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XRN1 stalling in the 5' UTR of Hepatitis C virus and Bovine Viral Diarrhea virus is associated with dysregulated host mRNA stability.

Moon SL, Blackinton JG, Anderson JR, Dozier MK, Dodd BJ, Keene JD, Wilusz CJ, Bradrick SS, Wilusz J - PLoS Pathog. (2015)

Bottom Line: We demonstrate that both Hepatitis C virus (HCV) and Bovine Viral Diarrhea virus (BVDV) contain regions in their 5' UTRs that stall and repress the enzymatic activity of the cellular 5'-3' exoribonuclease XRN1, resulting in dramatic changes in the stability of cellular mRNAs.In the context of HCV infection, we observed globally increased stability of mRNAs resulting in significant increases in abundance of normally short-lived mRNAs encoding a variety of relevant oncogenes and angiogenesis factors.These findings suggest that non-coding regions from multiple genera of the Flaviviridae interfere with XRN1 and impact post-transcriptional processes, causing global dysregulation of cellular gene expression which may promote cell growth and pathogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado, United States of America.

ABSTRACT
We demonstrate that both Hepatitis C virus (HCV) and Bovine Viral Diarrhea virus (BVDV) contain regions in their 5' UTRs that stall and repress the enzymatic activity of the cellular 5'-3' exoribonuclease XRN1, resulting in dramatic changes in the stability of cellular mRNAs. We used biochemical assays, virus infections, and transfection of the HCV and BVDV 5' untranslated regions in the absence of other viral gene products to directly demonstrate the existence and mechanism of this novel host-virus interaction. In the context of HCV infection, we observed globally increased stability of mRNAs resulting in significant increases in abundance of normally short-lived mRNAs encoding a variety of relevant oncogenes and angiogenesis factors. These findings suggest that non-coding regions from multiple genera of the Flaviviridae interfere with XRN1 and impact post-transcriptional processes, causing global dysregulation of cellular gene expression which may promote cell growth and pathogenesis.

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RNAs containing the 3’ or 5’ UTR of various members of the Flaviviridae are reversible inhibitors of XRN1 activity.Panel A. The effects of adding control (pGem-4 derived), JEV 3’ UTR, or DENV 3’ UTR competitor RNAs that contained a 5’ monophosphate on XRN-1 mediated decay of a radioactive reporter transcript in HeLa extract were assessed over the indicated time course of incubation. Products were analyzed on a 5% denaturing acrylamide gel and quantified by phosphorimaging. The average +/− standard deviation of the percent reporter RNA remaining from two independent experiments is shown below the gel. Asterisks indicate significant differences of percent reporter RNA remaining compared to controls. Panel B. Increasing amounts of recombinant XRN1 were incubated with a radioactive reporter transcript either in the absence of competitor RNA (No Competitor), with the DENV-2 3’ UTR competitor RNA (DENV-2 3’ UTR), or with a control competitor RNA (Control RNA). The rate at which the reporter RNA decayed in the presence of each competitor was calculated over the indicated range of XRN1 concentrations. The average +/− standard deviation of three independent experiments is shown with asterisks indicating significant differences between the reporter RNA decay rates compared to control at each time point. Panel C. Similar to panel A with the addition of the HCV and BVDV 5’ UTR RNAs included as competitors. Results were quantified as described for panel A and the average +/− standard deviation of three independent competition experiments is shown graphically. The significance of the differences in the half-life of the reporter RNA in the presence of DENV-2, BVDV, or HCV competitor RNAs compared to reactions containing the Control RNA competitor are indicated in the inset. Panel D. Total RNA from HCV replicon containing or HCV infected Huh7.5 cells was fractionated using an antibody to the methyl guanosine cap structure and qRT-PCR performed to detect TUT1 and FOS mRNA abundances in the uncapped fraction using the endogenous 7SL transcript as a reference gene. The uncapped fraction was normalized to 10% of the input RNA. The average +/− standard deviation of RNAs from three independent infections is shown. Significance was determined by t-test. For all panels *p≤0.05, **p≤0.01 and ***p≤0.001.
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ppat.1004708.g003: RNAs containing the 3’ or 5’ UTR of various members of the Flaviviridae are reversible inhibitors of XRN1 activity.Panel A. The effects of adding control (pGem-4 derived), JEV 3’ UTR, or DENV 3’ UTR competitor RNAs that contained a 5’ monophosphate on XRN-1 mediated decay of a radioactive reporter transcript in HeLa extract were assessed over the indicated time course of incubation. Products were analyzed on a 5% denaturing acrylamide gel and quantified by phosphorimaging. The average +/− standard deviation of the percent reporter RNA remaining from two independent experiments is shown below the gel. Asterisks indicate significant differences of percent reporter RNA remaining compared to controls. Panel B. Increasing amounts of recombinant XRN1 were incubated with a radioactive reporter transcript either in the absence of competitor RNA (No Competitor), with the DENV-2 3’ UTR competitor RNA (DENV-2 3’ UTR), or with a control competitor RNA (Control RNA). The rate at which the reporter RNA decayed in the presence of each competitor was calculated over the indicated range of XRN1 concentrations. The average +/− standard deviation of three independent experiments is shown with asterisks indicating significant differences between the reporter RNA decay rates compared to control at each time point. Panel C. Similar to panel A with the addition of the HCV and BVDV 5’ UTR RNAs included as competitors. Results were quantified as described for panel A and the average +/− standard deviation of three independent competition experiments is shown graphically. The significance of the differences in the half-life of the reporter RNA in the presence of DENV-2, BVDV, or HCV competitor RNAs compared to reactions containing the Control RNA competitor are indicated in the inset. Panel D. Total RNA from HCV replicon containing or HCV infected Huh7.5 cells was fractionated using an antibody to the methyl guanosine cap structure and qRT-PCR performed to detect TUT1 and FOS mRNA abundances in the uncapped fraction using the endogenous 7SL transcript as a reference gene. The uncapped fraction was normalized to 10% of the input RNA. The average +/− standard deviation of RNAs from three independent infections is shown. Significance was determined by t-test. For all panels *p≤0.05, **p≤0.01 and ***p≤0.001.

Mentions: In addition to stalling XRN1, we previously demonstrated that XRN1 enzymatic activity is repressed by the generation of sfRNA from WNV or DENV-2 3’ UTRs [41]. These observations are now extended to include sfRNA generation from the 3’ UTR of JEV in Fig. 3A. Briefly, a radiolabeled reporter RNA containing a 5’ mono-phosphate was efficiently degraded by XRN1 in HeLa cytoplasmic extracts in the absence of any competitor RNA (‘none’ lanes) or in the presence of a control RNA competitor (‘Control RNA’ lanes). However, RNA competitors containing either the 3’ UTR of JEV or the 3’ UTR of DENV-2 dramatically repressed XRN1 activity and this sfRNA-mediated repression was reversible (Fig. 3B). When the rate of RNA decay was measured relative to increasing concentrations of XRN1 enzyme, the rate of the reaction slowed in the presence of an sfRNA generating competitor RNA relative to control conditions, but the enzyme was not effectively titrated away by the RNA inhibitor as would be expected for an irreversible competitor.


XRN1 stalling in the 5' UTR of Hepatitis C virus and Bovine Viral Diarrhea virus is associated with dysregulated host mRNA stability.

Moon SL, Blackinton JG, Anderson JR, Dozier MK, Dodd BJ, Keene JD, Wilusz CJ, Bradrick SS, Wilusz J - PLoS Pathog. (2015)

RNAs containing the 3’ or 5’ UTR of various members of the Flaviviridae are reversible inhibitors of XRN1 activity.Panel A. The effects of adding control (pGem-4 derived), JEV 3’ UTR, or DENV 3’ UTR competitor RNAs that contained a 5’ monophosphate on XRN-1 mediated decay of a radioactive reporter transcript in HeLa extract were assessed over the indicated time course of incubation. Products were analyzed on a 5% denaturing acrylamide gel and quantified by phosphorimaging. The average +/− standard deviation of the percent reporter RNA remaining from two independent experiments is shown below the gel. Asterisks indicate significant differences of percent reporter RNA remaining compared to controls. Panel B. Increasing amounts of recombinant XRN1 were incubated with a radioactive reporter transcript either in the absence of competitor RNA (No Competitor), with the DENV-2 3’ UTR competitor RNA (DENV-2 3’ UTR), or with a control competitor RNA (Control RNA). The rate at which the reporter RNA decayed in the presence of each competitor was calculated over the indicated range of XRN1 concentrations. The average +/− standard deviation of three independent experiments is shown with asterisks indicating significant differences between the reporter RNA decay rates compared to control at each time point. Panel C. Similar to panel A with the addition of the HCV and BVDV 5’ UTR RNAs included as competitors. Results were quantified as described for panel A and the average +/− standard deviation of three independent competition experiments is shown graphically. The significance of the differences in the half-life of the reporter RNA in the presence of DENV-2, BVDV, or HCV competitor RNAs compared to reactions containing the Control RNA competitor are indicated in the inset. Panel D. Total RNA from HCV replicon containing or HCV infected Huh7.5 cells was fractionated using an antibody to the methyl guanosine cap structure and qRT-PCR performed to detect TUT1 and FOS mRNA abundances in the uncapped fraction using the endogenous 7SL transcript as a reference gene. The uncapped fraction was normalized to 10% of the input RNA. The average +/− standard deviation of RNAs from three independent infections is shown. Significance was determined by t-test. For all panels *p≤0.05, **p≤0.01 and ***p≤0.001.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4352041&req=5

ppat.1004708.g003: RNAs containing the 3’ or 5’ UTR of various members of the Flaviviridae are reversible inhibitors of XRN1 activity.Panel A. The effects of adding control (pGem-4 derived), JEV 3’ UTR, or DENV 3’ UTR competitor RNAs that contained a 5’ monophosphate on XRN-1 mediated decay of a radioactive reporter transcript in HeLa extract were assessed over the indicated time course of incubation. Products were analyzed on a 5% denaturing acrylamide gel and quantified by phosphorimaging. The average +/− standard deviation of the percent reporter RNA remaining from two independent experiments is shown below the gel. Asterisks indicate significant differences of percent reporter RNA remaining compared to controls. Panel B. Increasing amounts of recombinant XRN1 were incubated with a radioactive reporter transcript either in the absence of competitor RNA (No Competitor), with the DENV-2 3’ UTR competitor RNA (DENV-2 3’ UTR), or with a control competitor RNA (Control RNA). The rate at which the reporter RNA decayed in the presence of each competitor was calculated over the indicated range of XRN1 concentrations. The average +/− standard deviation of three independent experiments is shown with asterisks indicating significant differences between the reporter RNA decay rates compared to control at each time point. Panel C. Similar to panel A with the addition of the HCV and BVDV 5’ UTR RNAs included as competitors. Results were quantified as described for panel A and the average +/− standard deviation of three independent competition experiments is shown graphically. The significance of the differences in the half-life of the reporter RNA in the presence of DENV-2, BVDV, or HCV competitor RNAs compared to reactions containing the Control RNA competitor are indicated in the inset. Panel D. Total RNA from HCV replicon containing or HCV infected Huh7.5 cells was fractionated using an antibody to the methyl guanosine cap structure and qRT-PCR performed to detect TUT1 and FOS mRNA abundances in the uncapped fraction using the endogenous 7SL transcript as a reference gene. The uncapped fraction was normalized to 10% of the input RNA. The average +/− standard deviation of RNAs from three independent infections is shown. Significance was determined by t-test. For all panels *p≤0.05, **p≤0.01 and ***p≤0.001.
Mentions: In addition to stalling XRN1, we previously demonstrated that XRN1 enzymatic activity is repressed by the generation of sfRNA from WNV or DENV-2 3’ UTRs [41]. These observations are now extended to include sfRNA generation from the 3’ UTR of JEV in Fig. 3A. Briefly, a radiolabeled reporter RNA containing a 5’ mono-phosphate was efficiently degraded by XRN1 in HeLa cytoplasmic extracts in the absence of any competitor RNA (‘none’ lanes) or in the presence of a control RNA competitor (‘Control RNA’ lanes). However, RNA competitors containing either the 3’ UTR of JEV or the 3’ UTR of DENV-2 dramatically repressed XRN1 activity and this sfRNA-mediated repression was reversible (Fig. 3B). When the rate of RNA decay was measured relative to increasing concentrations of XRN1 enzyme, the rate of the reaction slowed in the presence of an sfRNA generating competitor RNA relative to control conditions, but the enzyme was not effectively titrated away by the RNA inhibitor as would be expected for an irreversible competitor.

Bottom Line: We demonstrate that both Hepatitis C virus (HCV) and Bovine Viral Diarrhea virus (BVDV) contain regions in their 5' UTRs that stall and repress the enzymatic activity of the cellular 5'-3' exoribonuclease XRN1, resulting in dramatic changes in the stability of cellular mRNAs.In the context of HCV infection, we observed globally increased stability of mRNAs resulting in significant increases in abundance of normally short-lived mRNAs encoding a variety of relevant oncogenes and angiogenesis factors.These findings suggest that non-coding regions from multiple genera of the Flaviviridae interfere with XRN1 and impact post-transcriptional processes, causing global dysregulation of cellular gene expression which may promote cell growth and pathogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado, United States of America.

ABSTRACT
We demonstrate that both Hepatitis C virus (HCV) and Bovine Viral Diarrhea virus (BVDV) contain regions in their 5' UTRs that stall and repress the enzymatic activity of the cellular 5'-3' exoribonuclease XRN1, resulting in dramatic changes in the stability of cellular mRNAs. We used biochemical assays, virus infections, and transfection of the HCV and BVDV 5' untranslated regions in the absence of other viral gene products to directly demonstrate the existence and mechanism of this novel host-virus interaction. In the context of HCV infection, we observed globally increased stability of mRNAs resulting in significant increases in abundance of normally short-lived mRNAs encoding a variety of relevant oncogenes and angiogenesis factors. These findings suggest that non-coding regions from multiple genera of the Flaviviridae interfere with XRN1 and impact post-transcriptional processes, causing global dysregulation of cellular gene expression which may promote cell growth and pathogenesis.

Show MeSH
Related in: MedlinePlus