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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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Determination of the 5’ ends of the HCV and BVDV 5’ UTR XRN1-mediated decay intermediates.Unlabeled RNAs containing either the HCV (Panel A) or BVDV 5’ UTR (Panel D) were incubated with recombinant XRN1. Decay intermediates were identified by SYBR green staining on a 5% denaturing acrylamide gel. The indicated bands were excised, circularized, and the 5’-3’ junction fragment was cloned and sequenced. Panels C and F: The position of the 5’-most nucleotide in each decay intermediates (1, 2, and 3) is indicated by the arrows. The percent of observed 5’ ends in the six sequenced clones from each RNA intermediate are indicated. Panel B and E. HCV and BVDV 5’UTR structure diagrams (modified from [46]) with arrows indicating the sites where XRN1 stalls. The numbers indicate which intermediate corresponds to the gels in panels A and D.
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ppat.1004708.g002: Determination of the 5’ ends of the HCV and BVDV 5’ UTR XRN1-mediated decay intermediates.Unlabeled RNAs containing either the HCV (Panel A) or BVDV 5’ UTR (Panel D) were incubated with recombinant XRN1. Decay intermediates were identified by SYBR green staining on a 5% denaturing acrylamide gel. The indicated bands were excised, circularized, and the 5’-3’ junction fragment was cloned and sequenced. Panels C and F: The position of the 5’-most nucleotide in each decay intermediates (1, 2, and 3) is indicated by the arrows. The percent of observed 5’ ends in the six sequenced clones from each RNA intermediate are indicated. Panel B and E. HCV and BVDV 5’UTR structure diagrams (modified from [46]) with arrows indicating the sites where XRN1 stalls. The numbers indicate which intermediate corresponds to the gels in panels A and D.

Mentions: To formally test the hypothesis that XRN1 stalls in the 5’ UTR of HCV and BVDV, we created RNA substrates that have a 5’ mono-phosphate (and are therefore susceptible to XRN1 digestion) and contained either an HCV/BVDV 5’ UTR or control sequences. These RNAs were incubated in XRN1 exonuclease assays either using HeLa cytoplasmic extract (Fig. 1B upper panel) or purified recombinant XRN1 (Fig. 1B, lower panel) using incubation times to optimize 5’-3’ exonucleolytic decay. HeLa cytoplasmic extracts were used in these studies due to their well-characterized usage in RNA decay studies (e.g. [41]). However it should be noted that they may also contain XRN2 activity and this 5’-3’ exonuclease may also be contributing to the decay observed in these assays [45]. As seen in Fig. 1B, RNA substrates that contained either the HCV or BVDV 5’ UTRs generated XRN1-mediated decay intermediates. For HCV, three degradation intermediates were identified by cloning and sequencing with 5’ ends at approximately 5, 45 and 123 bases from the normal 5’ end of the genomic RNA. BVDV RNA degradation yielded two decay intermediates, a strong band at ∼70 nt and a weaker but reproducible stop at ∼137 nt from the 5’ end of the genome. The positions of these XRN1 decay stall sites on the HCV and BVDV 5’ UTRs are indicated in Fig. 2. Notably, the stall sites are all positioned at or near predicted sites of structural landmarks in both RNAs [46]. Importantly, no XRN1 decay intermediates are seen with numerous control RNAs in these assays (Figs. 1B, S1A and [47]). The observation of decay intermediates when using purified XRN1 and purified RNA implies that the RNA by itself is sufficient to stall XRN1 and no additional RNA-protein interactions are needed.


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)

Determination of the 5’ ends of the HCV and BVDV 5’ UTR XRN1-mediated decay intermediates.Unlabeled RNAs containing either the HCV (Panel A) or BVDV 5’ UTR (Panel D) were incubated with recombinant XRN1. Decay intermediates were identified by SYBR green staining on a 5% denaturing acrylamide gel. The indicated bands were excised, circularized, and the 5’-3’ junction fragment was cloned and sequenced. Panels C and F: The position of the 5’-most nucleotide in each decay intermediates (1, 2, and 3) is indicated by the arrows. The percent of observed 5’ ends in the six sequenced clones from each RNA intermediate are indicated. Panel B and E. HCV and BVDV 5’UTR structure diagrams (modified from [46]) with arrows indicating the sites where XRN1 stalls. The numbers indicate which intermediate corresponds to the gels in panels A and D.
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Related In: Results  -  Collection

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

ppat.1004708.g002: Determination of the 5’ ends of the HCV and BVDV 5’ UTR XRN1-mediated decay intermediates.Unlabeled RNAs containing either the HCV (Panel A) or BVDV 5’ UTR (Panel D) were incubated with recombinant XRN1. Decay intermediates were identified by SYBR green staining on a 5% denaturing acrylamide gel. The indicated bands were excised, circularized, and the 5’-3’ junction fragment was cloned and sequenced. Panels C and F: The position of the 5’-most nucleotide in each decay intermediates (1, 2, and 3) is indicated by the arrows. The percent of observed 5’ ends in the six sequenced clones from each RNA intermediate are indicated. Panel B and E. HCV and BVDV 5’UTR structure diagrams (modified from [46]) with arrows indicating the sites where XRN1 stalls. The numbers indicate which intermediate corresponds to the gels in panels A and D.
Mentions: To formally test the hypothesis that XRN1 stalls in the 5’ UTR of HCV and BVDV, we created RNA substrates that have a 5’ mono-phosphate (and are therefore susceptible to XRN1 digestion) and contained either an HCV/BVDV 5’ UTR or control sequences. These RNAs were incubated in XRN1 exonuclease assays either using HeLa cytoplasmic extract (Fig. 1B upper panel) or purified recombinant XRN1 (Fig. 1B, lower panel) using incubation times to optimize 5’-3’ exonucleolytic decay. HeLa cytoplasmic extracts were used in these studies due to their well-characterized usage in RNA decay studies (e.g. [41]). However it should be noted that they may also contain XRN2 activity and this 5’-3’ exonuclease may also be contributing to the decay observed in these assays [45]. As seen in Fig. 1B, RNA substrates that contained either the HCV or BVDV 5’ UTRs generated XRN1-mediated decay intermediates. For HCV, three degradation intermediates were identified by cloning and sequencing with 5’ ends at approximately 5, 45 and 123 bases from the normal 5’ end of the genomic RNA. BVDV RNA degradation yielded two decay intermediates, a strong band at ∼70 nt and a weaker but reproducible stop at ∼137 nt from the 5’ end of the genome. The positions of these XRN1 decay stall sites on the HCV and BVDV 5’ UTRs are indicated in Fig. 2. Notably, the stall sites are all positioned at or near predicted sites of structural landmarks in both RNAs [46]. Importantly, no XRN1 decay intermediates are seen with numerous control RNAs in these assays (Figs. 1B, S1A and [47]). The observation of decay intermediates when using purified XRN1 and purified RNA implies that the RNA by itself is sufficient to stall XRN1 and no additional RNA-protein interactions are needed.

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