - PubMed

Affiliation: CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences (CAS), Beijing 100101, China.

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Silencing mvtRNA by inhaled aerosolized ASOs decreases the susceptibility of mice to IAV infection. (A, B) The C57BL/6 mice that inhaled aerosolized ASOs targeting mvtRNA or GFP for 24 h were infected with WSN (1 × 104 PFU) for another 96 h. Then mice were sacrificed and the lungs were dissected and lysed. The efficiency of ASO-based knockdown of mvtRNA was determined by RT-PCR (A) and qRT-PCR (B). Plotted are the average levels from three independent experiments. The error bars represent the SE, *P < 0.05. (C) Shown is the body weight change of mvtRNA knockdown and control mice intranasally inoculated with WSN or PBS (control). Body weight was measured every 24 h. The dashed line indicates the endpoint of 25% weight loss. (D) Shown are survival rates of mvtRNA knockdown mice and control mice inoculated intranasally with WSN (8–10 mice/group). Mice were monitored for a period of 144 h. (E) Shown are viral titers in the lungs of mvtRNA knockdown and control mice inoculated intranasally with WSN for 96 h. The viral titer was measured by plaque assay. Plotted are the average levels from three independent experiments. The error bars represent the SE, **P < 0.01. (F) mvtRNA knockdown and control mice were intranasally inoculated with WSN or PBS for 96 h. Then mice were sacrificed and the lungs and spleens were collected. Shown are representative images from three independent experiments. (G) Experiments were performed as described in (F). Shown are representative micrographs of lung sections of the musvtRNA knockdown and control mice stained with hematoxylin and eosin (HE).
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Figure 4: Silencing mvtRNA by inhaled aerosolized ASOs decreases the susceptibility of mice to IAV infection. (A, B) The C57BL/6 mice that inhaled aerosolized ASOs targeting mvtRNA or GFP for 24 h were infected with WSN (1 × 104 PFU) for another 96 h. Then mice were sacrificed and the lungs were dissected and lysed. The efficiency of ASO-based knockdown of mvtRNA was determined by RT-PCR (A) and qRT-PCR (B). Plotted are the average levels from three independent experiments. The error bars represent the SE, *P < 0.05. (C) Shown is the body weight change of mvtRNA knockdown and control mice intranasally inoculated with WSN or PBS (control). Body weight was measured every 24 h. The dashed line indicates the endpoint of 25% weight loss. (D) Shown are survival rates of mvtRNA knockdown mice and control mice inoculated intranasally with WSN (8–10 mice/group). Mice were monitored for a period of 144 h. (E) Shown are viral titers in the lungs of mvtRNA knockdown and control mice inoculated intranasally with WSN for 96 h. The viral titer was measured by plaque assay. Plotted are the average levels from three independent experiments. The error bars represent the SE, **P < 0.01. (F) mvtRNA knockdown and control mice were intranasally inoculated with WSN or PBS for 96 h. Then mice were sacrificed and the lungs and spleens were collected. Shown are representative images from three independent experiments. (G) Experiments were performed as described in (F). Shown are representative micrographs of lung sections of the musvtRNA knockdown and control mice stained with hematoxylin and eosin (HE).

Mentions: It is reported that respirable ASOs represent a novel therapeutic approach for the treatment of lung diseases (46,47). To further define the functional relevance of vtRNA to IAV infection in vivo, C57BL/6 mice were treated with aerosolized ASOs targeting mvtRNA (ASO-mvtRNA) or GFP (ASO-GFP) for 24 h, and then infected with WSN for 96 h. As shown in Figure 4A and B, ASO-mvtRNA significantly downregulated mvtRNA expression in lungs of the mice, as compared with the ASO-GFP control. The mvtRNA knockdown mice displayed slower body weight loss and higher survival rate than the control group during viral infection (Figure 4C and D). All control mice died within 96 h p.i., whereas ∼33% of mvtRNA knockdown mice remained alive within 120 h p.i.. Consistent with these observations, the plaque assay indicated that the viral titers in lung tissues of mvtRNA knockdown mice were significantly lower than that in the control group (Figure 4E), suggesting that silencing mvtRNA inhibits the IAV replication in vivo. In addition, mvtRNA knockdown mice exhibited less-severe organ damage caused by IAV infection, while the control group showed a greater degree of acute lung injury and spleen atrophy (Figure 4F). Consistently, pathologic examination by hematoxylin and eosin (HE) staining displayed less severe edema and reduced infiltration of inflammatory cells across the interalveolar septum in the lungs of infected mice treated with ASOs targeting mvtRNA (Figure 4G). These results indicate that disruption of mvtRNA expression in mice decreases the susceptibility of the animals to IAV infection.


Robust expression of vault RNAs induced by influenza A virus plays a critical role in suppression of PKR-mediated innate immunity.

Li F, Chen Y, Zhang Z, Ouyang J, Wang Y, Yan R, Huang S, Gao GF, Guo G, Chen JL - Nucleic Acids Res. (2015)

Silencing mvtRNA by inhaled aerosolized ASOs decreases the susceptibility of mice to IAV infection. (A, B) The C57BL/6 mice that inhaled aerosolized ASOs targeting mvtRNA or GFP for 24 h were infected with WSN (1 × 104 PFU) for another 96 h. Then mice were sacrificed and the lungs were dissected and lysed. The efficiency of ASO-based knockdown of mvtRNA was determined by RT-PCR (A) and qRT-PCR (B). Plotted are the average levels from three independent experiments. The error bars represent the SE, *P < 0.05. (C) Shown is the body weight change of mvtRNA knockdown and control mice intranasally inoculated with WSN or PBS (control). Body weight was measured every 24 h. The dashed line indicates the endpoint of 25% weight loss. (D) Shown are survival rates of mvtRNA knockdown mice and control mice inoculated intranasally with WSN (8–10 mice/group). Mice were monitored for a period of 144 h. (E) Shown are viral titers in the lungs of mvtRNA knockdown and control mice inoculated intranasally with WSN for 96 h. The viral titer was measured by plaque assay. Plotted are the average levels from three independent experiments. The error bars represent the SE, **P < 0.01. (F) mvtRNA knockdown and control mice were intranasally inoculated with WSN or PBS for 96 h. Then mice were sacrificed and the lungs and spleens were collected. Shown are representative images from three independent experiments. (G) Experiments were performed as described in (F). Shown are representative micrographs of lung sections of the musvtRNA knockdown and control mice stained with hematoxylin and eosin (HE).
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Figure 4: Silencing mvtRNA by inhaled aerosolized ASOs decreases the susceptibility of mice to IAV infection. (A, B) The C57BL/6 mice that inhaled aerosolized ASOs targeting mvtRNA or GFP for 24 h were infected with WSN (1 × 104 PFU) for another 96 h. Then mice were sacrificed and the lungs were dissected and lysed. The efficiency of ASO-based knockdown of mvtRNA was determined by RT-PCR (A) and qRT-PCR (B). Plotted are the average levels from three independent experiments. The error bars represent the SE, *P < 0.05. (C) Shown is the body weight change of mvtRNA knockdown and control mice intranasally inoculated with WSN or PBS (control). Body weight was measured every 24 h. The dashed line indicates the endpoint of 25% weight loss. (D) Shown are survival rates of mvtRNA knockdown mice and control mice inoculated intranasally with WSN (8–10 mice/group). Mice were monitored for a period of 144 h. (E) Shown are viral titers in the lungs of mvtRNA knockdown and control mice inoculated intranasally with WSN for 96 h. The viral titer was measured by plaque assay. Plotted are the average levels from three independent experiments. The error bars represent the SE, **P < 0.01. (F) mvtRNA knockdown and control mice were intranasally inoculated with WSN or PBS for 96 h. Then mice were sacrificed and the lungs and spleens were collected. Shown are representative images from three independent experiments. (G) Experiments were performed as described in (F). Shown are representative micrographs of lung sections of the musvtRNA knockdown and control mice stained with hematoxylin and eosin (HE).
Mentions: It is reported that respirable ASOs represent a novel therapeutic approach for the treatment of lung diseases (46,47). To further define the functional relevance of vtRNA to IAV infection in vivo, C57BL/6 mice were treated with aerosolized ASOs targeting mvtRNA (ASO-mvtRNA) or GFP (ASO-GFP) for 24 h, and then infected with WSN for 96 h. As shown in Figure 4A and B, ASO-mvtRNA significantly downregulated mvtRNA expression in lungs of the mice, as compared with the ASO-GFP control. The mvtRNA knockdown mice displayed slower body weight loss and higher survival rate than the control group during viral infection (Figure 4C and D). All control mice died within 96 h p.i., whereas ∼33% of mvtRNA knockdown mice remained alive within 120 h p.i.. Consistent with these observations, the plaque assay indicated that the viral titers in lung tissues of mvtRNA knockdown mice were significantly lower than that in the control group (Figure 4E), suggesting that silencing mvtRNA inhibits the IAV replication in vivo. In addition, mvtRNA knockdown mice exhibited less-severe organ damage caused by IAV infection, while the control group showed a greater degree of acute lung injury and spleen atrophy (Figure 4F). Consistently, pathologic examination by hematoxylin and eosin (HE) staining displayed less severe edema and reduced infiltration of inflammatory cells across the interalveolar septum in the lungs of infected mice treated with ASOs targeting mvtRNA (Figure 4G). These results indicate that disruption of mvtRNA expression in mice decreases the susceptibility of the animals to IAV infection.

Bottom Line: Importantly, silencing vtRNA in A549 cells significantly inhibited IAV replication, whereas overexpression of vtRNAs markedly promoted the viral replication.The vtRNA knockdown animals exhibited significantly enhanced resistance to IAV infection, as evidenced by attenuated acute lung injury and spleen atrophy and consequently increased survival rates.In addition, increased expression of vtRNAs was required for efficient suppression of PKR by NS1 during IAV infection.

View Article: PubMed Central - PubMed

Affiliation: CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences (CAS), Beijing 100101, China.

Show MeSH
Related in: MedlinePlus