Inhibition of the host 5'-3' RNA decay pathway is a novel mechanism by which flaviviruses influence cellular gene expression
Date
2014
Authors
Moon, Stephanie L., author
Wilusz, Jeffrey, advisor
Wilusz, Carol, advisor
Schenkel, Alan, committee member
Curthoys, Norman, committee member
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Abstract
Host gene expression is an intricate process that requires many levels of regulation to allow the cell to react properly to a given stimulus or maintain homeostasis. One mechanism by which RNA viruses perturb host gene expression and potentially favor the allocation of host cell resources for viral proliferation is through interfering with cellular post-transcriptional processes. Furthermore, because viral RNAs must persist in the host cell cytoplasm to allow translation of viral proteins and ultimately viral replication, the same post-transcriptional processes that regulate host messenger RNAs (mRNAs) likely act on viral RNAs as well. The general RNA decay machinery in the cell serves as an important regulatory step for proper gene expression at the post-transcriptional level. Many RNA viruses have evolved unique mechanisms for dealing with the cellular RNA decay machinery to preserve their transcripts and ensure a productive infection. Flaviviruses contain positive-sense, single-stranded RNA genomes that are not polyadenylated. Therefore, these viral RNAs are likely recognized by the host cell as deadenylated, incongruous mRNAs and are likely substrates for the general cellular RNA decay machinery. Remarkably, flaviviruses including the dengue viruses (DENV) and West Nile virus (WNV) produce an abundant non-coding subgenomic RNA (sfRNA) during infection that is generated through incomplete degradation of the viral genome by the host 5'-3' exoribonuclease 1 (XRN1). We demonstrate that human and mosquito XRN1 stalls on highly structured, conserved elements in the 3' untranslated region of flaviviral RNAs, resulting in sfRNA formation. Furthermore, we determined that these sfRNAs act as competitive, reversible inhibitors of XRN1. Infected cells display several signs of sfRNA-dependent XRN1 dysfunction, including the accumulation of uncapped transcripts and an overall stabilization of host mRNAs. Additionally, sfRNA acts as a weak inhibitor of the host cell RNA interference (RNAi) pathway. We propose that sfRNA likely acts as a sponge for Argonaute-2 (AGO2) and DICER, and have determined that siRNA-mediated decay is suppressed in an sfRNA-dependent fashion in flavivirus-infected human cells. This suppression of the RNAi pathway appears to alter host gene expression to a limited extent, and may be especially important for viral replication in the mosquito vector. Other flaviviruses, including hepatitis C virus (HCV) and bovine viral diarrhea virus (BVDV) do not form an sfRNA from their 3' untranslated regions, but they do contain highly structured 5' untranslated regions. Herein we show that aside from acting as internal ribosome entry sites, the 5' UTRs of HCV and BVDV also stall and inhibit XRN1. Therefore, flaviviruses, pestiviruses and hepaciviruses appear to inhibit a major mRNA decay pathway by suppressing XRN1 activity via highly structured viral RNAs. Consequences of XRN1 suppression during viral infection include the stabilization and upregulation of short-lived transcripts including those encoding oncogenes, angiogenic factors, and pro-inflammatory factors. Furthermore, we present evidence that WNV sfRNA may dysregulate the coordination between mRNA stability and transcription. Therefore, the suppression of XRN1 may potentially act as an important mechanism by which diverse viruses in the Flaviviridae induce pathogenesis by dysregulating cellular gene expression.
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Subject
mRNA stability
RNA interference
XRN1
virus-host interaction
flavivirus
mRNA decay