Norovirus RNA Synthesis Is Modulated by an Interaction between theViral RNA-Dependent RNA Polymerase and the Major CapsidProtein, VP1

Chennareddy V. Subba-Reddy,a Muhammad Amir Yunus,b Ian G. Goodfellow,b and C. Cheng Kaoa

Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, Indiana, USA,a and Section of Virology, Department of Medicine, Imperial CollegeLondon, London, United Kingdomb

Using a cell-based assay for RNA synthesis by the RNA-dependent RNA polymerase (RdRp) of noroviruses, we previously ob-served that VP1, the major structural protein of the human GII.4 norovirus, enhanced the GII.4 RdRp activity but not that of therelated murine norovirus (MNV) or other unrelated RNA viruses (C. V. Subba-Reddy, I. Goodfellow, and C. C. Kao, J. Virol. 85:13027–13037, 2011). Here, we examine the mechanism of VP1 enhancement of RdRp activity and the mechanism of mouse noro-virus replication. We determined that the GII.4 and MNV VP1 proteins can enhance cognate RdRp activities in a concentration-dependent manner. The VP1 proteins coimmunoprecipitated with their cognate RdRps. Coexpression of individual domains ofVP1 with the viral RdRps showed that the VP1 shell domain (SD) was sufficient to enhance polymerase activity. Using SD chime-ras from GII.4 and MNV, three loops connecting the central �-barrel structure were found to be responsible for the species-spe-cific enhancement of RdRp activity. A differential scanning fluorimetry assay showed that recombinant SDs can bind to the puri-fied RdRps in vitro. An MNV replicon with a frameshift mutation in open reading frame 2 (ORF2) that disrupts VP1 expressionwas defective for RNA replication, as quantified by luciferase reporter assay and real-time quantitative reverse transcription-PCR (qRT-PCR). Trans-complementation of VP1 or its SD significantly recovered the VP1 knockout MNV replicon replication,and the presence or absence of VP1 affected the kinetics of viral RNA synthesis. The results document a regulatory role for VP1in the norovirus replication cycle, further highlighting the paradigm of viral structural proteins playing additional functionalroles in the virus life cycle.

Noroviruses (genus Norovirus, family Caliciviridae) are re-sponsible for more than 90% of all epidemic nonbacterial

gastroenteritis outbreaks in the United States (1), and they arenow recognized as the second leading cause of deaths due to gas-troenteritis (24). Currently, noroviruses are divided into 5 geno-groups (GI to GV) based on sequence similarity (22). Humannoroviruses (HuNoV) belong to GI and GII and are subsequentlysubdivided into a number of genotypes. GII genotype 4 (GII.4)HuNoVs are responsible for 70 to 80% of norovirus (NoV) out-breaks worldwide (16). Despite extensive efforts, HuNoVs haveyet to be efficiently propagated in cell culture or animal modelsand, hence have been difficult to study and to manipulate for thedevelopment of therapeutics (17, 5). The discovery that the mu-rine norovirus (MNV; genogroup V) replicates in cell culture andmice has made MNV an attractive model for the studies of NoVmolecular biology (47). Studies with MNV have already yieldedinsights into the molecular mechanism of translation, replication,and the immune response to infection (9, 28, 33, 43, 48). NoVshave a nonenveloped T�3 icosahedral capsid that encapsidates avirus protein, genome-linked VPg, single-stranded, positive-senseRNA genome (25, 27). The RNA genomes of NoVs are about 7.7kb and are typically organized into three major open readingframes (ORFs) (22). ORF1 encodes six or seven nonstructuralproteins, including an RNA-dependent RNA polymerase (RdRp)(15). ORF2 and ORF3 encode the major and minor capsid pro-teins VP1 and VP2, respectively (22, 20). MNV, but not HuNoV,also encodes an alternative reading frame overlapping the VP1coding region (33). The genomic RNA serves as a template forsynthesis of the nonstructural polyprotein, while the subgenomicRNAs are used to translate the VP1 and VP2 proteins (27). In vitro

and in cells, the NoV RdRps can initiate RNA synthesis by both aVPg-dependent and a VPg-independent (de novo) manner, sug-gesting that VPg may have distinct roles in genomic and antige-nomic RNA synthesis (2, 7, 19, 41, 44).

VP1 contains two major domains, the shell domain (SD) and aprotruding domain (PD), and these are linked by a flexible hinge(25, 39). The PD is further organized into two subdomains: P1 andP2 (39, 43). P2 contains the receptor binding sites and is an im-portant determinant of virulence (4, 30). It is also recognized byneutralizing antibodies and has been demonstrated to mutate at ahigh frequency (8, 16). The SD contains an eight-stranded antipa-rallel �-sandwich that is commonly found in viral capsid proteinsand forms the icosahedral shell that contains the genomic RNA(39). The SDs can undergo localized conformational changes tomaintain essentially the same interactions between the opposingSDs in dimers (39). Recombinant SDs can self-assemble intosmooth virus-like particles of ca. 30 nm in diameter (3).

To study RNA synthesis of GII.4 RdRp, we established a cell-based assay wherein the GII.4 RdRp products are recognized bythe innate immune receptors RIG-I and MDA5 to activate re-porter expression (44). This so-called NoV-5BR assay is able to

Received 14 May 2012 Accepted 3 July 2012

Published ahead of print 11 July 2012

Address correspondence to C. Cheng Kao, ckao@indiana.edu, or Chennareddy V.Subba-Reddy, vchennar@indiana.edu.

Copyright © 2012, American Society for Microbiology. All Rights Reserved.

doi:10.1128/JVI.01208-12

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detect VPg-primed RNA synthesis as well as the de novo-initiatedRNA products generated by both the GII.4 and MNV RdRps. Fur-ther, coexpression of the GII.4 and the MNV VP1 proteins withtheir respective RdRps enhanced RNA synthesis reproducibly by40 to 60%. In this study, we sought to elucidate how the structuralprotein VP1 may affect RdRp activity and contribute to a biolog-ically relevant activity during virus replication. The observation ofstructural proteins playing nonstructural roles in the viral life cy-cle is increasingly evident. Using VP1 truncations and mutations,the SD of VP1 was found to be sufficient to enhance NoV poly-merase activity in a species-specific manner. Furthermore, anMNV replicon defective for VP1 expression was debilitated forreplication, but the expression of the cognate VP1 or its SD couldrescue replication by trans-complementation. The results showthat, apart from virion formation, VP1 has a regulatory role inNoV genome replication.

MATERIALS AND METHODSPlasmid constructs and manipulations. The cDNA clones of GII.4HuNoV RdRp, VP1, and VPg (NoV Hu/GII.4/MD-2004/2004/US; Gen-Bank accession number DQ658413) were as reported earlier (44). MNVRdRp, VP1, and VPg were PCR amplified from a cDNA clone of MNV-1strain CW1 (GenBank accession number DQ285629.1) (7, 47) and clonedinto the pUNO vector (InvivoGen, San Diego, CA). Plasmid pUNO-hRIGwas from InvivoGen (San Diego, CA). The plasmid containing the fireflyluciferase reporter gene driven by the beta interferon (IFN-�) promoter(IFN-�-Luc) was used as a reporter, and pRL-TK containing herpes sim-plex virus thymidine kinase (TK) promoter-driven Renilla reniformis lu-ciferase was used to monitor and standardize the efficacy of transfection(Promega, Madison, WI).

VP1 truncations were generated by PCR amplification using sense andantisense primers containing the AgeI and NheI sites, respectively, andthese were cloned into the pUNO vector. VP1 SD chimeras were customsynthesized (Bio Basic Canada, Inc.) with AgeI and NheI sites and clonedinto the pUNO vector. For construction of the Escherichia coli expressionvectors pBAD-GII.4 RdRp, pBAD GII.4 VP1 S, and pBAD MNV VP1 SD,their respective genes were amplified from mammalian expression con-structs by using sense and antisense primers containing PstI and HindIIIrestriction sites, respectively, and cloned into the multiple cloning site ofthe pBAD/Myc-His A vector (Invitrogen) digested with the same restric-tion enzymes. The expression plasmid for the production of recombinantMNV NS7 was generated by cloning the NS7 sequence into the pET26Ub-His plasmid containing a T7 polymerase promoter and the ubiquitin genefrom Saccharomyces cerevisiae, followed by a C-terminal polyhistidine tag(21, 49). The N-terminal ubiquitin fusion is subsequently removed bycoexpression in E. coli with a ubiquitin-specific protease to produce theMNV NS7 with a C-terminal histidine tag. The sequences of all constructsused in this study were confirmed by sequencing with the BigDye Termi-nator v3.1 cycle sequencing kit (Applied Biosystems).

Construction of luciferase-expressing WT and VP1 knockout MNVreplicons. Luciferase-expressing wild-type (WT) and VP1 knockoutMNV replicons were constructed using the MNV infectious clone namedpT7:Mflc that contains the MNV CW1 genome under the control of theT7 RNA polymerase promoter (10). The resulting replicon (Mflc), con-struction and primer details of which are available upon request, containsthe Renilla luciferase inserted immediately after the VP1 coding sequenceunder the control of the MNV TURBS sequence (34, 35). Luciferase wasfollowed by the foot-and-mouth disease virus (FMDV) 2A protease se-quence (NFDLLKLAGDVESNPGP) and the MNV VP2-coding sequence.Translational chain termination on the FMDV2A sequence between theC-terminal glycine-proline resulted in the addition of a proline residue tothe N terminus of VP2. The sequence of the subgenomic region was con-firmed prior to use. A similar replicon in which the RdRp active siteYGDD sequence was changed to YGGG (MflcGGG-R) was also generated

by overlapping PCR mutagenesis. This mutation was found to ablate virusrecovery when introduced into the MNV full-length infectious clone(data not shown).

To introduce a �1 frameshift into the VP1 ORF, a single nucleotidewas inserted at position 5070 using QuikChange mutagenesis of a SexAI-SacII fragment encompassing nucleotides (nt) 4276 to 5767 of Mflc-R.This fragment was subsequently reintroduced into the MNV repliconMflc-R, and the sequence was confirmed prior to use. The resulting plas-mid with the �1 frameshift was designated Mflc-Rfs. The sequences of allconstructs were confirmed prior to use.

In vitro transcription of MNV replicons. Plasmids Mflc, Mflc-R, andMflc-Rfs were linearized with NheI, and capped RNA transcripts weresynthesized from the linearized templates using the AmpliCap-Max T7high-yield message maker kit (Epicenter Biotechnologies). The reactionswere performed according to the manufacturer’s instructions. In vitrotranscripts were purified by ammonium acetate precipitation and ana-lyzed by electrophoresis in a 1% agarose gel.

Mammalian cell cultures. Human embryonic kidney cells (HEK293T)were cultured in Dulbecco modified Eagle medium (DMEM) and GlutaMAXhigh-glucose medium (Gibco) supplemented with 10% fetal bovine serum(FBS). The murine macrophage cell line, RAW264.7, was cultured in DMEMsupplemented with 10% FBS, penicillin (100 U/ml), and streptomycin (100mg/ml). All the cell cultures were grown and maintained at 37°C and 5% CO2.

Luciferase reporter assays. The NoV-5BR luciferase reporter assayswere essentially performed as described in Subba-Reddy et al. (44). Plas-mids expressing RdRp and VP1 were cotransfected with plasmids express-ing RIG-I as well as firefly and Renilla luciferase reporters. All transfec-tions were performed with Lipofectamine 2000 according to themanufacturer’s instructions (Invitrogen, Carlsbad, CA). Twenty-fourhours prior to transfection, 0.5 � 105 cells were seeded into each well ofCostar 96-well plates in DMEM containing 10% FBS. Cells were thentypically transfected at 75% confluence. A typical transfection used 20 ngof IFN-�-Luc, 5 ng of pRL-TK, 0.5 ng of the plasmid expressing the RIG-I,and 50 ng of the plasmid expressing the viral polymerase. Where neces-sary, the vector plasmid (pUNO-MCS) was used to maintain a constantamount of total plasmid DNA per well. At 36 h after transfection, lucifer-ase activity was measured using the Dual-Glo luciferase assay system (Pro-mega, Madison, WI) in a Synergy 2 microplate reader (BioTek, Winooski,VT). The ratios of firefly to Renilla luciferase activity were calculated foreach well, and the values of the samples were normalized to that of thecontrol. When used, the exogenous RIG-I agonist was a 60-nt hairpintriphosphorylated RNA (shR9) and was transfected at a 10 nM final con-centration. The cells were assayed for luciferase levels 18 to 22 h aftertransfection of exogenous agonists.

The Renilla luciferase activity assay to quantify the MNV replicon used0.5 � 105 RAW264.7 cells seeded into each well of a Costar 96-well plate.The cells at 80% confluence were transfected with 100 ng of in vitro tran-scripts made from Mflc-R and Mflc-Rfs using Lipofectamine 2000 as avehicle according to the manufacturer’s instructions (Invitrogen, Carls-bad, CA). The in vitro transcripts of Mflc-R (100 ng) that did not expressluciferase were transfected as a background control. At 36 h after trans-fection, the cells were washed once with 1� phosphate-buffered saline(PBS), and the cells were lysed in 20 �l of 1� passive lysis buffer. Theluciferase activity was measured using the Renilla luciferase assay system(Promega, Madison, WI).

Protein expression analysis. To determine the expression of recom-binant proteins, about 1 � 105 293T cells per well were transfected with100 ng of each plasmid in 48-well plates (BD Falcon). To determine theexpression of MNV replicon proteins, about 1 � 105 RAW264.7 cells perwell were transfected with 100 ng of each in vitro transcript in 48-well cellculture plates (BD Falcon). After 24 h, cells were washed with 1� PBS (pH7.4) and harvested into 1� SDS-PAGE sample buffer. Lysates were re-solved on a 4 to 12% NuPage Novex Bis-Tris gel and electrophoreticallytransferred onto polyvinylidene difluoride (PVDF) membranes (Invitro-gen, Carlsbad, CA). Membranes were incubated in blocking buffer (5%

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nonfat milk in Tris-buffered saline) supplemented with antibodies.RdRps were detected using a mouse monoclonal anti-FLAG antibody(Sigma). VP1, VP1 truncations, VP1 chimeras, and VPg were probedusing a goat anti-HA polyclonal antibody (Abcam). MNV replicon-ex-pressed RdRp and VP2 proteins were detected by rabbit polyclonal anti-bodies as described previously (7). MNV VP1 was detected using a mousemonoclonal antibody specific to the norovirus capsid protein (Abcam).Membranes were probed with horseradish peroxidase (HRP)-conjugatedsecondary antibodies and developed using the ECL Plus Western blottingdetection system (Amersham, United Kingdom).

Coimmunoprecipitation assays. Coimmunoprecipitation (co-IP)assays to assess protein complex formation used 106 HEK293T cells perwell in 6-well cell culture plates (BD Falcon). These were cotransfectedwith 1 �g of plasmid expressing FLAG-tagged RdRp and 100 ng of plas-mid expressing hemagglutinin (HA)-tagged VP1, VP1 truncations, orVP1 chimeras. Twenty-four hours after transfection, the cell lysates wereprepared in nondenaturing lysis buffer (20 mM Tris-HCl [pH 8], 137 mMNaCl, 10% glycerol, 1% Nonidet P-40, 2 mM EDTA) mammalian cellprotease inhibitor cocktail (Sigma) at 10 �l/ml of lysate. The FLAG-tagged RdRps were immunoprecipitated using anti-FLAG tag monoclo-nal antibody (Sigma) covalently linked to Dynabeads M-270 epoxy resinaccording to the instructions of the manufacturer and as reported (44).Samples were subsequently resolved by 4 to 12% NuPage Novex Bis-Trisgels using MOPS (morpholinepropanesulfonic acid)-SDS running buffer(Invitrogen, Carlsbad, CA), transferred to PVDF membranes, and de-tected by a Western blot analysis using the appropriate antibodies.

Recombinant protein expression and purification. Overnight cul-tures of E. coli TOP 10 cells harboring pBAD-GII.4 RdRp, pBAD GII.4VP1 S, and pBAD MNV VP1 S were diluted 1:250 in 1 liter of Luria-Bertani (LB) medium containing ampicillin (50 ng/1 ml LB medium).The cultures were grown with vigorous shaking to an optical density at600 nm (OD600) of �0.5, and L-arabinose was added to a 0.02% finalconcentration. After 5 h of growth at 37°C with shaking, the cells wereharvested by centrifugation, and the pellet was suspended in 50 ml of 50mM Tris-HCl (pH 8.0), 150 mM NaCl, and 0.25 mM EDTA (buffer 1).The cell suspension was sonicated, and the supernatant, recovered aftercentrifugation at 15,000 rpm for 30 min in a Sorvall SS-34 rotor, wasmixed with 1 ml of Talon metal affinity resin (Clontech Laboratories) andadsorbed by shaking for 1 h at 4°C. Following the absorption step, theresin was washed five times with 5 ml of buffer 1 containing 40 mMimidazole. After the final wash, the protein was eluted in buffer 1 contain-ing 300 mM imidazole. All eluted proteins were further purified with asecond Talon resin. C-terminally His-tagged MNV RdRp was expressedand purified essentially as described above, with minor modifications.Briefly, cultures were induced with IPTG (isopropyl-�-D-thiogalactopy-ranoside), initially purified by nickel affinity chromatography (GEHealthcare), and washed with 20 mM imidazole, followed by specific elu-tion with 500 mM imidazole. The resulting protein was diluted to 100 mMNaCl and passed through a phosphocellulose P11 column (Whatman) toremove any nonspecific proteins. The protein that did not bind to the P11column was further purified by nickel affinity chromatography as de-scribed before. Protein purity was checked by SDS-PAGE, and concentra-tions were estimated by using the Quick Start Bradford protein assay kit(Bio-Rad) with bovine serum albumin (BSA) as the standard. In vitroRNA synthesis by the recombinant RdRps was performed using the pro-tocol and template RNAs described in Chinnaswamy et al. (13).

Differential scanning fluorimetry. Thermal melting curves of GII.4RdRp and VP1 SD were obtained in 96-well plates using the StratageneMx3005P quantitative PCR (qPCR) system (Agilent Technologies) andthe fluorescent dye Sypro Orange (Invitrogen, Carlsbad, CA). Differentconcentrations of expressed VP1 SD were mixed with 20 �M purifiedGII.4 RdRp and a 5� concentration of Sypro Orange. A heating rate of1.0°C per min was used from 25 to 80°C, and fluorescence intensity wasread at excitation and emission wavelengths of 470 and 550 nm, respec-tively.

VPg electrophoretic mobility shift assay. About 1 � 106 HEK293Tcells were seeded in each well of 6-well cell culture plates (BD Falcon) andcotransfected with 1 �g of recombinant plasmid expressing FLAG-taggedRdRp and 100 ng of plasmid expressing HA-tagged VPg. Twenty-fourhours later, VPg was immunoprecipitated using anti-HA tag polyclonalantibody covalently linked to Dynabeads M-270 epoxy resin. Sampleswere resolved by 4 to 12% NuPage Novex Bis-Tris gel using MOPS-SDSrunning buffer (Invitrogen, Carlsbad, CA) and transferred to PVDFmembranes. The VPg and RNA-linked VPg were detected by a Westernblot analysis using anti-HA antibodies.

Quantitative RT-PCR. Strand-specific quantification of genomic andantigenomic RNAs by reverse transcription-PCR (qRT-PCR) was per-formed as described by Vashist et al. (45). To prepare standard curves forqRT-PCR quantification of genomic and antigenomic RNAs, T7 pro-moter sequences were added to genomic- and antigenomic-sense strandsby PCR. The genomic-sense strands were amplified by PCR using a for-ward primer with a T7 promoter sequence (5=-GCGTAATACGACTCACTATAG TGGACAACGTGGTGAAGGAT-3=, with the T7 promoter se-quence underlined) (corresponding to nt 1678 to 1697) and a reverseprimer (5=-CAAACATCTTTCCCTTGTTC-3=) (corresponding to nt1760 to 1779). An antigenomic product was amplified by PCR using areverse primer with a T7 promoter sequence (5=-GCGTAATACGACTCACTATAGCAAACATCTTTCCCTTGTTC-3=, with the T7 promoter se-quence underlined) and a forward primer (5=-TGGACAACGTGGTGAAGGAT-3=). The PCR products were purified by gel extraction and used astemplates for in vitro RNA synthesis by using the AmpliCap-Max T7 kit(Epicenter Biotechnologies) as described previously.

About 106 RAW264.7 cells per well in 6-well cell culture plates (BDFalcon) were transfected with 1 �g of each in vitro transcript. Cells wereharvested at different time points posttransfection, and total RNAs wereprepared using the TRIzol reagent (Ambion, Carlsland, CA) according tothe manufacturer’s instructions. The RNA preparations were treated withDNase I (RNase-free) (New England BioLabs) for 30 min at 37°C andagain purified using TRIzol reagent as described above. A total of 100 ng ofeach RNA sample was used for cDNA synthesis using SuperScript IIIreverse transcriptase (Invitrogen) with either a genomic (5=-CAAACATCTTTCCCTTGTTC-3=) or antigenomic (5=-TGGACAACGTGGTGAAGGAT-3=) specific reverse primer at 55°C for 30 min, followed by heatinactivation at 90°C for 5 min. The assays targeted regions within thegenomic RNA, specifically within ORF1 (nt 1678 to 1779). Quantitativereal-time RT-PCR was performed using the Power SYBR green PCR mas-ter mix (Applied Biosystems, Warrington, United Kingdom) and theEppendorf Mastercycler (Eppendorf AG, Hamburg).

Statistical analysis. The data are shown as the means and the rangesfor one standard error. Data sets of three or more groups were comparedby the Student t test using GraphPad Prism 5 software. In all analyses, Pvalues of �0.05 were considered statistically significant.

RESULTSVP1 can modulate RdRp activity. Using the NoV-5BR assay, weinvestigated the effect of VP1 expression on the activities of theRdRps from GII.4 NoV and MNV. A typical assay used HEK293Tcells transfected to express the viral RdRp, RIG-I, firefly luciferaseexpressed from the IFN-� promoter, and constitutive Renilla lu-ciferase. The ratio of firefly to Renilla luciferase reports on theRNA products synthesized by the RdRp and subsequently de-tected by RIG-I. The presence of the GII.4 VP1 expression plasmidat 10 ng resulted in a 50 to 60% increase in the activity of thecognate RdRp but not that of the MNV RdRp (Fig. 1A) (P �0.001). A similar species-specific VP1-RdRp interaction was alsofound with the MNV proteins (P � 0.003) (Fig. 1A). VP1 expres-sion did not affect RIG-I-mediated signaling via a short triphos-phorylated RNA agonist, shR9 (40) (Fig. 1B). These observations

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confirmed that the enhancement of RdRp activity by VP1 is spe-cies specific and acts through the RdRp.

Given that the ratio of RdRp to VP1 is likely to vary during theviral life cycle as a function of subgenomic RNA synthesis, weexamined the effect of increasing levels of the GII.4 or MNV VP1proteins on RdRp activity. A Western blot analysis confirmed thatincreasing plasmid concentrations resulted in a corresponding in-crease in VP1 accumulation, although detection of expression re-quired a minimum of 5 ng of plasmid (Fig. 1C). Transfection of 10ng of the GII.4 VP1 expression plasmid into cells exhibited opti-mal enhancement, while higher levels did not enhance as well,without obviously inhibiting RdRp activity (Fig. 1D). Impor-tantly, the concentration-dependent effect of VP1 was species spe-cific for the MNV and GII.4 VP1 proteins (Fig. 1D and E).

The S domain of VP1 is necessary and sufficient to modulatethe RdRp activity. VP1 contains two domains, the shell domain(SD) that includes an N-terminal tail of 45 residues and a protrud-ing domain (PD) (Fig. 2A) (39). We constructed four VP1 trun-cations that consist of VP1 lacking the N-terminal tail (�NT), theSD, the PD, or the SD lacking the N-terminal tail (S�NT) (Fig. 2B)and tested for their effect on RdRp activity. The GII.4 RdRp activ-ity was enhanced by the GII.4 �NT, SD, and S�NT but not by thePD (Fig. 2C). Comparable results were observed with MNV RdRp(Fig. 2D). The enhancement of RdRp activity by the SD was notonly species specific but also concentration dependent (Fig. 2E

and F). The SD of VP1 without the NT was sufficient to modulatethe RdRp activity in a manner comparable to that of VP1.

Loops in the VP1 S domain are critical for interaction withRdRp. We sought to map further the motifs within the VP1 SDsrequired to modulate RdRp activity. The SD contains a central8-stranded �-barrel structure that contains 8 major loops. We willrefer to the �-strands as the “core” and the loops as loops 1 to 8(Fig. 3A). Notably, loops 1, 3, 5, and 7 are located on one surface ofthe core, while loops 2, 4, 6, and 8 are on the other surface (39). Sixchimeras (C1 to C6) that mixed and matched the cores and loopsof the GII.4 and MNV SDs were tested (Fig. 3B). C1, which con-tains the GII.4 SD core and all eight loops from the MNV SD [NCore-M(L1-L8)] (Fig. 3B), considerably enhanced the MNVRdRp activity (P � 0.007) but not the GII.4 RdRp activity (Fig. 3Band D). Similarly, C2, which contains the MNV SD core and alleight of the GII.4 SD loops, significantly enhanced only the GII.4RdRp activity (P � 0.009) (Fig. 3B). Species-specific interactionwith the RdRp segregated with the loops in the SD.

Chimeras C3 and C5, which contain loops 2, 4, 6, and 8 andthe core from the same species but heterologous loops 1, 3, 5,and 7, failed to enhance the homologous RdRp activity (Fig. 3Band data not shown). However, C4 and C6, which contain ho-mologous cores and loops 1, 3, 5, and 7 but heterologous loops2, 4, 6, and 8, enhanced the cognate RdRp (P � 0.013) (Fig. 3B).These results reveal that loops 1, 3, 5, and 7, together with the

FIG 1 VP1 modulates RdRp activity. (A) Activities of the NoV RdRps are enhanced by coexpression of the homologous VP1 proteins. The results used theNoV-5BR assay format assessed in HEK293T cells. A plus symbol below the x axis shows the presence of a plasmid encoding the protein of interest. Ratios of firefly(FF) to Renilla (Ren) luciferase activities (Luc.) were determined after 36 h of transfection and are shown on the y axis. Each bar represents the means of threeindependent experiments, and the standard errors are shown above the bar. (B) VP1 did not affect RIG-I signaling induced by exogenously provided agonists.The concentrations of the relevant construct transfected into cells are shown on the x axis. The RIG-I agonist, shR9, was transfected at a final concentration of 10nM 24 h after the transfection of the expression plasmids (40). The ratios of firefly luciferase to Renilla luciferase activities were determined after 12 to 16 h of shR9transfection. The data are the means and standard errors of two independent experiments, each of which had three independent samples. (C) The expression ofMNV and GII.4 VP1 depended on the concentration of transfected plasmids. (D) GII.4 VP1 stimulates its RdRp in a concentration-dependent manner. Theconcentrations of the VP1-expressing plasmid used are shown on the x axis. The plasmid expressing the GII.4 RdRp was kept at 50 ng per transfection. Luciferaseactivities were assessed 36 h after transfection. (E) MNV VP1 stimulates its RdRp in a concentration-dependent manner. The format of the experiment is the sameas in panel D.

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�-strands in the SD core, are important for species-specificRdRp interaction.

We examined whether the chimeras were altered in their con-centration-dependent enhancement of RdRp activity. C2, whichcontains all of the loops from the GII.4 SD but has the MNV core,enhanced the GII.4 RdRp activity in a concentration-dependentmanner (Fig. 3C), although the overall enhancement of RdRpactivity was lower than that of the WT SD. Similarly, C1, whichcontains all of the loops from the MNV SD, exhibited a concen-tration-dependent enhancement. Interestingly, the three chime-ras that had loops derived from different NoV species failed toaffect RdRp activity in a concentration-dependent manner (Fig.3C and D). Altogether, results with the chimeras indicate thatloops 1, 3, 5, and 7 contribute to enhancing the RdRp activity;

however, loops 2, 4, 6, and 8 on the other side of the core structurecontribute to the suppression of enhancement. Given that the loopsof the SD are involved in the interactions between SD subunits thatlead to virus-like particle (VLP) formation, we hypothesize that oli-gomerization of SD and VP1 molecules prevents interaction with thecognate RdRps and the observed enhanced activity.

A comparison of the sequences of loops 1, 3, 5, and 7 from theGII.4 and MNV SDs is presented in Fig. 3B. Loop 3 had identicalsequences, loops 1 and 5 differed by only a single residue, and loop7 differed by five residues. We further examined whether chimeraswith a swap of only loop 1, 5, or 7 affected RdRp activity and foundthat none did (data not shown). These results indicate that two ormore of loops 1, 5, and/or 7 are required to functionally interactwith the RdRp.

FIG 2 The VP1 S domain modulates RdRp activity. (A) Ribbon structure showing the domains in GII.4 VP1. The amino-terminal tail (NT), the SD, and the PDare connected by flexible loops. In this orientation of the VP1, the right-hand side of the PD is involved in dimeric contacts (39). (B) Schematic of VP1 and itstruncations. The full-length VP1 was labeled as WT. The amino acid numbers represent those of the GII.4 genotype used in the present study (GenBank accessionnumber DQ658413). VP1 with the N-terminal 45 residues deleted is named �NT. SD contains residues 1 to 216. PD contains residues 217 to 540. S�NT expressesresidues 45 to 216. (C) The SD is sufficient to enhance RdRp activity. Where present, the plasmids to express the GII.4 RdRp and VP1 constructs were at 50ng and 10 ng per well of cells, respectively. A plus symbol below the x axis shows the presence of the respective plasmid. The ratios of luciferase activitieswere determined 36 h after plasmid transfection. The data are the means of two independent experiments with three replicates each, and the standarderrors are shown. (D) The SD is sufficient to enhance RdRp activity. The reagents and format used in this experiment are identical to those in panel C. (E)The GII.4 SD has a concentration-dependent effect on GII.4 RdRp activity. GII.4 RdRp was transfected at a constant concentration of 50 ng. Theconcentrations of the GII.4 and MNV VP1 SD transfected, in ng of plasmid per well of cells, are shown on the x axis. (F) The GII.4 SD has aconcentration-dependent effect on GII.4 RdRp activity.

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The S domain can bind to RdRp in a species-specific manner.Immunoprecipitation assays were performed to determinewhether VP1 and its derivatives can bind the viral RdRp. FLAG-tagged RdRp and HA-tagged VP1 proteins were confirmed to befunctional in the NoV-5BR assays and were expressed to detect-able levels in cells (Fig. 4A). Western blots of the immunoprecipi-tates revealed that all the RdRps were pulled down by anti-FLAGantibody (Fig. 4A, top panel), but only the homologous VP1 pro-teins were able to be coprecipitated with the RdRps, i.e., the GII.4RdRp could coprecipitate its cognate VP1 but not the MNV VP1,and vice versa (Fig. 4A, middle panel). VP1, �NT, and the SD werecoimmunoprecipitated with the homologous GII.4 and MNVRdRps, but the PD was not (Fig. 4C and E). These results confirmthat the SDs of the VP1 proteins can form a complex with theircognate RdRps. However, while chimeras C1 to C6 were expressedat levels comparable to that of the WT SD, none were detectablycoimmunoprecipitated with the RdRp (Fig. 4F). These resultssuggest that in addition to the specific interaction between the SDand RdRp requiring loops 1, 3, 5, and 7, the core �-barrel structuremay be needed to stabilize the interaction with the RdRp and toenable the complex to be immunoprecipitated.

Differential scanning fluorimetry (DSF) assays were used todetermine whether recombinant VP1 SD can bind RdRp in vitro(Fig. 5). DSF analyzes the thermal denaturation (Tm) of pro-teins that can be altered by ligand binding (37). The denatur-ation of the protein is detected by the binding of the dye SyproOrange, which fluoresces upon contact with hydrophobic por-tions of polypeptides. The SD had minimal signal, likely due toits small size (Fig. 5B). The preparations of the RdRps werecompetent for RNA synthesis in vitro (data not shown). TheGII.4 and MNV RdRps displayed a prominent change in fluo-rescence that was maximally evident at 43°C, which we willrefer to as the Tmapp (Fig. 5B). An equimolar solution (20 nM[each] GII.4 RdRp and VP1 SD) increased the Tmapp by 2.5°C(Fig. 5B). A 2:1 molar ratio of the SD to the RdRp resulted in a�Tm of 1.5°C, suggesting that interaction between the SD andthe RdRp preferred a lower ratio of the two molecules (Fig. 5C).Consistent with all of the results demonstrating a species-spe-cific interaction, the MNV SD did not alter the fluorescenceemission of the GII.4 RdRp and vice versa (Fig. 5B and C).Similar results were obtained using the MNV RdRp and SD(Fig. 5C). The addition of RNA to the DSF reaction did not

FIG 3 Loops in the S domain are critical for specific interactions with RdRp. (A) Amino acid sequence of the GII.4 VP1 SD. The amino acid numbers are thoseof the GII.4 VP1 (GenBank accession number DQ658413). The � strands that form the classical eight-stranded �-barrel structure are labeled �1 to �8. Loopsconnecting the eight-stranded �-sandwich structure are labeled L1 to L8 and highlighted in red. (B) Schematic depicting the SD chimeras with mixtures ofdifferent loop sequences. The color schemes for the GII.4 and MNV SD loops and �-barrel motifs are shown in the upper two constructs and used to denote themotifs present in the chimeras. A summary of the effects of the chimeras on GII.4 and MNV RdRp activity is shown beside the chimeras. The plus (�) symbolshows the enhancement of RdRp activity. The sequences of loops 1, 3, and 5 for the GII.4 are shown in one-letter amino acid codes. A dash denotes where theMNV residues are identical in these loops. (C) Effect of SD chimeras on GII.4 RNA synthesis in the NoV-5BR assay. GII.4 RdRp-expressing plasmid wastransfected at 50 ng, and the amounts of the plasmids for the SD constructs are shown on the x axis. Luciferase activities were read 36 h after transfection. (D)Concentration-dependent effects of the SD chimeras on MNV RNA synthesis. The format of the experiment is the same as in panel C.

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cause a further change to the Tmapp of the RdRp-SD complex(data not shown).

VP1 and VPg-primed RNA synthesis. Results from the NoV-5BR assay thus far illustrated that VP1 can enhance RNA synthesisin the absence of VPg. To examine whether VP1 can affect VPg-primed RNA synthesis, we expressed homologous and heterolo-gous combinations of VP1, RdRp, and VPg proteins in HEK293Tcells (Fig. 6). The coexpression of VPg with the RdRp increasedluciferase activity in the NoV-5BR assay by ca. 30 to 40% andproduced a covalently linked VPg-RNA complex that can be dis-tinguished from free VPg by its electrophoretic mobility (44). Co-expression of 10 ng of either the MNV or GII.4 VP1 with theircognate RdRps and VPgs increased luciferase activity (Fig. 6A andB). To investigate whether the combination of three proteins af-fected VPg-primed RNA synthesis, we immunoprecipitated thetagged VPg and performed a Western blot analysis of VPg prod-ucts as previously described (44). With the expression of all three

proteins, the VPg-RNA complex was detected (Fig. 6). The VPg-RNA was not observed in cells that did not express the RdRp.Notably, while the presence of VP1 increased the abundance ofboth VPg and VPg-RNA, the relative ratio of VPg to VPg-RNAwas not increased (Fig. 6C and D).

VP1 knockout significantly reduces MNV genome replica-tion. The results on VP1-RdRp interaction led us to hypothesizethat VP1 may regulate NoV RNA replication. To test this, weconstructed an MNV replicon, referred to as Mflc-R, that ex-presses a Renilla luciferase-FMDV 2A-VP2 fusion protein in placeof VP2 (Fig. 7A). In parallel, we constructed MflcGGG-R, which hasa mutation in the active site of the RdRp. Transfection of thecapped transcript of Mflc-R into RAW264.7 cells resulted in a 10-to 12-fold increase in Renilla levels compared to those of controltranscripts that did not express the reporter (Fig. 7B). MutantMflcGGG-R had 6-fold lower Renilla activity than Mflc-R, con-firming that replication of the WT replicon is responsible for the

FIG 4 VP1-RdRp interaction. (A) Coimmunoprecipitation of GII.4 and MNV VP1s with their cognate RdRps. HEK293T cells were cotransfected with 50 ng ofplasmid expressing a FLAG-tagged GII.4 or MNV RdRp and 10 ng of plasmid expressing HA-tagged VP1 from either GII.4 or MNV. The identities of the bandsare shown to the right of the Western blot images. (B) Expression of the GII.4 VP1 or its truncated derivatives present in the cell lysates used for theimmunoprecipitation assays. (C) Coimmunoprecipitation of GII.4 VP1 truncations with its RdRp. The expected positions of VP1 or its truncations are identifiedby asterisks to the right of the bands identified in the Western blots. The bottom Western blot image shows the amount of RdRp present in the precipitatedmaterial. (D) Western blot showing the expression of different truncations of MNV VP1. The proteins were analyzed by Western blotting using goat anti-HApolyclonal antibody (Abcam) to detect the expression of HA-tagged MNV VP1 and its truncations. (E) Amount of MNV VP1 or its truncations that coimmu-noprecipitated with the MNV RdRp. FLAG-tagged RdRps were immunoprecipitated using anti-FLAG mouse monoclonal antibody (Sigma), and immunopre-cipitates were analyzed by a Western blot using goat anti-HA polyclonal antibodies (Abcam) (top panel). (F) The WT SD, but not the SD chimeras, couldcoimmunoprecipitate with the GII.4 RdRp. The identities of the bands in the input (top panel) and the precipitated materials (middle and bottom panels) areshown to the right of the Western blot images. The relevant masses from the molecular mass standards are shown to the left of the Western blot image..

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readout and indicating that there is a sufficient window for theanalysis of the manipulation of the VP1 expression in the replicon.

To examine the effect of VP1 expression on viral replication,we made a single nucleotide insertion in the VP1 open readingframe of Mflc-R to generate Mflc-Rfs (Fig. 7). The insertion is 3= ofthe termination codon of the ORF1 polyprotein and was designedto abolish VP1 translation. Mflc-Rfs exhibited a significant de-crease in luciferase activity compared to that of Mflc-R (Fig. 7B). AWestern blot analysis of the proteins produced during infectionshowed that both Mflc-R and Mflc-Rfs expressed RdRp, but onlyMflc-R expressed VP1 (Fig. 7C). Mflc-Rfs did reduce RdRp accu-mulation to 50% of that of Mflc-R, likely due to decreased repli-cation (Fig. 7C). When trans-complemented with VP1, RdRp ex-pression by Mflc-Rfs recovered to 80% of that of Mflc-R (Fig. 7C).These results suggest that, despite VP1 being a structural protein,its proper expression is needed for optimal MNV replication.

Next, we determined the concentration- and species-specificeffects on Mflc-Rfs by expressing increasing concentrations of theMNV or GII.4 VP1 or SD in trans. Mflc-Rfs replication was en-hanced by cotransfection of the cells with 10 ng of the plasmidexpressing MNV VP1 (Fig. 7D). Higher levels of VP1 plasmidreduced Renilla levels expressed from the replicon, consistent withthe results from the NoV-5BR assay (Fig. 7D). Importantly, the

coexpression of the GII.4 VP1 did not increase the Renilla levels(Fig. 7D), thus ruling out the effects of nonspecific RNA bindingby VP1 being responsible for enhanced RNA levels. Expression ofthe MNV VP1 SD, but not the GII.4 SD, in trans also enhancedMflc-Rfs replication (Fig. 7E). All these findings are consistentwith VP1 SD playing a regulatory role in MNV RNA replication.

VP1 affects the kinetics of MNV RNA replication in cell cul-ture. We sought to examine further the kinetics of MNV replica-tion in response to exogenously expressed VP1 or SD. RAW264.7cells were transfected with in vitro-transcribed RNA of WT Mflc-Ror Mflc-Rfs along with plasmids to express either VP1 or the SDand harvested over a time course. Total RNAs were prepared, andthe accumulation of genomic and antigenomic RNAs was quan-tified by a strand-specific qRT-PCR assay. The copy numbers ofRNAs were extrapolated from a standard curve generated usingthe same qRT-PCR assay with in vitro-transcribed genomic andantigenomic RNA copy number controls. The antigenomic RNAwas found to increase, starting from 4 h posttransfection (hpt).Both the genomic and antigenomic RNAs increased up to 3 log10

by 16 hpt (Fig. 8A). Interestingly, the genomic and antigenomicRNAs from Mflc-Rfs increased more slowly than those from Mflc-R, resulting in a 1 log10 increase in the genomic RNA at 16 hpt (Fig.8A). When Mflc-Rfs was trans-complemented with VP1, thegenomic RNA copy number improved to nearly the same level asthat for Mflc-R (Fig. 8A). These results confirm that the accumu-lations of both antigenomic and genomic RNAs were expeditedand increased by the presence of VP1.

We further sought to examine whether the accumulation of thegenomic and antigenomic MNV replicon RNAs was affected byVP1 concentrations. VP1 was expressed at three concentrations oftransfected plasmids, and the level of VP1 expression affected boththe timing and the overall level of Mflc-Rfs genomic and antige-nomic RNAs (Fig. 8B). With the highest level of VP1, genomic andantigenomic RNAs were more abundant at 4 hpt. Interestingly,cells transfected to express the highest concentration of the VP1had reduced levels of antigenomic and genomic RNAs after 8 hpt,consistent with our previous observations that a higher abun-dance of VP1 fails to stimulate RNA synthesis by the RdRps (e.g.,see Fig. 1D and 1E). These results corroborate those from Fig. 7Dand E and show that VP1 concentration is an important factor inregulating NoV RNA synthesis.

Finally, we investigated whether the stimulatory effect of VP1or its SD on the MNV replicon is species specific. The GII.4 VP1,MNV VP1, and MNV SD proteins were individually coexpressedwith the Mflc-Rfs. The MNV VP1 and its SD were able to enhanceboth genomic and antigenomic RNA accumulation, although theMNV SD had a less robust stimulatory effect than full-length VP1.Consistent with the NoV-5BR assay and Renilla luciferase-ex-pressing replicon results, the GII.4 VP1 failed to enhance RNAaccumulation by the MNV replicon (Fig. 8C). The effect of theVP1 and its SD on NoV replicon replication was also species spe-cific.

DISCUSSION

Despite an emerging realization of their impact on human health,NoVs are one of the most poorly characterized groups of smallRNA viruses due to their failure to infect cultured cells. This studyidentified that VP1, the NoV major capsid protein, can modulateviral RNA-dependent RNA polymerase activity and MNV repli-cation. VP1 was able to increase RNA synthesis in the absence of

FIG 5 Recombinant VP1 S domains can interact with their RdRps in vitro. (A)SDS-PAGE analysis of purified RdRps and SD. E. coli purified GII.4 and MNVRdRps and VP1 SDs were resolved by a 4 to 12% NuPage Novex Bis-Tris gel(Invitrogen, Carlsbad, CA) and visualized by staining with Coomassie brilliantblue. (B) Differential scanning fluorimetry (DSF) profile of purified GII.4 andMNV RdRps in the presence of SDs. DSF was used to measure the stability ofpurified GII.4 RdRp in the presence of GII.4 or MNV SD. Each sample com-bination was tested in triplicate, and the results were duplicated in at least twoindependent assays. (C) Determination of thermal stability of GII.4 and MNVRdRps in the presence of their SDs by DSF. The differences between the Tms ofRdRp alone and RdRp plus SD were calculated (�Tm). Each sample combina-tion was tested in triplicate, and the results were duplicated in at least twoindependent assays. The data shown are the derivatives of the change in thefluorescence of the sample over time [-R= (T)].

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VPg and did not produce higher proportions of VPg-RNA mole-cules when VPg was coexpressed (Fig. 1 and 6). The shell domainsof GII.4 and MNV VP1s were able to stimulate RdRp activity ofthe homologous polymerases when expressed in trans (Fig. 3). Thelack of VP1 expression from an MNV replicon decreased replica-tion, while expression of VP1 or the SD of the replicon in trans wasable to partially restore replication (Fig. 2 and 8). VP1 and the SDcan form a coimmunoprecipitable complex with the RdRp, andrecombinant SD can bind RdRp in a DSF assay (Fig. 4 and 5).Finally, all interactions between VP1 and its derivatives with thereplication enzymes were species specific and depended on anoptimal level of VP1 expression (Fig. 2, 3, and 8).

Viral RNA replication requires a membrane-associated multi-subunit complex that has a number of modulatory factors to co-ordinate the rate and kinetics of synthesis as well as to avoid de-tection by cellular defenses (11, 14, 18). Our observation of a VP1-RdRp interaction is consistent with and extends a previousobservation of the feline calicivirus (FCV) polymerase (ProPol)interacting with VPg and VP1 in a yeast two-hybrid assay (29).Furthermore, the finding that VP1 can enhance both antigenomicand genomic RNA synthesis suggests that it may be an active com-ponent of the NoV replication complex. We speculate that VP1from the virion can gain access to the replicase during the trans-lational disassembly of the viral particle. However, as shown inFig. 8, additional VP1 proteins produced from the replicon afterthe initial translation of the nonstructural proteins remain com-petent to stimulate RNA synthesis. The stimulatory activity of VP1on RNA synthesis likely requires contact with the RdRp rather

than an indirect effect on the RNA template, given that the recom-binant SD can bind the RdRp in a species-specific manner. Wewant to emphasize that VP1’s activity is to enhance, but not acti-vate, RdRp and that RNA synthesis can take place, albeit at a lowerlevel, in the absence of VP1. Once RNA replication and sub-genomic RNA synthesis have been initiated, the production ofVP1 boosts the level of antigenomic RNA replication, thus pro-viding templates for genomic RNA synthesis.

The observation that the S domain of VP1 is sufficient for thestimulatory activity suggests that the stimulatory effect is impor-tant for viral infectivity. VP1 is one of the most rapidly evolvingproteins in human caliciviruses, with changes in the P domainbeing correlated with escape from neutralizing antibodies (8). Incontrast, the S domain is highly conserved. The degree of conser-vation may be related to the need for several loops in the SD topromote species-specific interactions with the RdRp. It is also ofinterest that some caliciviruses and sapoviruses express VP1 as afusion to ORF1. Furthermore, McCormick et al. (31) demon-strated that the bovine norovirus expressed VP1 using a transla-tional termination-reinitiation process and proposed that thismechanism was required for functions other than RNA encapsi-dation. All of these results support a role for VP1 to affect genomereplication.

The replicase initiates antigenomic RNA synthesis from thegenomic plus-strand for use as templates for the synthesis of bothgenomic and subgenomic RNAs. VP1 interaction with the RdRpmay provide temporal regulation of NoV RNA synthesis relativeto other processes needed for successful infection. An essential

FIG 6 VP1 has a modest effect on VPg-primed RNA synthesis. (A) Effect of GII.4 VP1 on its VPg-primed RNA synthesis in the 5BR assay. HEK293T cells werecotransfected with GII.4 RdRp, VPg, and VP1 along with other luciferase reporter plasmids of the NoV-5BR assay. A plus symbol (�) on the x axis denotes thepresence of the respective plasmids. Empty vector was added as necessary to ensure the transfection of equal amounts of the plasmids into the cells. At 36 hpt, thefirefly-to-Renilla luciferase ratios were measured, and they are denoted on the y axis. (B) Effect of MNV VP1 on its VPg-primed RNA synthesis. HEK293T cellswere cotransfected with MNV RdRp, VPg, and VP1 along with other luciferase reporter plasmids of 5BR assay. A plus symbol (�) on the x axis denotes thepresence of the respective plasmids. Empty vector was added as necessary to ensure the transfection of equal amounts of the plasmids into the cells. After 36 h oftransfection, the firefly-to-Renilla luciferase ratios were measured, and they are denoted on the y axis. (C) Western blot analysis of GII.4 VPg immunoprecipitatedfrom HEK293T cells expressing the GII.4 RdRp and VP1. HEK293T cells were cotransfected with RdRp, HA-tagged VP1, and FLAG-tagged VPg. At 24 hpt, thecell lysates were immunoprecipitated using anti-FLAG monoclonal antibody. The relevant masses from the molecular mass standards are shown to the left of theWestern blot image. “VPg-RNA” denotes a band shifted in molecular mass from the free VPg molecule that was previously characterized in Subba-Reddy et al.(44) that VPg covalently linked to RNA. (D) Western blot analysis of MNV VPg immunoprecipitated from HEK293T cells expressing the MNV RdRp and VP1.

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property of capsid proteins is their ability to form higher-orderoligomers during RNA encapsidation. In fact, the S domain issufficient for oligomerization and to form icosahedral virus-likeparticles (VLPs) in the absence of the P domain (2). The loops inthe SD that participate in species-specific interaction with theRdRp are also needed for VP1 subunit interactions to form VLPs.The formation of the VLPs may act to prevent dissociated VP1from interacting with the RdRp. Thus, when VP1 concentrationsare high and viral RNA encapsidation becomes the dominant ac-tivity in an infected cell, the propensity of VP1 to oligomerizewould be expected to promote virion production and, concomi-tantly, decrease the enhancement of viral RNA synthesis. Consis-tent with this novel mode of concentration-dependent regulation,an MNV replicon coexpressed with higher levels of VP1 resulted

in higher levels of antigenomic and genomic RNA synthesis inRAW264.7 cells at 8 h posttransfection than replicons that lackedVP1 expression in trans or had lower VP1 levels (Fig. 8B). We alsonote that the brome mosaic virus has a similar concentration-dependent regulatory activity on RNA translation and RNA syn-thesis (50, 51).

We observed that VP1 can enhance the polymerase activity inthe absence of VPg. Furthermore, while VP1 coexpression in-creased the overall level of VPg in the NoV-5BR assay, it did notaffect the ratio of VPg to VPg-RNA (Fig. 6C and D). While we donot have a definitive explanation for the observed increase in VPglevels, it is possible that an interaction between VPg and VP1, asobserved in FCV using a yeast two-hybrid assay (29), may simplystabilize or protect VPg from proteolytic degradation. Also, in the

FIG 7 VP1 expression is required for efficient MNV replicon RNA. (A) Schematic representation of the Renilla luciferase expressing the MNV replicon (Mflc-R),showing the positions of the ORFs, the T7 promoter at the 5= end, and hepatitis delta virus ribozyme (3=Rz) at the 3= end. All the nonstructural proteins, p48,NTPase, p22, VPg, Pro, and RdRp were encoded by ORF1. The major and minor structural proteins VP1 and VP2 were encoded by ORF2 and ORF3, respectively.The Renilla luciferase gene was cloned upstream of the VP2 ORF. A mutant replicon containing an RdRp active-site mutant is named MflcGGG-R. The locationof the insertion of a single adenine (A) to cause a frameshift in the VP1 ORF is denoted by the black triangle. (B) The Mflc-Rfs replicon can be trans-complemented by the MNV VP1. Cells were transfected with 100 ng capped in vitro transcripts of Mflc that did not express luciferase reporter and the Mflc-R,Mflc-RGGG, and Mflc-Rfs replicons. “�” on the x axis denotes cotransfection with the empty vector, and “�” denotes cotransfection with VP1. Renilla luciferaseactivity was determined after 24 hpt and is shown in relative light units (RLU). Each assay was performed in triplicate, and the means and standard errors of twoindependent assays are plotted. (C) Western blot analysis of RAW264.7 cells transfected with Mflc-R, MflcGGG-R, and Mflc-Rfs. RAW264.7 cells were transfectedwith 100 ng of capped in vitro transcripts. Mock cells did not contain in vitro transcripts. Cell lysates were harvested 24 hpt, separated by SDS-PAGE, and analyzedby a Western blot using antisera to RdRp and VP1. Nonspecific host proteins that reacted to the antisera in Western blots are shown as loading controls (LC). (D)Mflc-Rfs replicon replication is recovered by trans-complementation of MNV VP1 in a concentration-dependent way. The RAW264.7 cells were transfected withincreasing amounts of MNV or GII.4 VP1 expression plasmids, as shown on the x axis. At 12 hpt, the cells were transfected again with 100 ng of Mflc-Rfs in vitrotranscripts. The cells were lysed 24 h later for quantification of Renilla luciferase activity (in RLU). The signal from Mflc that did not express luciferase reporterwas used as the background control. (E) The MNV VP1 SD has a concentration-dependent effect on the replicon RNA replication in RAW264.7 cells. The formatof the experiment was identical to that in panel D.

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MNV replicon assays, VP1 increased antigenomic as well asgenomic RNA levels. In these assays, the increase in antigenomicRNA in turn provides an additional template for genomic RNAsynthesis. While our results cannot absolutely rule out that VP1 isalso stimulating VPg-dependent RNA synthesis during NoV rep-lication, the parsimonious interpretation of our collective obser-vations is that VP1 exerts a stimulatory effect of minus-strandRNA synthesis, which, in turn, affects the plus-strand genomicRNA synthesis.

Our observations of a regulatory role for VP1 on HuNoV andMNV RNA synthesis provide two additional examples of crosstalk between viral structural proteins and RNA synthesis. With the

positive-strand RNA viruses, the capsids from hepatitis C virusand the rubella virus can affect translation as well as RNA replica-tion (12, 26, 42, 49). Neeleman et al. (36) reported that the coatprotein (CP) of alfalfa mosaic virus (AMV), a member of the ge-nus Alfamovirus in the family Bromoviridae, can bind to the 3= endof viral RNA and enhance subgenomic RNA4 translation. TheAMV CP can regulate RNA synthesis by binding to the 3= ends ofAlfamovirus and Ilarvirus RNAs to activate genome replication (6,23). In rotavirus, VP2 can serve as a scaffold for the viral polymer-ase as well as act as a cofactor for VP1 to initiate genome replica-tion (32, 38). The CPs of the plant-infecting brome mosaic virusand the bacteriophage MS2 play regulatory roles in binding toRNA elements that regulate RNA synthesis (46, 50, 51).

In summary, we report a novel interaction between the NoVstructural protein and the RdRp that modulated viral RNA syn-thesis in a concentration-dependent manner. Our results illus-trate that the MNV serves as a useful model system for the regu-lation of NoV translation and replication. This work providesadditional evidence of viral structural proteins having regulatoryroles in viral RNA synthesis, an emerging theme for a number ofmammalian viruses.

ACKNOWLEDGMENTS

Research by C.C.K. was supported by the Indiana Economic Develop-ment Council. Research by I.G.G. was supported by the Wellcome Trust,and M.A.Y. was supported by the Malaysian Government.

We thank Dalan Bailey (Institute for Animal Health, Pirbright) for hisinput on the initial design and characterization of the MNV repliconsystem and our colleagues at Indiana University for many helpful andformative discussions.

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FIG 8 Quantification of Mflc-Rfs genomic and antigenomic RNAs by qRT-PCR. (A) qRT-PCR quantification of MNV genomic and antigenomic RNAs atmultiple time points. RAW264.7 cells were transfected with 1 �g of in vitrotranscripts each of the Mflc-R (WT) and Mflc-Rfs (Fs) replicons and Mflc-Rfswith MNV VP1. Cells were harvested at various time points posttransfection,washed, and lysed, and the total RNA was extracted. Total RNA was used toreverse transcribe to cDNA, followed by quantitative RT-PCR. All RT-PCRquantifications were performed in duplicate in two independent experiments,and the average copy number and standard deviation are plotted. (B) Concen-tration-dependent effect of VP1 on Mflc-Rfs genomic and antigenomic RNAs.RAW264.7 cells coexpressing 5, 10, or 20 ng of VP1 were transfected with 1 �gof transcripts of Mflc-Rfs. The samples were processed as described in panel B.(C) Species-specific effect of VP1 or SD on Mflc-Rfs genomic and antigenomicRNAs. RAW264.7 cells coexpressing MNV VP1, MNV VP1 SD, or GII.4 VP1were transfected with 1 �g in vitro transcripts of Mflc-Rfs. The paired Studentt test was used to determine the statistical difference between the tested sampleand the reference sample (identified by the asterisk). The P values for each pairof data are shown in parentheses. All samples analyzed were from the 16-h timepoint.

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VP1 Modulates Norovirus RNA Replication

September 2012 Volume 86 Number 18 jvi.asm.org 10149

RETRACTED

on March 17, 2018 by guest

http://jvi.asm.org/

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nloaded from

Retraction for Subba-Reddy et al.,“Norovirus RNA Synthesis Is Modulatedby an Interaction between the ViralRNA-Dependent RNA Polymerase andthe Major Capsid Protein, VP1”

Chennareddy V. Subba-Reddy,a Muhammad Amir Yunus,b Ian G. Goodfellow,b

C. Cheng Kaoa

Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, Indiana, USAa; Section ofVirology, Department of Medicine, Imperial College London, London, United Kingdomb

Volume 86, no. 18, p. 10138 –10149, 2012, https://doi.org/10.1128/JVI.01208-12. Weregret that we need to retract this article due to probable image manipulation in Fig.6C and D. The original images used in the paper are no longer available. Dr. Subba-Reddy assembled the figure and could not be reached. Drs. Yunus and Goodfellow didnot participate in making the problematic figure panels.

The paper’s overall conclusion that the norovirus major capsid protein could mod-ulate norovirus polymerase activity has been confirmed in both the Kao and theGoodfellow labs.

Citation Subba-Reddy CV, Yunus MA,Goodfellow IG, Kao CC. 2017. Retraction forSubba-Reddy et al., “Norovirus RNA synthesisis modulated by an interaction between theviral RNA-dependent RNA polymerase andthe major capsid protein, VP1.” J Virol91:e01708-17. https://doi.org/10.1128/JVI.01708-17.

Copyright © 2017 American Society forMicrobiology. All Rights Reserved.

C.V.S.-R. could not be reached when asked toagree to the Retraction.

RETRACTION

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December 2017 Volume 91 Issue 24 e01708-17 jvi.asm.org 1Journal of Virology