Inhibition of the host 5'-3' RNA decay pathway is a novel mechanism by which flaviviruses influence cellular gene expression

2014 Spring.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

Metabolomics based annotation of novel genes in Arabidopsis thaliana

Of the approximately 30,000 genes that are predicted to be in the Arabidopsis genome, the function of a large proportion is experimentally undetermined. The NSF Arabidopsis 2010 program has supported the development of community resources to identify the functions of these genes. As part of this effort, a consortium of metabolomics laboratories has been established to generate and evaluate metabolomics data as a tool for generating hypotheses concerning the metabolic and physiological function of genes of unknown function. In combination, this consortium has the ability to evaluate the relative abundance of nearly 2,000 Arabidopsis metabolites. This consortium is combining reverse genetics and metabolomics to evaluate the consequence of loss-of-function mutations on the Arabidopsis metabolome and provide information to formulate initial hypotheses about genes of unknown function that can be further explored by the research community

Framework for Implementation of a Partial Hospitalization Program

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The role of protein kinase R in dystonia

Dystonia is a progressive neurological motor disease with few treatment options and no cure. This review synthesizes the results of recent studies that implicate protein kinase R in mediating the molecular mechanisms of dystonia pathogenesis. Mutations in the PKR gene EIF2AK2 and the PKR activator protein PACT are associated with early-onset generalized dystonia. Protein kinase R (PKR) is important for neuronal function. Genetic depletion or inhibition of PKR is associated with increased long-term potentiation and memory, while also causing neuronal hyper-excitability and seizures in mouse models. PKR also senses double stranded RNA within cells and activates the integrated stress response (ISR). The ISR is a conserved signaling pathway that hinges on controlled translational suppression to remodel gene expression during stress. When PKR is activated through binding double stranded RNA or the PKR activator protein PACT, PKR dimerizes, autophosphorylates, and phosphorylates the translation initiation factor eIF2. Translation suppression by p-eIF2 causes stress granule formation and the upregulation of stress-induced genes. The ISR is thought to drive cellular resilience during acute stress. However, chronic ISR activation is associated with neurological diseases, traumatic brain injury, and aging. Neurodevelopmental and neurodegenerative diseases are associated with mutations in other integrated stress response genes, suggesting a critical role for ISR regulation in neuronal health. A growing body of work suggests the ISR is also dysfunctional in dystonia. Future research investigating the molecular mechanisms of the ISR in dystonia will likely reveal therapeutic targets and treatment strategies for this currently incurable disease

Characterization of Kaposi's sarcoma-associated herpesvirus (KSHV) K1 promoter activation by Rta

The K1 gene of Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a 46-kDa transmembrane glycoprotein that possesses transforming properties, initiates signaling pathways in B cells, and prevents apoptosis. Here, we demonstrate a mechanism for activation of the K1 promoter by the Rta transactivator. Electrophoretic mobility shift assay (EMSA) analysis of the K1 promoter demonstrated that purified Rta protein bound to the K1 promoter at three locations, independent of other DNA-binding factors. Transcriptional assays revealed that only two of these Rta DNA-binding sites are functionally significant, and that they could impart Rta responsiveness to a heterologous E4 TATA box promoter. In addition, TATA-binding protein (TBP) bound to a TATA box element located 25 bp upstream of the K1 transcription start site and was also shown to associate with Rta by coimmunoprecipitation analysis. Rta transactivation may therefore be mediated in part through recruitment of TBP to target promoters

Analysis of eIF2B bodies and their relationships with stress granules and P-bodies.

Eukaryotic cells respond to stress and changes in the environment in part by repressing translation and forming cytoplasmic assemblies called stress granules and P-bodies, which harbor non-translating mRNAs and proteins. A third, but poorly understood, assembly called the eIF2B body can form and contains the eIF2B complex, an essential guanine exchange factor for the translation initiation factor eIF2. Hypomorphic EIF2B alleles can lead to Vanishing White Matter Disease (VWMD), a leukodystrophy that causes progressive white matter loss. An unexplored question is how eIF2B body formation is controlled and whether VWMD alleles in EIF2B alter the formation of eIF2B bodies, stress granules, or P-bodies. To examine these issues, we assessed eIF2B body, stress granule, and P-body induction in wild-type yeast cells and cells carrying VWMD alleles in the EIF2B2 (GCD7) and EIF2B5 (GCD6) subunits of eIF2B. We demonstrate eIF2B bodies are rapidly and reversibly formed independently of stress granules during acute glucose deprivation. VWMD mutations had diverse effects on stress-induced assemblies with some alleles altering eIF2B bodies, and others leading to increased P-body formation. Moreover, some VWMD-causing mutations in GCD7 caused hyper-sensitivity to chronic GCN2 activation, consistent with VWMD mutations causing hyper-sensitivity to eIF2α phosphorylation and thereby impacting VWMD pathogenesis

Oxidative stress influences positive strand RNA virus genome synthesis and capping

AbstractFlaviviruses are 5′ capped positive-stranded RNA viruses that replicate their genomes within endoplasmic reticulum-derived vesicles. Flaviviruses are well known to induce oxidative stress late in infection but it is unknown if oxidative stress plays a positive role in the viral RNA replication cycle. We therefore examined how oxidation affects flavivirus RNA replication. We found that antioxidant treatment reduced virus production, reduced the viral positive-to-negative strand RNA ratio, and resulted in the accumulation of uncapped positive-sense viral RNAs. Treatment of the NS5 RNA capping enzyme in vitro with oxidizing agents enhanced guanylyltransferase activity, indicating that the guanylyltransferase function of the flavivirus NS5 RNA capping enzyme is activated by oxidative conditions. Antioxidant treatment also reduced alphavirus RNA replication and protein expression while enhancing nsP1 capping activity. These findings suggest that RNA viruses may utilize oxidative stress induced during infection to help temporally control genome RNA capping and genome replication

A Pilot Test of a Mobile App for Drug Court Participants

The U.S. criminal justice system refers more people to substance abuse treatment than any other system. Low treatment completion rates and high relapse rates among addicted offenders highlight the need for better substance use disorder treatment and recovery tools. Mobile health applications (apps) may fill that need by providing continuous support. In this pilot test, 30 participants in a Massachusetts drug court program used A-CHESS, a mobile app for recovery support and relapse prevention, over a four-month period. Over the course of the study period, participants opened A-CHESS on average of 62% of the days that they had the app. Social networking tools were the most utilized services. The study results suggest that drug court participants will make regular use of a recovery support app. This pilot study sought to find out if addicted offenders in a drug court program would use a mobile application to support and manage their recovery

Use of lip protecting agents in the prevention of Actinic cheilitis, Herpes labialis and cancer of lip: a systematic review

Background: Actinic cheilitis, herpes labialis and lip cancer are relatively common conditions presenting on the lips associated with exposure to periods of sun exposure and thereby ultraviolet radiation. Objective: This systematic review aimed to determine the efficacy of the application of sunscreen-containing lip-protecting agents (LPA) in the prevention of actinic cheilitis (AC), recurrent herpes labialis (RHL) and lip cancer (LC). Methods: This review was conducted in accordance with the PRISMA guidelines and registered with the PROSPERO database. A literature search was conducted using SCOPUS, Google Scholar, Medline (Ovid), Pubmed, CINAHL, Cochrane Library databases and manual search using search terms actinic cheilitis (AC), recurrent herpes labialis (RHL) and lip cancer (LC) along with lip protecting agents and their variations as keywords. A total of 1,567 papers were yielded. Of them, nine studies were eligible for qualitative data synthesis. Results: Nine articles (3 AC, 5 RHL, 1 LC) were deemed eligible and thus selected for qualitative synthesis. Three studies on AC identified approximately 21.7% lower prevalence of lesions when some form of lip protection was used. Eighty percent of studies on RHL identified that the application of LPA is effective in preventing RHL. Subjects who applied LPA more than once daily only had half the risk of having LC compared to those who applied once daily. Conclusion: This review of randomised controlled trials (RCTs) and observational studies supports the use of LPA as an effective method in preventing lip-associated lesions. Further, RCTs and observational studies should aim at determining a definitive LPA application regime and optimal SPF strength to prevent lip-associated lesions

A noncoding RNA produced by arthropod-borne flaviviruses inhibits the cellular exoribonuclease XRN1 and alters host mRNA stability

All arthropod-borne flaviviruses generate a short noncoding RNA (sfRNA) from the viral 3′ untranslated region during infection due to stalling of the cellular 5′-to-3′ exonuclease XRN1. We show here that formation of sfRNA also inhibits XRN1 activity. Cells infected with Dengue or Kunjin viruses accumulate uncapped mRNAs, decay intermediates normally targeted by XRN1. XRN1 repression also resulted in the increased overall stability of cellular mRNAs in flavivirus-infected cells. Importantly, a mutant Kunjin virus that cannot form sfRNA but replicates to normal levels failed to affect host mRNA stability or XRN1 activity. Expression of sfRNA in the absence of viral infection demonstrated that sfRNA formation was directly responsible for the stabilization of cellular mRNAs. Finally, numerous cellular mRNAs were differentially expressed in an sfRNA-dependent fashion in a Kunjin virus infection. We conclude that flaviviruses incapacitate XRN1 during infection and dysregulate host mRNA stability as a result of sfRNA formation