A genome-wide RNAi screen reveals that mRNA decapping restricts bunyaviral replication by limiting the pools of Dcp2-accessible targets for cap-snatching

Hopkins, Kaycie C.; McLane, Laura M.; Maqbool, Tariq; Panda, Debasis; Gordesky-Gold, Beth; Cherry, Sara

doi:10.1101/gad.215384.113

Cited by 90 publications

(137 citation statements)

References 86 publications

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“…For bunyaviruses, a Drosophila melanogaster genome-wide siRNA screen was used to show that mRNA uncapping restricts bunyavirus replication (43). We describe here the results from the first human genome-wide siRNA screen for a bunyavirus and report the identity of 562 cellular factors in human cells with a potential role in UUKV entry and replication.…”

Section: Discussionmentioning

confidence: 99%

Genome-Wide Small Interfering RNA Screens Reveal VAMP3 as a Novel Host Factor Required for Uukuniemi Virus Late Penetration

Meier

Franceschini

Horváth

et al. 2014

J Virol

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The Bunyaviridae constitute a large family of enveloped animal viruses, many of which are important emerging pathogens. How bunyaviruses enter and infect mammalian cells remains largely uncharacterized. We used two genome-wide silencing screens with distinct small interfering RNA (siRNA) libraries to investigate host proteins required during infection of human cells by the bunyavirus Uukuniemi virus (UUKV), a late-penetrating virus. Sequence analysis of the libraries revealed that many siRNAs in the screens inhibited infection by silencing not only the intended targets but additional genes in a microRNA (miRNA)-like manner. That the 7-nucleotide seed regions in the siRNAs can cause a perturbation in infection was confirmed by using synthetic miRNAs (miRs). One of the miRs tested, miR-142-3p, was shown to interfere with the intracellular trafficking of incoming viruses by regulating the v-SNARE VAMP3, a strong hit shared by both siRNA screens. Inactivation of VAMP3 by the tetanus toxin led to a block in infection. Using fluorescence-based techniques in fixed and live cells, we found that the viruses enter VAMP3 ؉ endosomal vesicles 5 min after internalization and that colocalization was maximal 15 min thereafter. At this time, LAMP1 was associated with the VAMP3 ؉ virus-containing endosomes. In cells depleted of VAMP3, viruses were mainly trapped in LAMP1-negative compartments. Together, our results indicated that UUKV relies on VAMP3 for penetration, providing an indication of added complexity in the trafficking of viruses through the endocytic network. IMPORTANCEBunyaviruses represent a growing threat to humans and livestock globally. Unfortunately, relatively little is known about these emerging pathogens. We report here the first human genome-wide siRNA screens for a bunyavirus. The screens resulted in the identification of 562 host cell factors with a potential role in cell entry and virus replication. To demonstrate the robustness of our approach, we confirmed and analyzed the role of the v-SNARE VAMP3 in Uukuniemi virus entry and infection. The information gained lays the basis for future research into the cell biology of bunyavirus infection and new antiviral strategies. In addition, by shedding light on serious caveats in large-scale siRNA screening, our experimental and bioinformatics procedures will be valuable in the comprehensive analysis of past and future high-content screening data.

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Section: Discussionmentioning

confidence: 99%

Genome-Wide Small Interfering RNA Screens Reveal VAMP3 as a Novel Host Factor Required for Uukuniemi Virus Late Penetration

Meier

Franceschini

Horváth

et al. 2014

J Virol

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“…Decapping plays an important role in multiple aspects of eukaryotic cytoplasmic mRNA turnover Parker 2012) and is critical to numerous biological processes, including development (Xu et al 2006;Ma et al 2013), DNA replication (Mullen and Marzluff 2008;Schmidt et al 2011), stress response (Hilgers et al 2006;Xu and Chua 2012), synapse plasticity (Hillebrand et al 2010), retrotransposition (Dutko et al 2010), and viral replication (Hopkins et al 2013). In yeast, mRNA decapping is carried out by a single enzyme composed of a regulatory subunit (Dcp1) and a catalytic subunit (Dcp2), but also requires the functions of specific regulators commonly designated as "decapping activators" (Parker 2012).…”

Section: Introductionmentioning

confidence: 99%

Control of mRNA decapping by positive and negative regulatory elements in the Dcp2 C-terminal domain

Jacobson

2015

RNA

139

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Decapping commits an mRNA to complete degradation and promotes general 5 ′ to 3 ′ decay, nonsense-mediated decay (NMD), and transcript-specific degradation. In Saccharomyces cerevisiae, a single decapping enzyme composed of a regulatory subunit (Dcp1) and a catalytic subunit (Dcp2) targets thousands of distinct substrate mRNAs. However, the mechanisms controlling this enzyme's in vivo activity and substrate specificity remain elusive. Here, using a genetic approach, we show that the large C-terminal domain of Dcp2 includes a set of conserved negative and positive regulatory elements. A single negative element inhibits enzymatic activity and controls the downstream functions of several positive elements. The positive elements recruit the specific decapping activators Edc3, Pat1, and Upf1 to form distinct decapping complexes and control the enzyme's substrate specificity and final activation. Our results reveal unforeseen regulatory mechanisms that control decapping enzyme activity and function in vivo, and define roles for several decapping activators in the regulation of mRNA decapping.

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“…Their replication occurs exclusively in the host cell cytoplasm. Hantaviral RdRp initiates transcription by a unique cap-snatching mechanism to generate 5=-capped viral mRNAs (6)(7)(8). Despite their 5= caps, viral mRNAs must actively compete with host cell transcripts for the same translation machinery.…”

mentioning

confidence: 99%

Andes Virus Nucleocapsid Protein Interrupts Protein Kinase R Dimerization To Counteract Host Interference in Viral Protein Synthesis

Wang

Mir

2015

J Virol

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Pathogenic hantaviruses delay the type I interferon response during early stages of viral infection. However, the robust interferon response and induction of interferon-stimulated genes observed during later stages of hantavirus infection fail to combat the virus replication in infected cells. Protein kinase R (PKR), a classical interferon-stimulated gene product, phosphorylates the eukaryotic translation initiation factor eIF2␣ and causes translational shutdown to create roadblocks for the synthesis of viral proteins. The PKR-induced translational shutdown helps host cells to establish an antiviral state to interrupt virus replication. However, hantavirus-infected cells do not undergo translational shutdown and fail to establish an antiviral state during the course of viral infection. In this study, we showed for the first time that Andes virus infection induced PKR overexpression. However, the overexpressed PKR was not active due to a significant inhibition of autophosphorylation. Further studies revealed that Andes virus nucleocapsid protein inhibited PKR dimerization, a critical step required for PKR autophosphorylation to attain activity. The studies reported here establish a hantavirus nucleocapsid protein as a new PKR inhibitor. These studies provide mechanistic insights into hantavirus resistance to the host interferon response and solve the puzzle of the lack of translational shutdown observed in hantavirus-infected cells. The sensitivity of hantavirus replication to PKR has likely imposed a selective evolutionary pressure on hantaviruses to evade the PKR antiviral response for survival. We envision that evasion of the PKR antiviral response by NP has likely helped hantaviruses to exist during evolution and to survive in infected hosts with a multifaceted antiviral defense. IMPORTANCEProtein kinase R (PKR), a versatile antiviral host factor, shuts down the translation machinery upon activation in virus-infected cells to create hurdles for the manufacture of viral proteins. The studies reported here reveal that the hantavirus nucleocapsid protein counteracts the PKR antiviral response by inhibiting PKR dimerization, which is required for its activation. We report the discovery of a new PKR inhibitor whose expression in hantavirus-infected cells prevents the PKR-induced host translational shutdown to ensure the continuous synthesis of viral proteins required for efficient virus replication.

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A genome-wide RNAi screen reveals that mRNA decapping restricts bunyaviral replication by limiting the pools of Dcp2-accessible targets for cap-snatching

Cited by 90 publications

References 86 publications

Genome-Wide Small Interfering RNA Screens Reveal VAMP3 as a Novel Host Factor Required for Uukuniemi Virus Late Penetration

Genome-Wide Small Interfering RNA Screens Reveal VAMP3 as a Novel Host Factor Required for Uukuniemi Virus Late Penetration

Control of mRNA decapping by positive and negative regulatory elements in the Dcp2 C-terminal domain

Andes Virus Nucleocapsid Protein Interrupts Protein Kinase R Dimerization To Counteract Host Interference in Viral Protein Synthesis

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