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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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vtRNAs are significantly upregulated during IAV infection both in vitro and in vivo. (A) The differentially expressed ncRNAs in A549 cells infected with or without A/WSN/33 influenza virus were analyzed by a cDNA microarray (http://www.ncbi.nlm.nih.gov/geo/; GEO access number GSE58741). Shown are representative ncRNAs whose expressions were significantly changed. vtRNAs are indicated by red rectangle. (B, C) A549 cells were infected with or without WSN for 16 h. RT-PCR (B) and qRT-PCR (C) were performed to determine the expression of vtRNAs. (D) A549 cells were infected with WSN at indicated MOIs for 16 h. Northern blotting was performed to determine vtRNAs expression. 5S rRNA was used as a loading control. (E) The expression of vtRNAs in indicated human cell lines infected with or without WSN for 16 h was examined by RT-PCR. The viral nucleoprotein (NP) was examined by western blotting. (F) The mvtRNA expression in mouse cell lines infected with or without WSN for 18 h was examined by RT-PCR. The viral nucleoprotein (NP) was examined by Western blotting. (G) C57BL/6 mice intranasally infected with or without WSN (5 × 104 PFU) for 2 days were sacrificed, and the lungs were dissected and lysed, followed by qRT-PCR to examine the mvtRNA levels. Shown are representative results from three independent experiments. The error bars represent the SE, **P < 0.01.
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Figure 1: vtRNAs are significantly upregulated during IAV infection both in vitro and in vivo. (A) The differentially expressed ncRNAs in A549 cells infected with or without A/WSN/33 influenza virus were analyzed by a cDNA microarray (http://www.ncbi.nlm.nih.gov/geo/; GEO access number GSE58741). Shown are representative ncRNAs whose expressions were significantly changed. vtRNAs are indicated by red rectangle. (B, C) A549 cells were infected with or without WSN for 16 h. RT-PCR (B) and qRT-PCR (C) were performed to determine the expression of vtRNAs. (D) A549 cells were infected with WSN at indicated MOIs for 16 h. Northern blotting was performed to determine vtRNAs expression. 5S rRNA was used as a loading control. (E) The expression of vtRNAs in indicated human cell lines infected with or without WSN for 16 h was examined by RT-PCR. The viral nucleoprotein (NP) was examined by western blotting. (F) The mvtRNA expression in mouse cell lines infected with or without WSN for 18 h was examined by RT-PCR. The viral nucleoprotein (NP) was examined by Western blotting. (G) C57BL/6 mice intranasally infected with or without WSN (5 × 104 PFU) for 2 days were sacrificed, and the lungs were dissected and lysed, followed by qRT-PCR to examine the mvtRNA levels. Shown are representative results from three independent experiments. The error bars represent the SE, **P < 0.01.

Mentions: To address the relationship between ncRNAs and IAV infection, the differential expression patterns of ncRNAs in human alveolar epithelial cells (A549) infected with or without IAV were analyzed by an annotated cDNA microarray (http://www.ncbi.nlm.nih.gov/geo; GEO access number GSE58741). Interestingly, four vtRNA family members, including vtRNA1-1, vtRNA1-2, vtRNA1-3 and vtRNA2-1, were significantly upregulated upon infection with the influenza A/WSN/33 (H1N1) virus (WSN) (Figure 1A). This was confirmed by RT-PCR and qRT-PCR (Figure 1B, C, Supplementary Figure S1A and B). Furthermore, we observed that vtRNAs were upregulated in a virus dose-dependent manner by analysis of northern blot and qRT-PCR (Figure 1D and Supplementary Figure S1C). Moreover, these vtRNAs were found to be expressed in various human cell lines including A549, 293T, K562, MCF7, HeLa and Huh7 by using RT-PCR, and their expression levels were elevated by IAV infection in all examined cell lines susceptible to the infection (Figure 1E), except for HeLa cell line that is less permissive to IAV replication (45). The increased expression of vtRNA2–1 in IAV-infected host was further confirmed in A549, MCF7 and HEK293 cells by Northern blotting (Supplementary Figure S1D).


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)

vtRNAs are significantly upregulated during IAV infection both in vitro and in vivo. (A) The differentially expressed ncRNAs in A549 cells infected with or without A/WSN/33 influenza virus were analyzed by a cDNA microarray (http://www.ncbi.nlm.nih.gov/geo/; GEO access number GSE58741). Shown are representative ncRNAs whose expressions were significantly changed. vtRNAs are indicated by red rectangle. (B, C) A549 cells were infected with or without WSN for 16 h. RT-PCR (B) and qRT-PCR (C) were performed to determine the expression of vtRNAs. (D) A549 cells were infected with WSN at indicated MOIs for 16 h. Northern blotting was performed to determine vtRNAs expression. 5S rRNA was used as a loading control. (E) The expression of vtRNAs in indicated human cell lines infected with or without WSN for 16 h was examined by RT-PCR. The viral nucleoprotein (NP) was examined by western blotting. (F) The mvtRNA expression in mouse cell lines infected with or without WSN for 18 h was examined by RT-PCR. The viral nucleoprotein (NP) was examined by Western blotting. (G) C57BL/6 mice intranasally infected with or without WSN (5 × 104 PFU) for 2 days were sacrificed, and the lungs were dissected and lysed, followed by qRT-PCR to examine the mvtRNA levels. Shown are representative results from three independent experiments. The error bars represent the SE, **P < 0.01.
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Figure 1: vtRNAs are significantly upregulated during IAV infection both in vitro and in vivo. (A) The differentially expressed ncRNAs in A549 cells infected with or without A/WSN/33 influenza virus were analyzed by a cDNA microarray (http://www.ncbi.nlm.nih.gov/geo/; GEO access number GSE58741). Shown are representative ncRNAs whose expressions were significantly changed. vtRNAs are indicated by red rectangle. (B, C) A549 cells were infected with or without WSN for 16 h. RT-PCR (B) and qRT-PCR (C) were performed to determine the expression of vtRNAs. (D) A549 cells were infected with WSN at indicated MOIs for 16 h. Northern blotting was performed to determine vtRNAs expression. 5S rRNA was used as a loading control. (E) The expression of vtRNAs in indicated human cell lines infected with or without WSN for 16 h was examined by RT-PCR. The viral nucleoprotein (NP) was examined by western blotting. (F) The mvtRNA expression in mouse cell lines infected with or without WSN for 18 h was examined by RT-PCR. The viral nucleoprotein (NP) was examined by Western blotting. (G) C57BL/6 mice intranasally infected with or without WSN (5 × 104 PFU) for 2 days were sacrificed, and the lungs were dissected and lysed, followed by qRT-PCR to examine the mvtRNA levels. Shown are representative results from three independent experiments. The error bars represent the SE, **P < 0.01.
Mentions: To address the relationship between ncRNAs and IAV infection, the differential expression patterns of ncRNAs in human alveolar epithelial cells (A549) infected with or without IAV were analyzed by an annotated cDNA microarray (http://www.ncbi.nlm.nih.gov/geo; GEO access number GSE58741). Interestingly, four vtRNA family members, including vtRNA1-1, vtRNA1-2, vtRNA1-3 and vtRNA2-1, were significantly upregulated upon infection with the influenza A/WSN/33 (H1N1) virus (WSN) (Figure 1A). This was confirmed by RT-PCR and qRT-PCR (Figure 1B, C, Supplementary Figure S1A and B). Furthermore, we observed that vtRNAs were upregulated in a virus dose-dependent manner by analysis of northern blot and qRT-PCR (Figure 1D and Supplementary Figure S1C). Moreover, these vtRNAs were found to be expressed in various human cell lines including A549, 293T, K562, MCF7, HeLa and Huh7 by using RT-PCR, and their expression levels were elevated by IAV infection in all examined cell lines susceptible to the infection (Figure 1E), except for HeLa cell line that is less permissive to IAV replication (45). The increased expression of vtRNA2–1 in IAV-infected host was further confirmed in A549, MCF7 and HEK293 cells by Northern blotting (Supplementary Figure S1D).

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