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Intestinal epithelia activate anti-viral signaling via intracellular sensing of rotavirus structural components.

Frias AH, Vijay-Kumar M, Gentsch JR, Crawford SE, Carvalho FA, Estes MK, Gewirtz AT - Mucosal Immunol (2010)

Bottom Line: Using model human intestinal epithelia, we observed that RV-induced activation of signaling events and gene expression typically associated with viral infection was largely mimicked by administration of ultraviolet (UV)-inactivated RV.In contrast, RV-induction of nuclear factor-κB-mediated interleukin-8 expression was dependent on viral replication.The anti-viral gene expression induced by UV-RV was not significantly recapitulated by RV RNA or RV virus-like particles although the latter could enter IEC.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Emory University, Atlanta, Georgia, USA.

ABSTRACT
Rotavirus (RV), a leading cause of severe diarrhea, primarily infects intestinal epithelial cells (IECs) causing self-limiting illness. To better understand innate immunity to RV, we sought to define the extent to which IEC activation of anti-viral responses required viral replication or could be recapitulated by inactivated RV or its components. Using model human intestinal epithelia, we observed that RV-induced activation of signaling events and gene expression typically associated with viral infection was largely mimicked by administration of ultraviolet (UV)-inactivated RV. Use of anti-interferon (IFN) neutralizing antibodies revealed that such replication-independent anti-viral gene expression required type I IFN signaling. In contrast, RV-induction of nuclear factor-κB-mediated interleukin-8 expression was dependent on viral replication. The anti-viral gene expression induced by UV-RV was not significantly recapitulated by RV RNA or RV virus-like particles although the latter could enter IEC. Together, these results suggest that RV proteins mediate viral entry into epithelial cells leading to intracellular detection of RV RNA that generates an anti-viral response.

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RV, UV-RV, and VLP cell entry during early stages of infectionIntestinal epithelial monolayers (HT29) were grown on collagen-coated permeable supports and treated apically with RV and UV-RV (MOI 10), and an amount of VLPs roughly equivalent to the estimated protein concentration in the RV preparation. Control samples were apically treated with an equivalent amount of trypsin in SFM (Mock) for 4 hpi. At 1 and 4 hpi, cells were fixed, stained and examined via confocal fluorescence microscopy for presence of rotaviral proteins (green) and F-actin (red) in the sub-apical region of the cells (3 μm below the apical surface). Sub-apical images of cells stimulated with RV, UV-RV, and RV VLPs at 1 hpi, magnification 60X (A). Sub-apical images of cells treated with RV and UV-RV at 1 and 4 hpi, magnification 40X (B). Data in A and B are results of a single experiment and representative of 3 separate experiments. Scale reflects distance of 10 μm.
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Figure 7: RV, UV-RV, and VLP cell entry during early stages of infectionIntestinal epithelial monolayers (HT29) were grown on collagen-coated permeable supports and treated apically with RV and UV-RV (MOI 10), and an amount of VLPs roughly equivalent to the estimated protein concentration in the RV preparation. Control samples were apically treated with an equivalent amount of trypsin in SFM (Mock) for 4 hpi. At 1 and 4 hpi, cells were fixed, stained and examined via confocal fluorescence microscopy for presence of rotaviral proteins (green) and F-actin (red) in the sub-apical region of the cells (3 μm below the apical surface). Sub-apical images of cells stimulated with RV, UV-RV, and RV VLPs at 1 hpi, magnification 60X (A). Sub-apical images of cells treated with RV and UV-RV at 1 and 4 hpi, magnification 40X (B). Data in A and B are results of a single experiment and representative of 3 separate experiments. Scale reflects distance of 10 μm.

Mentions: To better understand the interaction of UV-RV and RV VLPs with polarized epithelia and activation of epithelial anti-viral signaling in response to RV, we examined the interaction of RV, UV-RV, and RV VLPs with model epithelia via confocal microscopy for up to 4 hpi (Figure 7A, B). RV and UV-RV (MOI 10), and RV VLPs roughly equivalent to the estimated protein concentration of the RV preparation, were applied to the apical surface of epithelia for 1 and 4 hpi, followed by washing off of non-adsorbed or non-adhered materials. As expected, planar images of IEC taken 3 μm below the apical surface revealed the presence of RV, UV-RV and RV VLPs in the sub-apical region within 1 hpi (Figure 10A). RV increased in abundance by 4 hpi, reflecting viral replication, while UV-RV levels decreased likely due to degradation of viral proteins and an inability to replicate (Figure 7B). Thus, consistent with models by which RV is internalized by IEC, RV’s primary structural proteins are sufficient to mediate its entry into IEC. Taken together, we interpret our results to suggest that RV activation of anti-viral signaling requires viral structural proteins to mediate entry of viral RNA into epithelial cells where it can be detected by host pattern-recognition receptor(s).


Intestinal epithelia activate anti-viral signaling via intracellular sensing of rotavirus structural components.

Frias AH, Vijay-Kumar M, Gentsch JR, Crawford SE, Carvalho FA, Estes MK, Gewirtz AT - Mucosal Immunol (2010)

RV, UV-RV, and VLP cell entry during early stages of infectionIntestinal epithelial monolayers (HT29) were grown on collagen-coated permeable supports and treated apically with RV and UV-RV (MOI 10), and an amount of VLPs roughly equivalent to the estimated protein concentration in the RV preparation. Control samples were apically treated with an equivalent amount of trypsin in SFM (Mock) for 4 hpi. At 1 and 4 hpi, cells were fixed, stained and examined via confocal fluorescence microscopy for presence of rotaviral proteins (green) and F-actin (red) in the sub-apical region of the cells (3 μm below the apical surface). Sub-apical images of cells stimulated with RV, UV-RV, and RV VLPs at 1 hpi, magnification 60X (A). Sub-apical images of cells treated with RV and UV-RV at 1 and 4 hpi, magnification 40X (B). Data in A and B are results of a single experiment and representative of 3 separate experiments. Scale reflects distance of 10 μm.
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Related In: Results  -  Collection

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Figure 7: RV, UV-RV, and VLP cell entry during early stages of infectionIntestinal epithelial monolayers (HT29) were grown on collagen-coated permeable supports and treated apically with RV and UV-RV (MOI 10), and an amount of VLPs roughly equivalent to the estimated protein concentration in the RV preparation. Control samples were apically treated with an equivalent amount of trypsin in SFM (Mock) for 4 hpi. At 1 and 4 hpi, cells were fixed, stained and examined via confocal fluorescence microscopy for presence of rotaviral proteins (green) and F-actin (red) in the sub-apical region of the cells (3 μm below the apical surface). Sub-apical images of cells stimulated with RV, UV-RV, and RV VLPs at 1 hpi, magnification 60X (A). Sub-apical images of cells treated with RV and UV-RV at 1 and 4 hpi, magnification 40X (B). Data in A and B are results of a single experiment and representative of 3 separate experiments. Scale reflects distance of 10 μm.
Mentions: To better understand the interaction of UV-RV and RV VLPs with polarized epithelia and activation of epithelial anti-viral signaling in response to RV, we examined the interaction of RV, UV-RV, and RV VLPs with model epithelia via confocal microscopy for up to 4 hpi (Figure 7A, B). RV and UV-RV (MOI 10), and RV VLPs roughly equivalent to the estimated protein concentration of the RV preparation, were applied to the apical surface of epithelia for 1 and 4 hpi, followed by washing off of non-adsorbed or non-adhered materials. As expected, planar images of IEC taken 3 μm below the apical surface revealed the presence of RV, UV-RV and RV VLPs in the sub-apical region within 1 hpi (Figure 10A). RV increased in abundance by 4 hpi, reflecting viral replication, while UV-RV levels decreased likely due to degradation of viral proteins and an inability to replicate (Figure 7B). Thus, consistent with models by which RV is internalized by IEC, RV’s primary structural proteins are sufficient to mediate its entry into IEC. Taken together, we interpret our results to suggest that RV activation of anti-viral signaling requires viral structural proteins to mediate entry of viral RNA into epithelial cells where it can be detected by host pattern-recognition receptor(s).

Bottom Line: Using model human intestinal epithelia, we observed that RV-induced activation of signaling events and gene expression typically associated with viral infection was largely mimicked by administration of ultraviolet (UV)-inactivated RV.In contrast, RV-induction of nuclear factor-κB-mediated interleukin-8 expression was dependent on viral replication.The anti-viral gene expression induced by UV-RV was not significantly recapitulated by RV RNA or RV virus-like particles although the latter could enter IEC.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Emory University, Atlanta, Georgia, USA.

ABSTRACT
Rotavirus (RV), a leading cause of severe diarrhea, primarily infects intestinal epithelial cells (IECs) causing self-limiting illness. To better understand innate immunity to RV, we sought to define the extent to which IEC activation of anti-viral responses required viral replication or could be recapitulated by inactivated RV or its components. Using model human intestinal epithelia, we observed that RV-induced activation of signaling events and gene expression typically associated with viral infection was largely mimicked by administration of ultraviolet (UV)-inactivated RV. Use of anti-interferon (IFN) neutralizing antibodies revealed that such replication-independent anti-viral gene expression required type I IFN signaling. In contrast, RV-induction of nuclear factor-κB-mediated interleukin-8 expression was dependent on viral replication. The anti-viral gene expression induced by UV-RV was not significantly recapitulated by RV RNA or RV virus-like particles although the latter could enter IEC. Together, these results suggest that RV proteins mediate viral entry into epithelial cells leading to intracellular detection of RV RNA that generates an anti-viral response.

Show MeSH
Related in: MedlinePlus