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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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SeV copy-back DVGs generated in situ during infection have a strong stimulatory ability.(A) TC-1 cells were either mock-infected or infected with a moi of 1.5 TCID50/cell of SeV strains Enders, 52, or Z. Total RNA was extracted at 2, 6, 12, and 24 h post-infection and analyzed for the presence of DVGs and standard virus genome (gSeV) by PCR. (B) BMDCs were infected with SeV strain Z or SeV Cantell LD and RNA was extracted at 2, 6, 12, and 24 h post-infection and analyzed for the presence of DVGs and gSeV. (C) Immunoblot of phosphorylated IRF3 in whole cell extracts from infected BMDCs and (D) Ifnb mRNA expression in infected BMDCs as determined by RT-qPCR. Gene expression is shown as copy number relative to the housekeeping genes Tuba1b and Rps11. Sequences from bands labeled with a star can be found in Fig. S5. Position of base pair size reference markers is indicated in each gel.
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ppat-1003703-g003: SeV copy-back DVGs generated in situ during infection have a strong stimulatory ability.(A) TC-1 cells were either mock-infected or infected with a moi of 1.5 TCID50/cell of SeV strains Enders, 52, or Z. Total RNA was extracted at 2, 6, 12, and 24 h post-infection and analyzed for the presence of DVGs and standard virus genome (gSeV) by PCR. (B) BMDCs were infected with SeV strain Z or SeV Cantell LD and RNA was extracted at 2, 6, 12, and 24 h post-infection and analyzed for the presence of DVGs and gSeV. (C) Immunoblot of phosphorylated IRF3 in whole cell extracts from infected BMDCs and (D) Ifnb mRNA expression in infected BMDCs as determined by RT-qPCR. Gene expression is shown as copy number relative to the housekeeping genes Tuba1b and Rps11. Sequences from bands labeled with a star can be found in Fig. S5. Position of base pair size reference markers is indicated in each gel.

Mentions: Based on the potent ability of SeV stocks containing a high content of copy-back DVGs to induce the host response to infection in vitro[23], [24], [25], [28] (Fig. 1) and on our prior reports of strong host responses to DVGs regardless of the presence of functional virus-encoded antagonists [23], [24], we hypothesized that DVGs that arise in situ during viral infections provide essential stimuli to initiate an antiviral immune response. To test this hypothesis, we first determined if SeV strains that accumulate copy-back DVGs early in infection induced faster Ifnb mRNA expression in vitro than viruses with delayed DVG accumulation. For these experiments we used SeV preparations that did not show immunostimulatory activity or evidence of copy-back DVG accumulation by 2 h post-infection and all the viruses were used at a multiplicity of infection of 1.5 TCID50/cell. While standard viral genomes of all the different SeV strains used were detected at all tested time points, copy-back DVGs of different sizes were detected starting at 6 h post-infection in cells infected with SeV Z and at later time points in cells infected with SeV 52, Enders, or Cantell LD in both murine lung epithelial cells (TC-1) and bone marrow-derived dendritic cells (BMDCs) (Fig. 3A and B and data not shown). Sequences of the starred PCR products confirming the amplification of copy-back DVGs are shown in Fig. S5. Remarkably, accumulation of DVGs was directly associated with phosphorylation of IRF3 (Fig. 3C) and with the expression of Ifnb mRNA (Fig. 3D), demonstrating that standard viral genomes alone are not sufficient to initiate this response during infection in vitro and strongly supporting a unique ability of naturally arising DVGs to initiate the cellular antiviral response.


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)

SeV copy-back DVGs generated in situ during infection have a strong stimulatory ability.(A) TC-1 cells were either mock-infected or infected with a moi of 1.5 TCID50/cell of SeV strains Enders, 52, or Z. Total RNA was extracted at 2, 6, 12, and 24 h post-infection and analyzed for the presence of DVGs and standard virus genome (gSeV) by PCR. (B) BMDCs were infected with SeV strain Z or SeV Cantell LD and RNA was extracted at 2, 6, 12, and 24 h post-infection and analyzed for the presence of DVGs and gSeV. (C) Immunoblot of phosphorylated IRF3 in whole cell extracts from infected BMDCs and (D) Ifnb mRNA expression in infected BMDCs as determined by RT-qPCR. Gene expression is shown as copy number relative to the housekeeping genes Tuba1b and Rps11. Sequences from bands labeled with a star can be found in Fig. S5. Position of base pair size reference markers is indicated in each gel.
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Related In: Results  -  Collection

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ppat-1003703-g003: SeV copy-back DVGs generated in situ during infection have a strong stimulatory ability.(A) TC-1 cells were either mock-infected or infected with a moi of 1.5 TCID50/cell of SeV strains Enders, 52, or Z. Total RNA was extracted at 2, 6, 12, and 24 h post-infection and analyzed for the presence of DVGs and standard virus genome (gSeV) by PCR. (B) BMDCs were infected with SeV strain Z or SeV Cantell LD and RNA was extracted at 2, 6, 12, and 24 h post-infection and analyzed for the presence of DVGs and gSeV. (C) Immunoblot of phosphorylated IRF3 in whole cell extracts from infected BMDCs and (D) Ifnb mRNA expression in infected BMDCs as determined by RT-qPCR. Gene expression is shown as copy number relative to the housekeeping genes Tuba1b and Rps11. Sequences from bands labeled with a star can be found in Fig. S5. Position of base pair size reference markers is indicated in each gel.
Mentions: Based on the potent ability of SeV stocks containing a high content of copy-back DVGs to induce the host response to infection in vitro[23], [24], [25], [28] (Fig. 1) and on our prior reports of strong host responses to DVGs regardless of the presence of functional virus-encoded antagonists [23], [24], we hypothesized that DVGs that arise in situ during viral infections provide essential stimuli to initiate an antiviral immune response. To test this hypothesis, we first determined if SeV strains that accumulate copy-back DVGs early in infection induced faster Ifnb mRNA expression in vitro than viruses with delayed DVG accumulation. For these experiments we used SeV preparations that did not show immunostimulatory activity or evidence of copy-back DVG accumulation by 2 h post-infection and all the viruses were used at a multiplicity of infection of 1.5 TCID50/cell. While standard viral genomes of all the different SeV strains used were detected at all tested time points, copy-back DVGs of different sizes were detected starting at 6 h post-infection in cells infected with SeV Z and at later time points in cells infected with SeV 52, Enders, or Cantell LD in both murine lung epithelial cells (TC-1) and bone marrow-derived dendritic cells (BMDCs) (Fig. 3A and B and data not shown). Sequences of the starred PCR products confirming the amplification of copy-back DVGs are shown in Fig. S5. Remarkably, accumulation of DVGs was directly associated with phosphorylation of IRF3 (Fig. 3C) and with the expression of Ifnb mRNA (Fig. 3D), demonstrating that standard viral genomes alone are not sufficient to initiate this response during infection in vitro and strongly supporting a unique ability of naturally arising DVGs to initiate the cellular antiviral response.

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