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Defective viral genomes arising in vivo provide critical danger signals for the triggering of lung antiviral immunity.

Tapia K, Kim WK, Sun Y, Mercado-López X, Dunay E, Wise M, Adu M, López CB - PLoS Pathog. (2013)

Bottom Line: Here we show that truncated forms of viral genomes that accumulate in infected cells potently trigger the sustained activation of the transcription factors IRF3 and NF-κB and the production type I IFNs through a mechanism independent of IFN signaling.Remarkably, the hallmark antiviral cytokine IFNβ is only expressed in lung epithelial cells containing DVGs, while cells within the lung that contain standard viral genomes alone do not express this cytokine.Together, our data indicate that DVGs generated during viral replication are a primary source of danger signals for the initiation of the host immune response to infection.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
The innate immune response to viruses is initiated when specialized cellular sensors recognize viral danger signals. Here we show that truncated forms of viral genomes that accumulate in infected cells potently trigger the sustained activation of the transcription factors IRF3 and NF-κB and the production type I IFNs through a mechanism independent of IFN signaling. We demonstrate that these defective viral genomes (DVGs) are generated naturally during respiratory infections in vivo even in mice lacking the type I IFN receptor, and their appearance coincides with the production of cytokines during infections with Sendai virus (SeV) or influenza virus. Remarkably, the hallmark antiviral cytokine IFNβ is only expressed in lung epithelial cells containing DVGs, while cells within the lung that contain standard viral genomes alone do not express this cytokine. Together, our data indicate that DVGs generated during viral replication are a primary source of danger signals for the initiation of the host immune response to infection.

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IAV deletion DVGs reduce virus virulence and are generated in vivo during infection.(A) PCR of standard and defective genomes in BMDCs infected for 6 h with a moi of 1.5 TCID50/cell IAV PR8 HD or LD. PCR strategies and sequences of starred product are shown in Fig. S7. (B) Weight loss of mice infected with 100 TCID50 IAV PR8 HD or LD (n = 5) (**p<0.01; Two-way ANOVA with Bonferroni's post hoc test). (C) Quantification of IAV Np mRNA and (D) Ifnb mRNA from total lung homogenates by RT-qPCR. (E) Lungs from mice infected with IAV PR8 LD were analyzed at day 1 (D1) and day 3 (D3) post-infection for the presence of DVGs and genomic fragments by PCR. Results for two different D3 mice are shown. Sequences for starred products can be seen in Fig. S8. (F) Expression of Ifnb mRNA in whole lung homogenates analyzed by RT-qPCR. Gene expression is shown as copy number relative to the housekeeping genes Tuba1b and Rps11. (*p<0.05, **p<0.01, ***p<0.001, ****p<0,0001; Unpaired, two tailed, t student test). Position of base pair size reference markers is indicated in each gel.
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ppat-1003703-g006: IAV deletion DVGs reduce virus virulence and are generated in vivo during infection.(A) PCR of standard and defective genomes in BMDCs infected for 6 h with a moi of 1.5 TCID50/cell IAV PR8 HD or LD. PCR strategies and sequences of starred product are shown in Fig. S7. (B) Weight loss of mice infected with 100 TCID50 IAV PR8 HD or LD (n = 5) (**p<0.01; Two-way ANOVA with Bonferroni's post hoc test). (C) Quantification of IAV Np mRNA and (D) Ifnb mRNA from total lung homogenates by RT-qPCR. (E) Lungs from mice infected with IAV PR8 LD were analyzed at day 1 (D1) and day 3 (D3) post-infection for the presence of DVGs and genomic fragments by PCR. Results for two different D3 mice are shown. Sequences for starred products can be seen in Fig. S8. (F) Expression of Ifnb mRNA in whole lung homogenates analyzed by RT-qPCR. Gene expression is shown as copy number relative to the housekeeping genes Tuba1b and Rps11. (*p<0.05, **p<0.01, ***p<0.001, ****p<0,0001; Unpaired, two tailed, t student test). Position of base pair size reference markers is indicated in each gel.

Mentions: To investigate whether the content of DVGs in IAV stocks affects virulence similar to SeV, we obtained IAV strain PR8 stocks with a high content of DVGs (HD) or lacking DVGs (LD). The stock of IAV PR8 HD produced two predominant DVGs derived from the PA and PB1 genomic segments in infected cells, while no DVGs were detected in cells infected with IAV PR8 LD (Fig. 6A) (Strategy for IAV detection and sequences for the IAV DVGs present in infected cells can be found in Fig. S7). Mice infected with IAV PR8 HD showed reduced morbidity compared to mice infected with IAV PR8 LD (Fig. 6B) despite similar levels of virus replication (Fig. 6C). Similar to SeV Cantell HD, reduced morbidity was associated with enhanced Ifnb mRNA expression in the lung (Fig. 6D).


Defective viral genomes arising in vivo provide critical danger signals for the triggering of lung antiviral immunity.

Tapia K, Kim WK, Sun Y, Mercado-López X, Dunay E, Wise M, Adu M, López CB - PLoS Pathog. (2013)

IAV deletion DVGs reduce virus virulence and are generated in vivo during infection.(A) PCR of standard and defective genomes in BMDCs infected for 6 h with a moi of 1.5 TCID50/cell IAV PR8 HD or LD. PCR strategies and sequences of starred product are shown in Fig. S7. (B) Weight loss of mice infected with 100 TCID50 IAV PR8 HD or LD (n = 5) (**p<0.01; Two-way ANOVA with Bonferroni's post hoc test). (C) Quantification of IAV Np mRNA and (D) Ifnb mRNA from total lung homogenates by RT-qPCR. (E) Lungs from mice infected with IAV PR8 LD were analyzed at day 1 (D1) and day 3 (D3) post-infection for the presence of DVGs and genomic fragments by PCR. Results for two different D3 mice are shown. Sequences for starred products can be seen in Fig. S8. (F) Expression of Ifnb mRNA in whole lung homogenates analyzed by RT-qPCR. Gene expression is shown as copy number relative to the housekeeping genes Tuba1b and Rps11. (*p<0.05, **p<0.01, ***p<0.001, ****p<0,0001; Unpaired, two tailed, t student test). Position of base pair size reference markers is indicated in each gel.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3814336&req=5

ppat-1003703-g006: IAV deletion DVGs reduce virus virulence and are generated in vivo during infection.(A) PCR of standard and defective genomes in BMDCs infected for 6 h with a moi of 1.5 TCID50/cell IAV PR8 HD or LD. PCR strategies and sequences of starred product are shown in Fig. S7. (B) Weight loss of mice infected with 100 TCID50 IAV PR8 HD or LD (n = 5) (**p<0.01; Two-way ANOVA with Bonferroni's post hoc test). (C) Quantification of IAV Np mRNA and (D) Ifnb mRNA from total lung homogenates by RT-qPCR. (E) Lungs from mice infected with IAV PR8 LD were analyzed at day 1 (D1) and day 3 (D3) post-infection for the presence of DVGs and genomic fragments by PCR. Results for two different D3 mice are shown. Sequences for starred products can be seen in Fig. S8. (F) Expression of Ifnb mRNA in whole lung homogenates analyzed by RT-qPCR. Gene expression is shown as copy number relative to the housekeeping genes Tuba1b and Rps11. (*p<0.05, **p<0.01, ***p<0.001, ****p<0,0001; Unpaired, two tailed, t student test). Position of base pair size reference markers is indicated in each gel.
Mentions: To investigate whether the content of DVGs in IAV stocks affects virulence similar to SeV, we obtained IAV strain PR8 stocks with a high content of DVGs (HD) or lacking DVGs (LD). The stock of IAV PR8 HD produced two predominant DVGs derived from the PA and PB1 genomic segments in infected cells, while no DVGs were detected in cells infected with IAV PR8 LD (Fig. 6A) (Strategy for IAV detection and sequences for the IAV DVGs present in infected cells can be found in Fig. S7). Mice infected with IAV PR8 HD showed reduced morbidity compared to mice infected with IAV PR8 LD (Fig. 6B) despite similar levels of virus replication (Fig. 6C). Similar to SeV Cantell HD, reduced morbidity was associated with enhanced Ifnb mRNA expression in the lung (Fig. 6D).

Bottom Line: Here we show that truncated forms of viral genomes that accumulate in infected cells potently trigger the sustained activation of the transcription factors IRF3 and NF-κB and the production type I IFNs through a mechanism independent of IFN signaling.Remarkably, the hallmark antiviral cytokine IFNβ is only expressed in lung epithelial cells containing DVGs, while cells within the lung that contain standard viral genomes alone do not express this cytokine.Together, our data indicate that DVGs generated during viral replication are a primary source of danger signals for the initiation of the host immune response to infection.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
The innate immune response to viruses is initiated when specialized cellular sensors recognize viral danger signals. Here we show that truncated forms of viral genomes that accumulate in infected cells potently trigger the sustained activation of the transcription factors IRF3 and NF-κB and the production type I IFNs through a mechanism independent of IFN signaling. We demonstrate that these defective viral genomes (DVGs) are generated naturally during respiratory infections in vivo even in mice lacking the type I IFN receptor, and their appearance coincides with the production of cytokines during infections with Sendai virus (SeV) or influenza virus. Remarkably, the hallmark antiviral cytokine IFNβ is only expressed in lung epithelial cells containing DVGs, while cells within the lung that contain standard viral genomes alone do not express this cytokine. Together, our data indicate that DVGs generated during viral replication are a primary source of danger signals for the initiation of the host immune response to infection.

Show MeSH
Related in: MedlinePlus