Norovirus Regulation of the Innate Immune Response and Apoptosis Occurs via the Product of the Alternative Open Reading Frame 4 methods
Aim. Evidence-backed execution summary for Norovirus Regulation of the Innate Immune Response and Apoptosis Occurs via the Product of the Alternative Open Reading Frame 4 methods from Norovirus Regulation of the Innate Immune Response and Apoptosis Occurs via the Product of the Alternative Open Reading Frame 4.
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mouse
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Analysis of CXCL10, ISG54 and IFN-Beta levels in infected RAW264.7 cells
reagent used in the protocol.
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- Analysis of mRNA levels in RAW264.7 cells was performed at low MOI (0.1 TCID 50 units/cell). Cells were seeded at 2 × 10 5 cells per well in a 24 well dish, grown overnight at 37°C before being infected. RNA was harvested from infected cells at 16, 20 and 24 hours post infection using the GenElute RNA extrac...
Analysis of CXCL10, ISG54 and IFN-Beta levels in infected RAW264.7 cells
Analysis of mRNA levels in RAW264.7 cells was performed at low MOI (0.1 TCID 50 units/cell). Cells were seeded at 2 × 10 5 cells per well in a 24 well dish, grown overnight at 37°C before being infected. RNA was harvested from infected cells at 16, 20 and 24 hours post infection using the GenElute RNA extrac...
- Use
- Analysis of mRNA levels in RAW264.7 cells was performed at low MOI (0.1 TCID 50 units/cell). Cells were seeded at 2 × 10 5 cells per well in a 24 well dish, grown overnight at 37°C before being infected. RNA was harvested from infected cells at 16, 20 and 24 hours post infection using the GenElute RNA extrac...
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VF1 localizes to mitochondria
To confirm the mitochondrial localization of VF1 during virus infection, mitochondria were purified from infected RAW264.7 cells at 15 hours post infection and analyzed for the presence of VF1 by western blot ( ). Whereas the well characterized host cell nucleic acid binding proteins PCBP1/2 were shown to be predominantly cytoplasmic as expected, VF1 was only detected in the mitochondrial fraction ( ). Apoptosis inducing factor 1, a predominantly mitochondrial protein was enriched in the mitochondrial fraction, confirming the validity of the purification procedure ( ).
VF1 production affects mitochondrial-dependent innate immune signalling
RNA viruses frequently encode proteins that antagonize the innate immune response to infection. Mitochondria play a significant role in signaling innate immune responses through the well characterized mitochondrial antiviral signaling protein (MAVS), an integral membrane protein found in the outer mitochondrial membrane -. MAVS is a key adapter protein in the sensing of viral RNA by RIG-I and MDA5 that, in part, leads to IRF3 and NFΚB activation and the upregulation of antiviral genes such as IFN-Beta, CXCL10 and ISG54. Given the mitochondrial localization of VF1 in infected cells we assessed the activation of this sub-section of the innate immune response in both M1 and WT infected cells. RAW264.7 cells infected at a low MOI (0.1 TCID 50 per cell) with the M1 VF1 knockout virus exhibited a greater induction of antiviral genes such as ISG54, CXCL10 and IFN-Beta in respons...
VF1 production affects mitochondrial-dependent innate immune signalling
(A) Time course of ISG54 and CXCL10 mRNA expression in RAW264.7 cells infected at an MOI of 0.1 TCID 50 per cell. mRNA levels were quantified by qPCR using an endogenous control gene (Hypoxanthine-guanine phosphoribosyltransferase, HPRT). Expression of the respective mRNAs was then calculated using the ΔΔCt method to compare infected and mock infected cells. Relative fold change was calculated using mock infected samples taken at comparable time points. Normalization was performed following MNV quantification in each sample. Infections were carried out in triplicate with each sample being subsequently analyzed in duplicate by qPCR. The error bars denote standard deviation from the mean. Statistical analysis was performed using an unpaired two tailed t test. (B) Time course of IFN-Beta mRNA and protein production in RAW264.7 cells infected as in (A). mRNA upregulation was cal...
Analysis of CXCL10, ISG54 and IFN-Beta levels in infected RAW264.7 cells
Analysis of mRNA levels in RAW264.7 cells was performed at low MOI (0.1 TCID 50 units/cell). Cells were seeded at 2 × 10 5 cells per well in a 24 well dish, grown overnight at 37°C before being infected. RNA was harvested from infected cells at 16, 20 and 24 hours post infection using the GenElute RNA extraction kit (Sigma) as detailed by the manufacturer. RNA was quantified and diluted to a standard concentration before being reverse transcribed using MuMLV RT enzyme (Promega) using an oligo dT primer. SYBR green based qPCR was performed using an ABI 7900 HT real time PCR machine. The MESA Blue (Eurogentec) SYBR master mix was combined with sample cDNAs and optimised high efficiency mouse specific primers for the following genes: HPRT, CXCL10, ISG54 and Interferon Beta (primer details available upon request). Relative mRNA fold change was calculated using the ΔΔCt m...
Supporting Information
The response of RAW264.7 cells to polyIC and UV inactivated viruses. IFN-Beta, CXCL10 and ISG54 mRNA and IFN-Beta protein analysis of RAW264.7 cells (A) infected with an equivalent MOI of 0.1 of UV inactivated M1 and WT viruses at 24hpi or (B) treated with 25 µg/ml polyIC for 24 hours. mRNA levels were quantified by qPCR using an endogenous control gene (Hypoxanthine-guanine phosphoribosyltransferase, HPRT). Expression of the respective mRNAs was then calculated using the ΔΔCt method to compare infected and mock infected cells. IFN-Beta protein secretion was quantified by murine IFN-B specific ELISA in the supernatants of treated/infected cells. Relative fold change was calculated using mock infected samples taken at comparable time points.
Supporting Information
CXCL10 and ISG54 responses in MNV infected STAT-/- mice at 3 days post infection. Tissues were harvested from STAT1-/- mice infected with 1000 TCID-50 of MNV-1 (CW1.P1) at 3 days post infection. Relative fold induction of the CXCL10 and ISG54 mRNAs was calculated for each tissue separately, through comparison with mRNA levels in tissues from a mock/uninfected mouse (represented by the green line). This analysis was performed using the ΔΔCt qPCR method with the cellular gene HPRT used as an endogenous control. The data shown is from two separate animals, with the bar referring to the mean fold induction detected in each instance.
Measurement outputs
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To confirm the mitochondrial localization of VF1 during virus infection, mitochondria were purified from infected RAW264.7 cells at 15 hours post infection and analyzed for the...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
RNA viruses frequently encode proteins that antagonize the innate immune response to infection. Mitochondria play a significant role in signaling innate immune responses through...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
(A) Time course of ISG54 and CXCL10 mRNA expression in RAW264.7 cells infected at an MOI of 0.1 TCID 50 per cell. mRNA levels were quantified by qPCR using an endogenous control...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Analysis of mRNA levels in RAW264.7 cells was performed at low MOI (0.1 TCID 50 units/cell). Cells were seeded at 2 × 10 5 cells per well in a 24 well dish, grown overnight a...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Analysis plan
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Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
inferred from protocolPreprocessing / cleaning
RNA viruses frequently encode proteins that antagonize the innate immune response to infection.
from paperScoring or quantification
Quantify the primary readouts for this experiment: To confirm the mitochondrial localization of VF1 during virus infection, mitochondria were purified from infected RAW264.7 cells at 15 hours post infection and analyzed for the...; RNA viruses frequently encode proteins that antagonize the innate immune response to infection. Mitochondria play a significant role in signaling innate immune responses through...; (A) Time course of ISG54 and CXCL10 mRNA expression in RAW264.7 cells infected at an MOI of 0.1 TCID 50 per cell. mRNA levels were quantified by qPCR using an endogenous control...; Analysis of mRNA levels in RAW264.7 cells was performed at low MOI (0.1 TCID 50 units/cell). Cells were seeded at 2 × 10 5 cells per well in a 24 well dish, grown overnight a....
from paperStatistical comparison
RNA viruses frequently encode proteins that antagonize the innate immune response to infection. Mitochondria play a significant role in signaling innate immune responses through...; (A) Time course of ISG54 and CXCL10 mRNA expression in RAW264.7 cells infected at an MOI of 0.1 TCID 50 per cell. mRNA levels were quantified by qPCR using an endogenous control...; Analysis of mRNA levels in RAW264.7 cells was performed at low MOI (0.1 TCID 50 units/cell). Cells were seeded at 2 × 10 5 cells per well in a 24 well dish, grown overnight a...
from paperReporting output
Report representative outputs alongside summary comparisons for To confirm the mitochondrial localization of VF1 during virus infection, mitochondria were purified from infected RAW264.7 cells at 15 hours post infection and analyzed for the..., RNA viruses frequently encode proteins that antagonize the innate immune response to infection. Mitochondria play a significant role in signaling innate immune responses through..., (A) Time course of ISG54 and CXCL10 mRNA expression in RAW264.7 cells infected at an MOI of 0.1 TCID 50 per cell. mRNA levels were quantified by qPCR using an endogenous control..., Analysis of mRNA levels in RAW264.7 cells was performed at low MOI (0.1 TCID 50 units/cell). Cells were seeded at 2 × 10 5 cells per well in a 24 well dish, grown overnight a....
inferred from protocolStructured statistical methods
RNA viruses frequently encode proteins that antagonize the innate immune response to infection. Mitochondria play a significant role in signaling innate immune responses through...; (A) Time course of ISG54 and CXCL10 mRNA expression in RAW264.7 cells infected at an MOI of 0.1 TCID 50 per cell. mRNA levels were quantified by qPCR using an endogenous control...; Analysis of mRNA levels in RAW264.7 cells was performed at low MOI (0.1 TCID 50 units/cell). Cells were seeded at 2 × 10 5 cells per well in a 24 well dish, grown overnight a...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (6)
To confirm the mitochondrial localization of VF1 during virus infection, mitochondria were purified from infected RAW264.7 cells at 15 hours post infection and analyzed for the presence of VF1 by western blot ( ). Whereas the well characterized host cell nucleic acid binding proteins PCBP1/2 were shown to be predominantly cytoplasmic as expected, VF1 was only detected in the mitochondrial fraction ( ). Apoptosis inducing factor 1, a predominantly mitochondrial protein was enriched in the mitochondrial fraction, confirming the validity of the purification procedure ( ).
RNA viruses frequently encode proteins that antagonize the innate immune response to infection. Mitochondria play a significant role in signaling innate immune responses through the well characterized mitochondrial antiviral signaling protein (MAVS), an integral membrane protein found in the outer mitochondrial membrane -. MAVS is a key adapter protein in the sensing of viral RNA by RIG-I and MDA5 that, in part, leads to IRF3 and NFΚB activation and the upregulation of antiviral genes such as IFN-Beta, CXCL10 and ISG54. Given the mitochondrial localization of VF1 in infected cells we assessed the activation of this sub-section of the innate immune response in both M1 and WT infected cells. RAW264.7 cells infected at a low MOI (0.1 TCID 50 per cell) with the M1 VF1 knockout virus exhibited a greater induction of antiviral genes such as ISG54, CXCL10 and IFN-Beta in response to viral infection than those infected with the WT virus ( and ). Alterations to the levels of mRNA were calculated relative to uninfected cells using the standard ΔΔCT method with hypoxanthine phosphoribosyltransferase 1 (HPRT) ( and ) or actin (data not shown) mRNA levels used as endog...
(A) Time course of ISG54 and CXCL10 mRNA expression in RAW264.7 cells infected at an MOI of 0.1 TCID 50 per cell. mRNA levels were quantified by qPCR using an endogenous control gene (Hypoxanthine-guanine phosphoribosyltransferase, HPRT). Expression of the respective mRNAs was then calculated using the ΔΔCt method to compare infected and mock infected cells. Relative fold change was calculated using mock infected samples taken at comparable time points. Normalization was performed following MNV quantification in each sample. Infections were carried out in triplicate with each sample being subsequently analyzed in duplicate by qPCR. The error bars denote standard deviation from the mean. Statistical analysis was performed using an unpaired two tailed t test. (B) Time course of IFN-Beta mRNA and protein production in RAW264.7 cells infected as in (A). mRNA upregulation was calculated as described for (A). Interferon Beta protein was quantified by murine IFN-B specific ELISA at 24 hpi from supernatant samples taken from infected cells. For the ELISA infections were carried out in sextuplate. The error bars denote standard deviation from the mean. (C) MEF cells transfected...
Analysis of mRNA levels in RAW264.7 cells was performed at low MOI (0.1 TCID 50 units/cell). Cells were seeded at 2 × 10 5 cells per well in a 24 well dish, grown overnight at 37°C before being infected. RNA was harvested from infected cells at 16, 20 and 24 hours post infection using the GenElute RNA extraction kit (Sigma) as detailed by the manufacturer. RNA was quantified and diluted to a standard concentration before being reverse transcribed using MuMLV RT enzyme (Promega) using an oligo dT primer. SYBR green based qPCR was performed using an ABI 7900 HT real time PCR machine. The MESA Blue (Eurogentec) SYBR master mix was combined with sample cDNAs and optimised high efficiency mouse specific primers for the following genes: HPRT, CXCL10, ISG54 and Interferon Beta (primer details available upon request). Relative mRNA fold change was calculated using the ΔΔCt method e.g. normalising target mRNA levels based on an endogenous control (HPRT) before comparison with mock infected cells at equivalent time points. Where appropriate this fold change was then normalised to the level of MNV RNA detected in each sample in order to provide statistical information on...
The response of RAW264.7 cells to polyIC and UV inactivated viruses. IFN-Beta, CXCL10 and ISG54 mRNA and IFN-Beta protein analysis of RAW264.7 cells (A) infected with an equivalent MOI of 0.1 of UV inactivated M1 and WT viruses at 24hpi or (B) treated with 25 µg/ml polyIC for 24 hours. mRNA levels were quantified by qPCR using an endogenous control gene (Hypoxanthine-guanine phosphoribosyltransferase, HPRT). Expression of the respective mRNAs was then calculated using the ΔΔCt method to compare infected and mock infected cells. IFN-Beta protein secretion was quantified by murine IFN-B specific ELISA in the supernatants of treated/infected cells. Relative fold change was calculated using mock infected samples taken at comparable time points.
CXCL10 and ISG54 responses in MNV infected STAT-/- mice at 3 days post infection. Tissues were harvested from STAT1-/- mice infected with 1000 TCID-50 of MNV-1 (CW1.P1) at 3 days post infection. Relative fold induction of the CXCL10 and ISG54 mRNAs was calculated for each tissue separately, through comparison with mRNA levels in tissues from a mock/uninfected mouse (represented by the green line). This analysis was performed using the ΔΔCt qPCR method with the cellular gene HPRT used as an endogenous control. The data shown is from two separate animals, with the bar referring to the mean fold induction detected in each instance.
Machine-readable layer
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