JVI Accepts, published online ahead of print on 12 October 2011 J. Virol. doi:10.1128/JVI.06191-11 Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.
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VPg-Primed RNA Synthesis by Norovirus RNA-Dependent RNA Polymerases Using a
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Novel Cell-based Assay
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Ch. V. Subba-Reddy1, Ian Goodfellow2*, and C. Cheng Kao1*
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1
Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, IN
47405 USA. 2
Section of Virology, Department of Medicine, Imperial College London, London W2 1PG, UK.
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* Co-corresponding authors: C. Kao, 205C Simon Hall, 212 S. Hawthorne Drive, Indiana
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University, Bloomington IN 47405. Phone: 812-855-7583. Fax: 812-856-5710. Email:
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[email protected].
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I. Goodfellow, Imperial College London, St Mary's Campus, Norfolk Place, London W2 1PG,
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UK. Phone: +44 2075942002. Fax: +44 207594 3973. Email:
[email protected]
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Running title: Norovirus RNA synthesis.
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ABSTRACT
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Molecular studies on human noroviruses (NoV) have been hampered by the lack of a
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permissive cell culture system. We have developed a sensitive and reliable mammalian cell-
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based assay for the human NoV GII.4 strain RNA-dependent RNA polymerase (RdRp). The
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assay is based on the finding that RNAs synthesized by transiently expressed RdRp can
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stimulate retinoic acid inducible gene-I (RIG-I)-dependent reporter luciferase production via
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the interferon β promoter. Comparable activities were observed for the murine norovirus
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(MNV) RdRp. RdRps with mutations at divalent metal ion binding residues did not activate
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RIG-I signaling. Furthermore, both NoV and MNV RdRp activities were stimulated by the co-
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expression of their respective VPg proteins, while mutations in the putative site of
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nucleotide linkage on VPg abolished most of their stimulatory effects. Sequencing of the
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RNAs linked to VPg revealed that the cellular TGOLN2 mRNA was the template for VPg
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primed RNA synthesis. siRNA knockdown of RNase L abolished the enhancement of
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signaling that occurred in the presence of VPg. Finally, the co-expression of each of the
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other NoV proteins revealed that the p48 (aka NS1-2) and VP1 enhanced and that VP2
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reduced the RdRp activity. The assay should be useful for the dissection of the
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requirements for NoV RNA synthesis as well as the identification of inhibitors of the NoV
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RdRp.
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INTRODUCTION
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Human noroviruses (NoV) (genus Norovirus, family Caliciviridae) are nonenveloped
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positive-strand RNA viruses, responsible for more than 90% of all epidemic viral
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gastroenteritis outbreaks in the United States (2). At least four epidemics of gastroenteritis
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have taken place between 1995 and 2009, with a single genotype, GII.4, being associated
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with these epidemics (9). Noroviruses are also highly contagious, extremely stable in the
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environment, resistant to common disinfectants, and associated with debilitating illness (33).
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Research in the development of prevention strategies has been impaired because NoV
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causing human disease lacks permissive cell-culture systems and robust animal models.
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The NoV genomic RNA is ~7.5 kb and the genome organization has some similarities to
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that of the related picornaviruses (Fig. 1A). The genome typically contains three open
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reading frames (ORFs). ORF2 and ORF3 encode the major and minor structural proteins
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VP1 and VP2 respectively (60). ORF1, located in the first two-thirds of the genome encodes
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a ~200 kDa polyprotein that is proteolytically processed by the virus-encoded 3C-like
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protease (3C Pro) to yield the non-structural proteins that are essential for the
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establishment of viral replication complexes and genome replication. Of particular note to
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this work, ORF1 contains NS5, also known as VPg, which is covalently linked to the 5′ end
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of the genome and NS7, the RNA-dependent RNA polymerase (RdRp). The key role that
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the NoV RdRp plays in viral genome replication and the fact that cells lack an equivalent,
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makes it an attractive target for development of norovirus-specific anti-viral therapies. The
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linkage of VPg to viral RNA is thought to occur during viral genome replication whereby VPg
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is attached as a protein primer to the 5’ terminus of the genomic RNAs (47). Biochemical
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data confirmed the ability of the NoV RdRp to transfer nucleotide to the VPg (47) and the
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covalent linkage of VPg to the 5’ end of NoV RNA has been shown to be essential for virus
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infectivity (24).
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Transcripts of the viral genome and dsRNA replication intermediates synthesized by the
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viral RdRp in a cell during viral replication are sensed by various innate immune receptors
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to activate the cellular defenses (62). The Retinoic acid Inducible Gene-I (RIG-I) and the
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Melanoma Differentiation–Associated gene 5 (MDA5) are the two important cytoplasmic
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innate immune receptors that contain DExD/H-box motifs characteristic of superfamily II
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helicases. Both proteins also contain two N-terminal caspase-recruitment domains (CARDs)
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that can interact with a CARD-domain in the adaptor protein, IPS-1 (also known as MAVS,
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VISA or Cardif) (67). IPS-1 relays the signal to the kinases TBK1 and IKK-ε, which
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phosphorylate the transcription factors, NF-kβ, IRF-3 and IRF-7. These transcription factors
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subsequently translocate to the nucleus to induce type-I interferon expression that further
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induces many interferon-stimulated genes (19, 27, 50, 53).
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We have developed a cell-based assay for human NoV RdRp (the NoV-5BR assay) that
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can assess RNAs synthesized by viral RdRps through the activation of RIG-I or MDA5,
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leading to the production of firefly luciferase from the IFNβ promoter (46). Using the NoV-
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5BR assay format, we demonstrate that the transiently-expressed RdRps from NoV GII.4,
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and related murine norovirus (MNV) are active for RNA synthesis. In the presence of co-
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expressed VPg, the NoV RdRp can produce a VPg-primed RNA that can be processed by
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the cellular endoribonuclease, RNase L. Furthermore, the NoV proteins p48, VP1 and VP2
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can modulate the GII.4 RdRp activity in a species-specific manner.
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MATERIALS AND METHODS Plasmid constructs and cell cultures. Each of the NoV genes p48 (NS1-2), NTPase
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(NS3), p22 (NS4), VPg (NS5), Pro (NS6), RdRp (NS7), VP1 and VP2 (Norovirus
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Hu/GII.4/MD-2004/2004/US, GenBank accession DQ658413) were custom synthesized
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(Origene, USA). Forward and reverse primers containing AgeI and NheI restriction enzyme
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sites, respectively, were used to amplify the cDNAs for subcloning into the pUNO vector.
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NoV RdRp with an N-terminal FLAG tag and NoV VPg with C-terminal FLAG- and
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hemagglutinin (HA)-tags were constructed using PCR. p48, NTPase, p22, 3C Pro, VP1 and
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VP2 contained HA tags at their C-termini. The sequences of all constructs used in this study
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were confirmed by use of the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied
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Biosystems, USA). MNV RdRp and VPg were PCR amplified from a cDNA clone of MNV-1
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strain CW1 (GenBank accession number: DQ285629.1; 64) and cloned into the pUNO
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vector (Invivogen Inc.).
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Plasmids containing the cDNAs of RIG-I (pUNO-hRIG) and MDA5 (pUNO-hMDA5) were
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from Invivogen (San Diego, CA). The TLR3 plasmid (pcDNA-TLR3) was previously
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described in Sun et al. (59). IFNβ-Luc plasmid containing firefly luciferase reporter gene
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driven by IFNβ promoter gene, was used as reporter plasmid. pRL-TK, expressing Renilla
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reniformis luciferase driven by the herpes simplex virus thymidine kinase (TK) promoter,
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was used to monitor and standardize the efficacy of transfection (Promega, Madison, WI,
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USA). For the assay in Huh-7 cells Renilla luciferase driven by CMV promoter was used.
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Dual luciferase reporter assays. Human embryonic kidney (HEK 293T) cells were
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cultured in DMEM GlutaMax high glucose medium (Gibco, USA) containing 10% fetal
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bovine serum (FBS) at 37°C with 5% CO2. Huh-7 cells were cultured in DMEM GlutaMax
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low glucose medium (Gibco, USA) supplemented with 10% FBS and 1x nonessential amino
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acids.
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The 5BR reporter assay was performed as per Ranjith-Kumar et al. (46). Briefly,
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plasmids expressing RdRp and VPg were co-transfected along with plasmids to express
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RIG-I or MDA5, as well as the firefly and Renilla luciferase reporters. All transfections in this
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work used Lipofectamine 2000 according to the manufacturer's instructions (Invitrogen,
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Carlsbad, CA). 24 h prior to transfection, 0.5 × 105 cells were seeded into each well of
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Costar 96-well plates in DMEM containing 10% FBS. Cells were then typically transfected at
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75% confluency. A typical transfection used 20 ng of the pIFNβ-Luc, 5 ng of pRL-TK, 0.5 ng
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of the pUNO-hRIG-I and 50 ng of the plasmid expressing the viral polymerase. Where
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necessary, the vector plasmid (pUNO-MCS) was used to maintain a constant amount of
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plasmid DNA per transfection. At 36 h after transfection, luciferase activity was measured
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using Dual-Glo Luciferase Assay System (Promega, Madison, WI) in a Synergy 2
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microplate reader (BioTek, Winooski, VT). The ratios of the firefly luciferase to the Renilla
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luciferase are usually shown in the results unless stated otherwise.
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When used, the exogenous RIG-I agonist was a 60-nt hairpin triphosphorylated RNA
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(shR9) and was transfected at a 10 nM final concentration into cells. The TLR3 assay was
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performed as described by Ranjith-Kumar et al. (45) using ISRE-Luc as the firefly reporter
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plasmid. TLR3 expressing cells were induced with poly(I:C) (500 ng/ml; Amersham
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Biosciences). The cells were assayed for luciferase levels 18–22 h after transfection of
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exogenous agonists.
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Protein expression analysis. 293T cells (1.0 x 105 cells per well) were transfected
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with 100 ng of each plasmid in 48 well cell culture plate (BD Falcon). After 24 h of
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transfection, cells were washed one with 1X PBS, pH 7.4, harvested by gentle scrapping
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into 1X SDS-PAGE sample buffer. Cell lysates were resolved on a 4-12% NuPAGE Novex
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Bis-Tris Gel and transferred to PVDF membranes (Invitrogen, Carlsbad, CA). Membranes
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were blocked with 5% nonfat milk in TBS-T (Tris-buffered saline Tween-20) for 2 h and
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incubated overnight in blocking buffer supplemented with respective primary antibody.
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Following washes in TBS-T, membranes were incubated in blocking buffer supplemented
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with HRP conjugated secondary antibody and developed using ECL Plus detection system
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(Amersham, UK). NoV RdRp and VPg were detected by using a mouse monoclonal anti-
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FLAG monoclonal antibody (Sigma Inc., St. Louis MO). RNase L was detected with anti-
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RNase L mouse monoclonal antibody (Millipore, Bedford MA) and β-actin with anti- β-actin
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mouse monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Expression
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analysis of MNV NS7 protein and GAPDH used rabbit polyclonal antibodies (Ambion, Austin
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TX). The blots were subsequently probed with goat anti-mouse DyLight 680 and goat anti-
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rabbit DyLight800 (Thermo Scientific) before reading in a Licor Odyssey.
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Electrophoretic mobility shift assays and VPg-linked RNA sequencing. To examine
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the VPg-linked RNAs, 293T cells (1.0 x 106) were seeded in each well of 6 well cell culture
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plate (BD Falcon) and 1 µg of recombinant plasmid expressing FLAG-tagged RdRp was co-
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transfected with 100 ng of recombinant plasmid expressing HA-tagged VPg. 24 h later, total
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RNA, along with any covalently linked VPg, were isolated using Qiagen RNeasy mini kit.
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The RNA preparations were subsequently resolved by 4-12% NuPAGE Novex Bis-Tris gel
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using MOPS-SDS running buffer (Invitrogen, Carlsbad, CA), transferred to PVDF
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membranes and the VPg-linked to RNAs was detected by Western blot using an anti-HA
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antibody.
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The RNAs linked to VPg were purified and prepared for DNA sequencing as follows.
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VPg was immunoprecipitated using anti-HA tag polyclonal antibody covalently linked to
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Dynabeads M-270 Epoxy resin. An RNA adapter (5’-AUCGUAUGCCGUCUUCUGCUUGU-
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3’) was ligated to the 3’-end of VPg-linked RNAs, digested with proteinase K to remove VPg
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linked to RNA, extracted with a 1:1 phenol:chloroform followed by ethanol precipitation.
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First-strand cDNAs were synthesized using a specific reverse primer that recognizes the 3’
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adapter sequence (5’-CAAGCAGAAGACGGCATAC-3’) and SuperScript III reverse
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transcriptase (Invitrogen, USA). Second-strand cDNAs were synthesized using E. coli
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RNase H (NEB, USA) and E. coli DNA polymerase I (NEB, USA), as described by Gubler
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and Hofman (23). The cDNAs were treated with T4 DNA polymerase (NEB, USA) and tailed
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with
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(Invitrogen, USA). Plasmids with inserts were sequenced using the BigDye Terminator v3.1
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Cycle Sequencing Kit (Applied Biosystems, USA).
Taq DNA polymerase (NEB, USA), prior to cloning into TOPO 2.1 PCR vector
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Northern hybridization. Northern blots were probed using the methods of Sambrook
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and Russell (49). The probe was an oligonucleotide corresponding to the sense-strand of
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the TGOLN2 sequence (5’-CTGACCTCATTTCTCCCCCGCAGGAGGAAG-3’) radiolabeled
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with T4 polynucleotide kinase and γ-32P ATP (NEB, MA), and the radioactive signal was
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imaged using Typhoon 9210 variable mode imager (Amersham Biosciences, USA).
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Knockdown of RNase L. HEK 293T cells seeded at 0.5 x 105 cells/well in a 96-well
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plate were transfected 5-6 h post seeding with 10, 50 and 100 nM of RNase L-specific
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siRNA (Invitrogen, USA) or control siRNAs. At 48 h after siRNA transfection, the cells were
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transfected with either empty vector or with plasmids expressing NoV RdRp, VPg, pRL-TK
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and IFNβ-Luc. Dual-luciferase quantification was performed at 24 h post transfection.
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Murine norovirus reverse genetics recovery. Reverse genetics recovery of MNV was
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performed essentially as previously described using the plasmid pT7MNV-3’Rz containing
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the MNV CW1 genome under the control of the T7 RNA polymerase promoter (14). BHK21
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cells were infected with fowl-pox expressing T7 RNA polymerase (14) and subsequently
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transfected with 1 μg of cDNA clone using Lipofectamine 2000 (Invitrogen). 24 h post
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transfection the infectious virus was released by freeze-thawing the samples and the virus
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titrated by TCID50 in RAW264.7 cells.
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RESULTS
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RNAs synthesized by NoV RdRp can induce signaling by RIG-I. We sought to
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establish a cell-based assay to examine RNA synthesis by the human NoV based on the
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5BR assay format used to detect the RNAs made by the HCV RdRp (46). An expression
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vector that contained the NS7 cDNA coding region of the NoV GII.4 was constructed along
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with a mutant NS7 where the active site GDD residues were changed to GAA (RpGAA).
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Western blots confirmed that both the WT and the RpGAA mutant RdRps were expressed
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and to comparable levels (Fig. 1B). For our assays, we expressed at least four constructs in
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cells: the viral RdRp to produce RNAs, RIG-I to detect RdRp products, a firefly luciferase
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construct driven by the IFNβ promoter that can be activated by the RNA-bound RIG-I, and a
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Renilla luciferase driven by the RIG-I–insensitive TK promoter to serve as an internal
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control. The ratios of the two luciferases thus indirectly report on the RNA products made
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by the NoV RdRp. In multiple experiments, a four- to eight-fold increase in the firefly
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luciferase activity was observed when the cells expressed the WT NoV RdRp, similar to the
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levels observed with a HCV genotype 1b NS5B RdRp (Fig. 1C). This increase was not
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observed with the RpGAA mutant (Fig. 1C). These results suggest that the NoV-5BR assay
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can detect RNAs synthesized by the NoV RdRp.
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The reactions conditions for the NoV-5BR assay have been optimized. The
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concentration of the plasmid encoding GII.4 RdRp with the highest signal to background
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was approximately 50 ng and the optimal time to assess luciferase levels was 36 h after
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plasmid transfection (data not shown). Furthermore, comparable results were observed in
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assays performed with 293T and Huh-7 cells (Fig. 1C and 1D). A mutant RIG-I defective in
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ligand binding (K888E; 37) resulted in only background levels of firefly luciferase activity
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from the IFNβ promoter (Fig. 1C). These results demonstrate that RIG-I binding of the
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RNAs produced by the NoV RdRp is required for the observed luciferase reporter
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production.
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The NoV RdRp can initiate RNA synthesis by a de novo mechanism in biochemical
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assays in the absence of VPg (10, 47). Furthermore, de novo initiated RNA synthesis by the
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NoV RdRp in vitro can be increased by adding Mn2+ to the reaction buffer (47). In our
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assay we found that supplementing the medium of cells with up to 50 µM MnCl2 increased
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luciferase levels by four-fold without an effect on Renilla luciferase levels (Fig. 1E). Higher
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levels of Mn2+ caused cytotoxicity, consistent with reports of Ranjith-Kumar et al. (46). MgCl2
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added to the medium at up to 200 µM had no effect on the output from the NoV-5BR assays
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(Fig. 1E). The results with the activation of RIG-I and the effects of Mn2+ are consistent with
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the GII.4 RdRp using a de novo-initiated RNA synthesis in the absence of the VPg protein.
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The NoV RdRp can activate signaling by MDA5, but not by TLR3. We next aimed to
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determine whether other innate immune receptors could substitute for RIG-I in the NoV-5BR
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assay. MDA5 is a member of the RIG-I-like receptor family that responds to longer dsRNAs
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than does RIG-I (35). Replacing RIG-I with MDA5 also led to signaling in the presence of
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catalytically active NoV RdRp, but not by an active site mutant of the NoV RdRp (Fig. 2A).
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The level of luciferase activity through MDA5 was usually lower than through RIG-I. The
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activation of both RIG-I and MDA5 suggests that at least some RNAs synthesized by the
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NoV RdRp have the length and terminal modifications for recognition by either receptor.
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No increase in luciferase production was observed with the innate immune receptor
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TLR3 that signals from acidic endosomes (Fig. 2B; 36). In these cells, TLR3 did respond to
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exogenously provided poly(I:C) ligand but not to catalytically active NoV RdRp. A TLR3
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mutant defective in ligand binding, H539E could not respond to poly(I:C) and since TLR3
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detects ligands in endosomes (36), the NoV RdRp products may not gain access to
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endosomes to induce TLR3 signaling.
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An assay for murine norovirus RdRp activity. MNV is the only fully infectious system
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currently available for any NoV and for which reverse genetic systems exist (14, 61). Having
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a parallel 5BR assay for the MNV RdRp could generate results that can be examined in an
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infectious virus system. In addition, having assays for both the MNV and NoV RdRps will
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allow us to determine whether requirements for RNA synthesis are specific to a polymerase.
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The WT and the active site mutant MNV RdRps were constructed and both were
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determined to express at comparable levels in Western blots (Fig. 3A). However, only the
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WT MNV RdRp was able to activate reporter expression and to levels comparable to those
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from the NoV GII.4 RdRp (Fig. 3B).
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NoV RNA synthesis during infection requires the protein primer, VPg (47). To examine
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whether our assay could detect the effect of VPg, we co-expressed the MNV RdRp along
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with the MNV VPg. Based on sequence homology and previously described reports on
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other caliciviruses (8, 14, 42), Y26 of MNV VPg is the most likely site for the linkage to viral
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RNA (Fig. 3C). However, Y117 has been reported to prime RNA synthesis in biochemical
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assays (25). Therefore, we made the Y26A and Y117A mutant VPgs and co-expressed
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them with the MNV polymerase. The WT and mutant VPgs were expressed to comparable
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levels in Western blots (Fig. 3A). In the presence of the WT VPg or the Y117A mutant, we
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consistently observed statistically-significant (p
