Article VPg Binds RNA through a Conserved -Terminal / Basic Patch

Alice . McSweeney, Vivienne . Young and Vernon K. Ward *

Department of Microbiology & Immunology, School of Biomedical Sciences, University of Otago, P.. Box 56, Dunedin 9054, New Zealand; [email protected] (A.M.M.); [email protected] (.L..) * Correspondence: [email protected]

Abstract: The viral protein -linked (VPg) of is a multi-functional protein that participates in essential roles during the cycle. Predictive analyses indicate that (MNV) VPg contains a disordered N-terminal region with RNA binding potential. VPg proteins were expressed with an N-terminal spidroin fusion protein in insect cells and the interaction with RNA investigated by electrophoretic mobility shift assays (EMSA) against a series of RNA probes (pentaprobes) representing all possible five nucleotide combinations. MNV VPg and human norovirus (HuNV) VPg proteins were directly bound to RNA in a non-specific manner. To identify amino acids involved in binding to RNA, all basic (K/R) residues in the first 12 amino acids of MNV VPg were mutated to alanine. Removal of the K/R amino acids eliminated RNA binding and is consistent with a K/R basic patch RNA binding motif within the disordered N-terminal region of norovirus VPgs. Finally, we show that mutation of the K/R basic patch required for RNA binding eliminates the ability of MNV VPg to induce a G0/G1 cell cycle arrest.

 Keywords: norovirus; calicivirus; RNA binding; VPg; spidroin 

Citation: McSweeney, A.M.; Young, V.L.; Ward, V.K. Norovirus VPg Binds RNA through a Conserved 1. Introduction N-Terminal K/R Basic Patch. Viruses Human noroviruses (HuNV) are a significant cause of and account for 2021, 13, 1282. https://doi.org/ approximately one-fifth of acute gastroenteritis across all age groups [1]. Recent studies 10.3390/v13071282 have demonstrated HuNV replication in a continuous human cell line, stem cell derived human enteroids or zebrafish larvae, significantly increasing our ability to directly study Academic Editor: Kim Y. Green HuNV [2–4]. However, implementation of these systems within the laboratory can be complex and there is considerable variation in replication between HuNV strains [5]. As a Received: 8 June 2021 result, murine norovirus (MNV) remains a commonly utilised model for studying Accepted: 26 June 2021 Published: 30 June 2021 the viral lifecycle. MNV replicates in laboratory cell culture lines and retains a similar genomic layout to

Publisher’ Note: MDPI stays neutral HuNV [6,7]. MNV possesses a positive-sense single-stranded RNA genome with four open with regard to jurisdictional claims in reading frames (ORF). ORF1 is translated as a large polyprotein, which is cleaved by the published maps and institutional affil- viral protease into the six non-structural proteins, NS1-2, NS3, NS4, NS5 (VPg), NS6 (Pro), iations. and NS7 (Pol, RdRp) [8]. ORF2 and ORF3 encode the major and minor proteins, respectively, while the fourth ORF of MNV encodes a virulence factor [9]. As with many viral proteins, the VPg proteins of noroviruses are multi-functional with a range of activities critical to the lifecycle of the virus. Nuclear magnetic resonance (NMR) analysis has shown that MNV VPg consists of a structured helical core flanked by Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. unstructured regions at each end of the protein (Figure1)[ 10]. During replication, VPg acts This article is an open access article as protein primer through the covalent addition of a nucleotide to a conserved tyrosine distributed under the terms and residue (Y26 in MNV) which then functions as the first base for extension by the RNA conditions of the Creative Commons polymerase during genome replication [11–13]. VPg is required for recruitment of the host Attribution (CC BY) license (https:// eukaryotic initiation factor 4G, facilitating translation of the viral genome [14–16]. VPg, creativecommons.org/licenses/by/ and in particular the N-terminal region of the protein, is also important for induction of a 4.0/). G0/G1 phase cell cycle arrest shown to promote viral replication [17–19].

Viruses 2021, 13, 1282. https://doi.org/10.3390/v13071282 https://www.mdpi.com/journal/viruses Viruses 2021, 13, FOR PEER REVIEW 2 of 18

Viruses 2021, 13, 1282 and in particular the N-terminal region of the protein, is also important for induction 2of of a 16 G0/G1 phase cell cycle arrest shown to promote viral replication [17–19].

Unstructured Helical core Unstructured

1 13 21 35 43 55 108 124 N Helix 1 Helix 2

NTP Y26 F123 binding eIF binding

Figure 1. Schematic of MNV VPg. The structural helices and helical core are indicated. Numbers Figure 1. Schematic of MNV VPg. The structural helices and helical core are indicated. Numbers represent the relative positions of amino acids. Amino acid Y26 is essential for nucleotidylylation represent the relative positions of amino acids. Amino acid Y26 is essential for nucleotidylylation and F123 is essential for translation. The unstructured N-terminal and C-terminal ends of the protein and F123 is essential for translation. The unstructured N-terminal and C-terminal ends of the protein areare indicated. indicated. The The first first 13 13 amino amino acids acids have have been been shown shown to to be be involved involved in in NTP NTP binding binding by by the the VPg VPg ofof HuNV. HuNV. VPg proteins are not unique to caliciviruses and have been identified in viruses in the VPg proteins are not unique to caliciviruses and have been identified in viruses in Picornaviridae and families, among others. The VPg proteins in these families the Picornaviridae and Potyviridae families, among others. The VPg proteins in these fami- share some similarities, including nucleotidylylation, however, additional functions and lies share some similarities, including nucleotidylylation, however, additional functions sequences of the proteins vary [20,21]. Non-calicivirus VPg proteins, either as mature and sequences of the proteins vary [20,21]. Non-calicivirus VPg proteins, either as mature or precursor proteins, have been shown to bind RNA [22–24] but binding of calicivirus or precursor proteins, have been shown to bind RNA [22–24] but binding of calicivirus VPg proteins to RNA is unknown. Despite an absence of canonical RNA binding motifs, VPg proteins to RNA is unknown. Despite an absence of canonical RNA binding motifs, MNV VPg has been shown to bind phosphocellulose [20], a characteristic of RNA binding MNV VPg has been shown to bind phosphocellulose [20], a characteristic of RNA binding proteins. The N-terminal region of HuNV VPg has also been shown to bind nucleotide proteins. The N-terminal region of HuNV VPg has also been shown to bind nucleotide triphosphates (NTPs) [25]. Recently, it has become apparent that the traditional view of triphosphates (NTPs) [25]. Recently, it has become apparent that the traditional view of canonical RNA binding motifs is too narrow, and disordered regions of proteins represent canonical RNA binding motifs is too narrow, and disordered regions of proteins represent a significant proportion of RNA binding proteins [26–28]. a significantWe identified proportion a region of RNA of MNV binding VPg proteins within [26–28]. the disordered N-terminus as a putative RNAWe binding identified site. a RNAregion electromobility of MNV VPg within shift assays the disordered (EMSA) showed N-terminus that MNV as a putative VPg and RNAHuNV binding VPg weresite. RNA able electromobility to directly bind shift to RNA assays in (EMSA) a non-specific showed manner that MNV and VPg we showand HuNVthat the VPg MNV were VPg able binds to directly to RNA bind through to RNA a in basic a non-specific patch of charged manner aminoand we acids show within that thethe MNV disordered VPg binds N-terminal to RNA region through that a is basic conserved patch inof noroviruses.charged amino Finally, acids we within show the that disorderedperturbation N-terminal of the N-terminal region that amino is conserve acids criticald in noroviruses. for RNA binding Finally, also we affects show that the ability per- turbationof VPg to of induce the N-terminal a cell cycle amino arrest. acids critical for RNA binding also affects the ability of VPg to induce a cell cycle arrest. 2. Materials and Methods 2. MaterialsVPg protein and Methods sequences for MNV and HuNV GII.4 strain Sydney 2012 were obtained fromVPg GenBank protein (accession sequences numbers for MNV DQ285629 and HuNV and JX459908,II.4 strain respectively).Sydney 2012 Thewere nucleic obtained acid frombinding GenBank potential (accession of MNV numbers VPg was DQ285629 assessed and using JX459908, the DRNAPred respectively). server The [29]. nucleic Protein acidsequences binding were potential submitted of MNV to PrDOSVPg was [30 assessed], PONDR using VL-XT the [DRNAPred31–33] and IUPred2Aserver [29]. [ 34Pro-,35] teinservers sequences for prediction were submitted of disorder to PrDOS in the [30], first PONDR 35 amino VL-XT acids of[31–33] MNV and VPg IUPred2A using the [34,35]default servers settings. for prediction of disorder in the first 35 amino acids of MNV VPg using the default settings. 2.1. Expression and Purification of Wild-Type and Mutant VPg Proteins 2.1. ExpressionSynthetic and genes Purification for MNV of Wild-Type VPg, including and Muta a K5A/K7A/R10Ant VPg Proteins triple mutant (TM), a mutantSynthetic with genes all K andfor MNV R residues VPg, including in the first a 12K5A/K7A/R10A amino acids of triple VPg mutant modified (TM), to alaninea mu- tant(K2-12), with all HuNV K and GII.4 R residues VPg and inE. the coli firstchloramphenicol 12 amino acids acetyltransferase of VPg modified (CAT)to alanine (GenBank (K2- 12),BBB37551) HuNV GII.4 were VPg synthesised and . bycoli Genscriptchloramphenicol (Piscataway, acetyltransferase , USA). All (CAT) constructs (GenBank were BBB37551)confirmed were by sequencing. synthesised Genes by Genscript were ligated (Pisca intotaway, a baculovirus NJ, USA). All transfer constructs vector were after con- a p10 firmedpromoter by sequencing. and recombinant Genesbaculovirus were ligated was into produced a baculovirus using transfer the Flashback vector Ultraafter systema p10 promoter(Oxford Expressionand recombinant Technologies, baculovirus Oxford, was UK). produced A solubility using tag,the spidroinFlashback NT* Ultra [36 ,system37], was engineered as a fusion protein to the N-terminus of VPg, which contained a N-terminal (Oxford Expression Technologies, Oxford, UK). A solubility tag, spidroin NT* [36,37], was His -Strep II tandem affinity tag, the spidroin NT* fusion partner, an enterokinase cleavage engineered6 as a fusion protein to the N-terminus of VPg, which contained a N-terminal site, and a flexible GGSRS linker, followed by the VPg or CAT protein. For protein expression, suspension cultures of Trichoplusia ni cells were infected with recombinant baculovirus expressing VPg proteins or CAT at a multiplicity of of one and incubated at 27 ◦C at 125 RPM for three days. Cells were harvested by centrifu-

Viruses 2021, 13, 1282 3 of 16

gation and lysed in 50 mM Tris pH 8.0, 150 mM NaCl, 10% glycerol, and 1% triton X-100. The protein was purified on Streptactin XT superflow beads (IBA Lifesciences, Gottingen, Germany) and eluted with 50 mM Tris pH 8, 150 mM NaCl, 10% glycerol, and 50 mM Biotin. Protein concentration was determined by absorbance at 280 nm on a NanoDrop One (Thermo Fisher Scientific, Waltham, MA, USA) and eluted proteins were stored at −80 ◦C. Expression and purification of recombinant proteins was confirmed by SDS-PAGE gel with Coomassie blue G250 staining.

2.2. Generation of RNA Probes and mRNA Transcripts The RNA binding potential of VPg was screened against a series of plasmids con- taining 12 pentaprobes (PP) gifted by Professor Joel Mackay (Addgene plasmid # 83326– 83337) [38]. Purified plasmid DNA was linearised with ApaI and single-stranded RNA (ssRNA) was transcribed for each pentaprobe sequence under control of a T7 promoter with incorporation of fluorescent rUTP ATTO 680 (Jena Biosciences, Jena, Germany) for detection. The reaction consisted of 4 µL of 5× Transcription Optimised buffer (Promega Corporation, Madison, WI, USA), 1 µg of linear DNA template, 0.5 mM rATP, 0.5 mM rCTP, 0.5 mM rGTP, 12.5 µM rUTP (Promega), 20 µM rUTP ATTO 680, and 40 U T7 RNA polymerase (Promega), in a final reaction volume of 20 µL with nuclease free water. The transcription reaction was incubated in the dark at 37 ◦C for two hours. The DNA was degraded with DNase (Promega) and the RNA purified using a MEGAclearTM Transcription Clean-Up kit according to the manufacturer’s instructions (Ambion, Austin, TX, USA). Labelled RNA probes were eluted in nuclease free water and stored at −80 ◦C. RNA probes, approximately 150–200 bp, were designed to represent the 50 and 30 ends of the positive and negative-sense MNV RNA. The constructs were designed to include prominent RNA secondary structure elements near the 50 and 30 termini of the genomic and anti-genomic RNA. DNA templates for RNA probes were isolated by PCR and cloned into a pUC57 vector. All primers included a T7 promoter sequence and EcoRV restriction enzyme site, in addition to EcoRI and BamHI sites for of the PCR products into the pUC57 plasmid (Table1).

Table 1. Primer sequences for generation of RNA probes corresponding to the viral genome.

Forward Primer Sequence (50–30) a Reverse Primer Sequence (50–30) b GAATTCTAATACGACTCACTATAGTGAAA- GGATCCGATATCAGGGGTCATGTAATTAAT- 50pos TGAGGATGGCAACG TTCGTC GAATTCTAATACGACTCACTATAGACACA- GGATCCGATATCTTTTTTTTTTAAAATGCAT- 30pos TCCCCTCTACCGA CTAACT GAATTCTAATACGACTCACTATAGTTTTTTT- GGATCCGATATCGACACATCCCCTCTACCG- 50neg TTTAAAATGCATCTAACTACCAC ATCTC GAATTCTAATACGACTCACTATAGAGGGG- GGATCCGATATCGTGAAATGAGGATGGCA- 30neg TCATGTAATTAATTTCGTC ACG a T7 promoter sequence is underlined; b EcoRV restriction site is shown in bold.

The 50pos probe corresponding to the 50 end of the positive-sense RNA represents nucleotides 1–155 of the MNV-1 genome and includes the 50UTR and three stem loop RNA structures present at the 50 end of the ORF1 sequence. The 30neg probe corresponds to the 30 end of the negative-sense RNA and is the mirror of the 50pos probe. The 30pos probe corresponds to the 30 end of the positive-sense RNA, representing nucleotides 7158–7392 of the MNV-1 genome. The 30pos probe contains three prominent stem-loops, the 30 UTR and a short poly(A) tail. The 50neg probe corresponding to the 50 end of the negative-sense RNA is the complement of the 30pos. The recombinant plasmids carrying the terminal sequences were linearised with EcoRV and RNA produced as described for the pentaprobes. Generation of messenger RNA transcripts for transfection of RAW-Blue cells was performed as described previously [18]. Briefly, plasmids were linearised at the 50 end using EcoRI and mRNA transcripts were produced using the mMessage mMachine T7 Viruses 2021, 13, 1282 4 of 16

ultra transcription kit (Ambion). RNA was purified using the MEGAclearTM Transcription Clean-Up kit according to the manufacturer’s instructions (Ambion). Unlabelled DNA for competition assays was either produced from a single-stranded DNA template (TATGCGGGCGAAACTTCTGGGAATAGTCCTGACAACCCCTATTGC- GATCAGCTCTTTTGCTAGCTCG) or a complementary sequence was annealed to the single-stranded DNA to produce double-stranded DNA.

2.3. RNA Binding Assays Purified VPg proteins were buffer exchanged into RNA binding buffer (10 mM MOPS ◦ pH 7.0, 50 mM KCl, 5 mM MgCl2 and 10% glycerol). Pentaprobe RNA was heated to 95 C for one minute and then immediately cooled on ice prior to use, viral RNA probes were not heat denatured prior to the addition of VPg. For RNA binding reactions, ~100 nM of ATTO680 labelled RNA was added to protein samples at the indicated concentrations. The reaction was incubated in RNA binding buffer at 4 ◦C for 30 min. For DNA com- petition assays, a 100-fold molar excess of unlabelled DNA probe was also added to the binding reaction. Binding reactions were loaded onto a 5% native TBE acrylamide gel (29:1 acrylamide: bis-acrylamide) and electrophoresed in 0.5× TBE buffer at 120 V for approximately 40 min at room temperature. Gels were imaged on an Odyssey Fc imager in the 700 nm channel.

2.4. Cell Cycle Analysis Cell cycle analysis was performed as described previously [18,19]. Briefly, 1 × 106 RAW-Blue cells were transfected with 4–5 µg of in vitro transcript mRNA using an Invit- rogen Neon transfection system (Thermo Fisher Scientific). Cells were incubated for 12 and harvested in 70% ethanol, washed in PBS (Dulbecco’s A), the RNA degraded with 0.1 mg/mL RNase A, and DNA stained with 50 µg/mL propidium iodide. The percentage of cells in each phase of the cell cycle was measured by fluorescence-activated cell sorting and the phases of the cell cycle assigned using MODfit LT 5.0 software (Verity Software House; Topsham, ME, USA). Expression of VPg proteins was confirmed by western blot using the appropriate primary and secondary ; polyclonal rabbit anti-MNV-1 VPg [39], goat anti-actin (I-19) (sc1616; Santa Cruz, CA, United States), DyLight 800 donkey anti-rabbit IgG (SA5-10050; Thermo Fisher Scientific), and DyLight 680 donkey anti-goat IgG (SA5-10090; Thermo Fisher Scientific). Cell cycle data presents the mean and standard deviation of three independent ex- periments. Results were analysed by one-way ANOVA with a Dunnett’s post-test, and p values of ≤0.05 were considered statistically significant.

3. Results 3.1. MNV VPg Is an RNA Binding Protein Analysis of the MNV VPg sequence for predicted interactions with RNA and DNA using DRNAPred indicates that MNV VPg has potential for RNA binding, despite the absence of canonical RNA binding motifs such as a zinc finger domain or RNA recognition motif. In particular, the first 12 amino acids have probabilities between 0.91–0.83 for an interaction with RNA, whereas no regions of MNV VPg were predicted to interact with DNA (Figure2a). The VPg protein of the plant , potato A virus (PVA), has been shown to bind RNA [23]. An alignment with the RNA binding motif of PVA VPg identified that the 20 N-terminal amino acids of MNV VPg have some conservation with several basic and glycine residues (Figure2c), suggesting that this region may have RNA binding potential. Bioinformatic analysis indicates that MNV VPg contains regions of disorder, consistent with NMR data and disorder predictions of other norovirus VPg proteins [10,40]. The N-terminal 20 amino acids have a disorder tendency of >0.5 and are therefore likely to be intrinsically disordered (Figure2b). Residues 20–35 in this analysis have low disorder predictions and correspond to the first alpha helix of VPg, as determined by NMR [10]. Viruses 2021, 13, x FOR PEER REVIEW 5 of 18

DNA (Figure 2a). The VPg protein of the plant potyvirus, potato A virus (PVA), has been shown to bind RNA [23]. An alignment with the RNA binding motif of PVA VPg identi- fied that the 20 N-terminal amino acids of MNV VPg have some conservation with several basic and glycine residues (Figure 2c), suggesting that this region may have RNA binding potential. Bioinformatic analysis indicates that MNV VPg contains regions of disorder, con- sistent with NMR data and disorder predictions of other norovirus VPg proteins [10,40]. Viruses 2021, 13, 1282 The N-terminal 20 amino acids have a disorder tendency of >0.5 and are therefore likely5 of 16 to be intrinsically disordered (Figure 2b). Residues 20–35 in this analysis have low disor- der predictions and correspond to the first alpha helix of VPg, as determined by NMR [10].The combinedThe combined RNA RNA binding binding and disorder and disorder predictions predictions along along with thewith absence the absence of canonical of ca- nonicalRNA binding RNA binding motifs raises motifs the raises possibility the possibility that the N-terminal that the N-terminal 20 amino acids20 amino of MNV acids VPg of MNVrepresents VPg represents a disordered a disordered RNA binding RNA element. binding element.

Figure 2. Predictions of nucleic acid binding and disorder of MNV VPg.VPg. ( a) DRNAPred graph depicting predicted RNA (black) andand DNADNA (red) (red) binding binding probabilities probabilities of MNVof MNV VPg. VPg. (b) Predictions(b) Predictions for propensity for propensity of disorder of disorder of the firstof the 35 N-terminalfirst 35 N- terminalamino acids amino of MNV acids VPgof MNV using VPg PrDOS using (blue), PrDOS PONDR (blue), VLXT PONDR (red), VLXT and IUPred2A(red), and (red)IUPred2A servers. (red) Regions servers. with Regions predictions with predictionsabove 0.5 were above considered 0.5 were disorderedconsidered [disordered33] and are [33] indicated and are by indicated the dotted by line. the dotted (c) Alignment line. (c) ofAlignment the first 35of N-terminalthe first 35 N-terminal amino acids of MNV VPg and the RNA binding region of PVA VPg. An asterisk (*) indicates fully conserved amino acids of MNV VPg and the RNA binding region of PVA VPg. An asterisk (*) indicates fully conserved amino acids amino acids and a colon (:) indicates amino acids of a similar nature. and a colon (:) indicates amino acids of a similar nature.

AsAs RNA RNA binding binding by by norovirus norovirus VPg proteins was not known, we sought to determine thethe RNA RNA binding binding potential potential of of MNV MNV VPg VPg and and whether whether it itwas was sequence sequence specific. specific. We We used used a seriesa series of RNA of RNA probes, probes, termed termed pentaprobes pentaprobes [38], which [38], which were algorithm-designed were algorithm-designed to predict to everypredict possible every possible five nucleotide five nucleotide long RNA long bi RNAnding binding motif. motif.These Theseprobes, probes, expressed expressed as 12 probes,as 12 probes, each 100 each nucleotides 100 nucleotides in length, in length, allowed allowed testing testing for binding for binding to RNA to across RNA acrossa broad a rangebroad of range sequences of sequences and to andexplore to explore specificity specificity of the interaction, of the interaction, as represented as represented by linear by RNAlinear sequences. RNA sequences. RecombinantRecombinant MNV VPg VPg protein protein was expressed expressed and purified purified with an an N-terminal N-terminal NT* NT* fusionfusion partner partner and and the the resulting resulting protein protein term termeded NT*MNV VPg (Figure 3 3a).a). TheThe NT*NT* fusionfusion partner is derived from spider silk protein and acts to reduce protein aggregation within thethe spider silk glandgland andand hashas provenproven effective effective as as a a fusion fusion partner partner for for proteins proteins expressed expressed in inE. coliE. coli[36 [36].]. Production Production ofMNV of MNV VPg VPg with with the NT*the NT* fusion fusion partner partner generating generating approximately approxi- mately40 mg/L 40 ofmg/L soluble, of soluble, purified puri proteinfied protein illustrates illustrates that NT* that is NT an* effective is an effective fusion fusion partner part- for nerbaculovirus for baculovirus expression expr ofession proteins. of proteins. TheThe expression andand purity purity of of recombinant recombinan proteinst proteins used used for RNAfor RNA binding binding experiments experi- mentswas confirmed was confirmed by SDS-PAGE by SDS-PAGE gel with gel Coomassie with Coomassie blue staining blue (Figure staining3b). (Figure The interaction 3b). The of NT*MNV VPg with all 12 pentaprobes was analysed by EMSA, to determine the ability of MNV VPg to bind RNA (Figure3c). Increasing concentrations of NT*MNV VPg were incubated with 100 nM of pentaprobe RNA and analysed for an upward shift of the

RNA, indicative of binding. FOX-1, a known RNA binding protein, previously shown to interact with all 12 pentaprobes [38,41,42], was included as a positive control. FOX-1 induced a mobility shift, or completely abolished RNA migration of the target RNA for all pentaprobes. Upon incubation with NT*MNV VPg, an RNA mobility shift was observed for each pentaprobe (Figure3c). Viruses 2021, 13, x FOR PEER REVIEW 6 of 18

interaction of NT*MNV VPg with all 12 pentaprobes was analysed by EMSA, to determine the ability of MNV VPg to bind RNA (Figure 3c). Increasing concentrations of NT*MNV VPg were incubated with 100 nM of pentaprobe RNA and analysed for an upward shift of the RNA, indicative of binding. FOX-1, a known RNA binding protein, previously shown to interact with all 12 pentaprobes [38,41,42], was included as a positive control. Viruses 2021, 13, 1282 FOX-1 induced a mobility shift, or completely abolished RNA migration of the target RNA6 of 16 for all pentaprobes. Upon incubation with NT*MNV VPg, an RNA mobility shift was ob- served for each pentaprobe (Figure 3c).

FigureFigure 3. 3. BindingBinding of of NT*MNV NT*MNV VPg VPg to to RNA RNA pentaprobes pentaprobes.. ( (aa)) Schematic Schematic of of the the NT*MNV NT*MNV VPg, VPg, NT*CAT NT*CAT and and FOX-1 FOX-1 proteins proteins used in RNA binding experiments. The construct contained a His6 and Strep II affinity tag, the NT* fusion partner, and used in RNA binding experiments. The construct contained a His6 and Strep II affinity tag, the NT* fusion partner, and the theprotein protein to be to expressed.be expressed. An An enterokinase enterokinase cleavage cleavage site site (EK) (EK) was was included included for for cleavage cleavage and an removald removal of of the the fusion fusion partner. part- ner. (b) Coomassie blue stained SDS-PAGE gel of purified protein (3 μg) used for RNA binding experiments. 1; NT*MNV (b) Coomassie blue stained SDS-PAGE gel of purified protein (3 µg) used for RNA binding experiments. 1; NT*MNV VPg, VPg, 2; NT*CAT, 3; FOX-1. (c) RNA EMSA of purified NT*MNV VPg incubated with 100 nM UTP ATTO 680 labelled 2; NT*CAT, 3; FOX-1. (c) RNA EMSA of purified NT*MNV VPg incubated with 100 nM UTP ATTO 680 labelled pentaprobe pentaprobe RNA. The concentration of NT*MNV VPg was increased from 0.5 μM to 8 μM in 2-fold increments and is indicatedRNA. The by concentration a gradient triangle of NT*MNV from low VPg conc wasentration increased to fromhigh 0.5concµMentration. to 8 µM NT*CAT in 2-fold (8 increments μM) and FOX-1 and is (8 indicated μM) were by includeda gradient astriangle negative from and lowpositive concentration controls, respectively. to high concentration. NT*CAT (8 µM) and FOX-1 (8 µM) were included as negative and positive controls, respectively. In some instances, a second shift band was apparent, which could indicate higher orderIn structures some instances, or binding a second of multiple shift band VPg was proteins apparent, to whicha single could molecule indicate of higherRNA. orderAs a negativestructures control or binding protein, of multiple NT*CAT VPg showed proteins no RNA to a single binding molecule activity of across RNA. all As 12 a negativepentap- robescontrol when protein, compared NT*CAT with showed the RNA no RNAonly control, binding confirming activity across that all a protein-RNA 12 pentaprobes binding when eventcompared was not with a theconsequence RNA only of control, the NT* confirming fusion tag. that Removal a protein-RNA of NT* using binding enterokinase event was not a consequence of the NT* fusion tag. Removal of NT* using enterokinase resulted in resulted in MNV VPg still binding to RNA, confirming that NT* did not cause the binding MNV VPg still binding to RNA, confirming that NT* did not cause the binding of VPg of VPg to pentaprobes (Figure S1). Cleavage of VPg from the NT* tag with enterokinase to pentaprobes (Figure S1). Cleavage of VPg from the NT* tag with enterokinase showed multiple non-specific cleavage sites (Figure S1). Given that RNA binding still occurs with the N-terminal fusion, future experiments were undertaken with NT*MNV VPg. Overall, the RNA EMSA assays show that MNV VPg is a non-specific RNA binding protein. As the protein was bound to all pentaprobe RNA targets, a subgroup of pentaprobes (PP4, PP7 and PP9) were used for subsequent analyses. Predictive analyses of VPg indicated a low probability for DNA binding (Figure2a ). The specificity for RNA over DNA was investigated by a competition EMSA assay. To achieve this, unlabelled single-stranded or double-stranded DNA was added to the reaction at a 100-fold molar excess over labelled PP9 RNA. An RNA mobility shift was observed Viruses 2021, 13, x FOR PEER REVIEW 7 of 18

showed multiple non-specific cleavage sites (Figure S1). Given that RNA binding still oc- curs with the N-terminal fusion, future experiments were undertaken with NT*MNV VPg. Overall, the RNA EMSA assays show that MNV VPg is a non-specific RNA binding pro- tein. As the protein was bound to all pentaprobe RNA targets, a subgroup of pentaprobes (PP4, PP7 and PP9) were used for subsequent analyses. Predictive analyses of VPg indicated a low probability for DNA binding (Figure 2a). Viruses 2021, 13, 1282 The specificity for RNA over DNA was investigated by a competition EMSA assay.7 of To 16 achieve this, unlabelled single-stranded or double-stranded DNA was added to the reac- tion at a 100-fold molar excess over labelled PP9 RNA. An RNA mobility shift was ob- whenserved NT*MNV when NT*MNV VPg was VPg incubated was incubated with the with labelled the labelled PP9 RNA PP9 pentaprobe RNA pentaprobe and either and ssDNAeither ssDNA or dsDNA or dsDNA (Figure 4(Figure). Despite 4). aDespite molar excessa molar of DNA,excess NT*MNVof DNA, NT*MNV VPg still bound VPg still to RNA,bound indicating to RNA, indicating a strong preferencea strong preferen for RNAce for and RNA no evidence and no evidence for DNA for binding DNA binding in this competitionin this competition assay. assay.

. FigureFigure 4.4. NT*MNVNT*MNV VPgVPg preferentiallypreferentially bindsbinds RNA.RNA. NT*MNVNT*MNV VPgVPg waswas incubatedincubated withwith 100100 nMnM UTPUTP ATTOATTO 680680 labelledlabelled pentaprobepentaprobe RNARNA andand a 100-fold molar excess excess of of either either sing single-strandedle-stranded or or double-stranded double-stranded unlabelled unlabelled DNA DNA probe probe for for 30 30min. min. The The concentration concentration of of NT*M NT*MNVNV VPg VPg was was increased increased from from 0.5 0.5 μµMM to to 8 8 μµMM in in 2-fold 2-fold increments, as shown byby aa gradientgradient triangle.triangle. NT*CATNT*CAT (8 (8µ μM)M) and and FOX-1 FOX-1 (8 (8µ μM)M) were were included included as as negative negative and and positive positive controls, controls, respectively. respectively.

VPgVPg is essential for for replication replication of of the the viral viral RNA RNA and and is covalently is covalently linked linked to the to the5′ end 50 endof the of viral the viral RNA RNA genome genome [13,43]. [13 ,RNA43]. RNA probes probes were weredesigned designed to represent to represent the 5′ theor 3 5′ 0ter-or 3mini0 termini of the of viral the positive-sense viral positive-sense or negative-s or negative-senseense RNA, to RNA, determine to determine if VPg could if VPg interact could interactwith these with genomic these genomic ends (Figure ends 5). (Figure These5 ).probes These were probes designed were designed to incorporate to incorporate the 5′ and the3′ UTRs 50 and of 3 0theUTRs genomic of the RNA genomic along RNA with along several with major several stem-loop major stem-loop structures. structures. Labelled LabelledRNA probes RNA were probes synthesized, were synthesized, purified, purified,and the interaction and the interaction of NT*MNV of NT*MNV VPg with VPg the with5′pos, the 3′pos, 50pos, and 3 03pos,′neg and probes 30neg was probes examined was examined(Figure 5c). (Figure By EMSA5c). NT*MNV By EMSA VPg NT*MNV bound 0 VPgto each bound of these to each RNA of probes. these RNA We were probes. unable We were to produce unable RNA to produce for the RNA5′neg forprobe, the 5corre-neg 0 probe,sponding corresponding to the 5′ end to of the the 5 negative-senseend of the negative-sense RNA. This confirms RNA. This that confirms VPg can that bind VPg to can the bindterminal to the regions terminal of the regions genomic of the and genomic antigenomi andc antigenomicRNA; however, RNA; there however, is no evidence there is that no evidencethis is specific that this or enhanced is specific orin enhancedcomparison in to comparison the binding to theaffinity binding shown affinity for pentaprobe shown for pentaprobeRNA. RNA. DueDue to the the conservation conservation in in sequence sequence an andd common common functions functions shown shown for norovirus for norovirus VPg VPgproteins, proteins, we tested we tested if RNA if RNA binding binding would would be a beconserved a conserved function function of other of other norovirus norovirus VPg VPgproteins. proteins. A recombinant A recombinant baculo baculovirusvirus was generated was generated to expres to expresss GII.4 GII.4Sydney Sydney 2012 strain 2012 strainHuNV HuNV VPg with VPg an with N-terminal an N-terminal NT*fusion NT*fusion protein. protein. Purity Purity of NT*HuNV of NT*HuNV VPg VPgwas wascon- confirmedfirmed by SDS-PAGE by SDS-PAGE gel gel(Figure (Figure 6a)6 anda) and the the interaction interaction with with PP4, PP4, PP7, PP7, and and PP9 PP9 RNA RNA was wasinvestigated investigated by EMSA by EMSA (Figure (Figure 6b).6 b).As Aswith with previous previous experiments, experiments, there there was was no no shift shift in inthe the unbound unbound RNA RNA band band upon upon incubation incubation with with NT*CAT NT*CAT and and a completecomplete disappearancedisappearance withwith FOX-1.FOX-1. AdditionAddition ofof NT*HuNVNT*HuNV VPgVPg showedshowed aa gelgel shiftshift withwith PP4,PP4, PP7,PP7, and PP9 RNA. TheThe shiftshift waswas identifiedidentified byby aa decreasedecrease inin thethe unboundunbound RNARNA bandband and,and, forfor PP4PP4 andand PP7,PP7, thethe appearanceappearance ofof aa shiftedshifted bandband visiblevisible whenwhen RNARNA waswas incubatedincubated withwith NT*HuNVNT*HuNV VPg.VPg. Similar to MNV VPg, the binding of HuNV VPg to multiple probes is interpreted as binding to RNA in a non-specific manner.

Viruses 2021, 13, x FOR PEER REVIEW 8 of 18

Viruses 2021, 13, x FOR PEER REVIEW 8 of 18

Viruses 2021, 13, 1282 8 of 16 Similar to MNV VPg, the binding of HuNV VPg to multiple probes is interpreted as bind- ingSimilar to RNA to MNV in a non-specificVPg, the binding manner. of HuNV VPg to multiple probes is interpreted as bind- ing to RNA in a non-specific manner.

Figure 5. ViralViral RNA probes probes.. ( a)) Schematic Schematic of of the the positive-sense positive-sense viral RNA probes (50′pos and 3 ′0pos). The The genome genome positions ofFigure nucleotides nucleotides 5. Viral for RNA for the the probes positive-sense positive-sense. (a) Schematic RNA RNA probes of probes the positive-senseare are indicated. indicated. (viralb) Schematic (b RNA) Schematic probes of the of(5 negative-sense′ thepos negative-senseand 3′pos). viral The RNA viralgenome RNAprobes positions probes 3′neg andof30neg nucleotides 5′neg. and 5The0neg. for negative-sense Thethe positive-sense negative-sense RNA probesRNA RNA probes probes are the are are complements indicated. the complements (b of) Schematic the positive-sense of the of positive-sense the negative-sense RNA probes. RNA probes.viral RNA RNA secondary RNA probes secondary struc- 3′neg turesandstructures 5 ′(neg.a,b) The (werea,b )negative-sense werepredicted predicted using RNA using mfold probes mfold [44,45]. are [44 the(,c45) complementsNT*MNV]. (c) NT*MNV VPg of was VPg the incubatedpositive-sense was incubated with RNA labelled with probes. labelled 5′pos, RNA 5 30pos,′pos secondary 3or0pos 3′neg or struc- 3RNA0neg probes and analysed by EMSA. The concentration of NT*MNV VPg was increased from 0.5 μM to 8 μM in 2-fold incre- turesRNA ( probesa,b) were and predicted analysed using by EMSA. mfold The [44,45]. concentration (c) NT*MNV ofNT*MNV VPg was incubated VPg was increasedwith labelled from 5′ 0.5pos,µ M3′pos to 8orµ M3′neg in 2-foldRNA ments.probes NT*CATand analysed (8 μM) by and EMSA. FOX-1 The (8 concentrat μM) wereion included of NT*MNV as negative VPg wasand increasedpositive controls, from 0.5 respectively. μM to 8 μM in 2-fold incre- increments. NT*CAT (8 µM) and FOX-1 (8 µM) were included as negative and positive controls, respectively. ments. NT*CAT (8 μM) and FOX-1 (8 μM) were included as negative and positive controls, respectively.

Figure 6. HuNV VPg binds RNA. (a) Coomassie blue stained SDS-PAGE gel of purified protein (3 μg) used for RNA bindingFigure 6. experiments. HuNVHuNV VPg VPg 1;binds binds NT*HuNV RNA RNA.. (VPg, (aa)) Coomassie Coomassie 2; NT*CAT, blue blue 3; FOX-1.stained stained ( bSDS-PAGE SDS-PAGE) RNA EMSA gel gel ofof of NT*HuNVpurified purified protein protein VPg with (3 (3 μµ pentaprobeg) used for RNA.RNA Proteins were incubated with 100 nM UTP ATTO 680 labelled pentaprobe RNA for 30 min, separated on a 5% TBE gel, binding experiments. 1; 1; NT*HuNV NT*HuNV VPg, VPg, 2; 2; NT*CAT, NT*CAT, 3; FOX-1. ( (bb)) RNA RNA EMSA EMSA of of NT*HuNV NT*HuNV VP VPgg with with pentaprobe pentaprobe RNA. RNA. Proteins were incubatedincubated withwith 100100 nM nM UTP UTP ATTO ATTO 680 680 labelled labelled pentaprobe pentaprobe RNA RNA for for 30 min,30 min, separated separated on aon 5% a TBE5% TBE gel, andgel, directly imaged in the 700 nm channel. The concentration of NT*HuNV VPg was increased from 0.5 µM to 8 µM in 2-fold

increments. NT*CAT (8 µM) was included as a negative control and FOX- 1 (8 µM) as a positive control.

Viruses 2021, 13, x FOR PEER REVIEW 9 of 18

Viruses 2021, 13, 1282 9 of 16 and directly imaged in the 700 nm channel. The concentration of NT*HuNV VPg was increased from 0.5 μM to 8 μM in 2- fold increments. NT*CAT (8 μM) was included as a negative control and FOX- 1 (8 μM) as a positive control.

3.2.3.2. The The Basic Basic Amino Amino Acid Acid Patch Patch Near Near the the N-Terminus N-Terminus of MNV VPg VPg Is Is Required Required for for Binding Binding to RNA.to RNA TheThe N-terminus N-terminus of of norovirus norovirus VPg VPg contains contains a a highly highly conserved conserved epitope epitope rich rich in in posi- posi- tivelytively charged charged lysinelysine andand argininearginine residues residues (Figure (Figure7). 7). Predictive Predictive analyses analyses indicate indicate that that the thedisordered disordered N-terminus N-terminus of MNV of MNV VPg VPg containing containing the the conserved conserved epitope epitope may may be responsiblebe respon- siblefor binding for binding to RNA to RNA (Figures (Figure1 and 17 and). Figure 7).

FigureFigure 7. 7. AlignmentAlignment of of VPg VPg sequences. sequences. An An asterisk asterisk (*) (*) indicates indicates identical identical residues residues and and a a colon colon (:) (:) indicatesindicates residues residues that that are are similar. similar. Conserved Conserved am aminoino acids acids of of the the N-terminal N-terminal region region are are shown shown in in red.red. The The tyrosine tyrosine residue residue (Y) (Y) corre correspondingsponding to to Y26 Y26 of of MNV MNV VPg VPg is is shown shown in in black black in in bold. bold. The The fully fully conservedconserved amino acids surroundingsurrounding the the Y26 Y26 are are shown shown in in blue blue and and similar similar amino amino acids acids in green. in green. The The represented genogroups are GI Norwalk virus VPg (AAC64602), GII Sydney 2012 VPg represented genogroups are GI Norwalk virus VPg (AAC64602), GII Sydney 2012 VPg (JX459908), (JX459908), GIII Jena virus VPg (CAA90480) and GIV Lake Macquarie VPg (AFJ21375). GIII Jena virus VPg (CAA90480) and GIV Lake Macquarie VPg (AFJ21375).

TwoTwo MNV MNV VPg VPg constructs constructs were were designed designed to to test test the the importance importance of of positively positively charged charged aminoamino acids acids within within the the conserved conserved sequence sequence regi regionon for for binding binding to to RNA. RNA. The The first first construct construct createdcreated mutations, toto alanine,alanine, at at residues residues K5, K5, K7, K7, and and R10 R10 of the of conservedthe conserved epitope epitope of MNV of MNVVPg (MNV VPg (MNV VPg TM)VPg TM) (Figure (Figure8a). The8a). secondThe seco constructnd construct generated generated alanine alanine mutations mutations at atall all lysine lysine and and arginine arginine residues residues within within the the first first 12 12 aminoamino acidsacids ofof MNVMNV VPg (MNV VPg VPg K2-12).K2-12). Proteins Proteins were expressed withwith anan N-terminalN-terminal NT* NT* fusion fusion protein protein and and purification purification of ofNT*MNV NT*MNV VPg VPg TM TM and and NT*MNV NT*MNV VPg VPg K2-12 K2-12 was was confirmed confirmed by by SDS-PAGE SDS-PAGE gel gel (Figure (Figure8b). 8b).The The interactions interactions with with PP4, PP4, PP7, PP7, and PP9and werePP9 were analysed analysed by EMSA by EMSA (Figure (Figure8c,). Incubation 8c and d). Incubationof NT*MNV of NT*MNV VPg TM withVPg TM RNA with resulted RNA resulted in a gel in shift a gel for shift all for three all pentaprobes.three pentaprobes. This Thisindicates indicates that MNVthat MNV VPg VPg TM isTM still is capablestill capable of binding of binding to RNA to RNA and mutationand mutation of just of three just threeamino amino acids acids did not did eliminate not eliminate RNA RNA binding. binding. In contrast, In contrast, addition addition of NT*MNV of NT*MNV VPg K2-12 VPg K2-12had no had effect no oneffect the on mobility the mobility of pentaprobe of pentaprobe RNA atRNA the proteinat the protein concentrations concentrations tested. tested.This suggests This suggests that the that seven the lysine sevenand lysine arginine and arginine residues residues within thewithin conserved the conserved N-terminal N- terminalregion of region MNV VPgof MNV may VPg form may a basic form patch a basic and actpatch co-operatively and act co-operatively to facilitate non-specificto facilitate non-specificRNA binding. RNA binding. The conserved N-terminal region of norovirus VPg proteins is involved in binding of NTPs, nucleotidylation, and cell cycle manipulation [11,17,25]. We were interested in determining if mutations that eliminate RNA binding have any effect on the cell cycle, as previously this has only been investigated in relation to single point mutations or truncations of the motif [17]. To determine the importance of positively charged amino acids for a G0/G1 cell cycle arrest, mRNA transcripts encoding MNV VPg TM and MNV VPg K2-12 were transfected into asynchronous RAW-Blue cells and the percentage of cells in each phase of the cell cycle measured by flow cytometry (Figure9). Expression of VPg constructs was confirmed by western blot and each construct was shown to express to similar levels as wild-type MNV VPg (Figure9c,e).

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Figure 8. N-terminal lysine and arginine amino acids are important for binding to RNA. (a) Schematic of the first 20 amino Figure 8. N-terminal lysine and arginine amino acids are important for binding to RNA. (a) Schematic of the first 20 amino acids of MNV VPg, wild-type amino acids are shown in blue. Amino acids mutated to alanine are shown in red for the acids of MNV VPg, wild-type amino acids are shown in blue. Amino acids mutated to alanine are shown in red for the NT*MNV VPg and NT*MNV VPg K2-12 constructs, respectively. (b) Coomassie blue stained SDS-PAGE gel of purified NT*MNV VPg and NT*MNV VPg K2-12 constructs, respectively. (b) Coomassie blue stained SDS-PAGE gel of purified µ protein (3 μg) used for RNA binding experiments. 1; 1; NT*MNV NT*MNV VP VPgg TM; TM; NT*CAT, NT*CAT, 3; NT* MNV VPg K2-12 and 4; 4; FOX-1. FOX-1. (c,,d) RNARNA EMSAEMSA ofof NT*MNVNT*MNV VPgVPg K2-12K2-12 andand NT*MNVNT*MNV VPg TMTM withwith pentaprobepentaprobe RNA.RNA. ProteinsProteins were incubatedincubated with 100 nMnM UTPUTP ATTOATTO 680 680 labelled labelled pentaprobe pentaprobe RNA RNA for for 30 30 min, min, separated separated on on a 5%a 5% TBE TBE gel gel and and directly directly imaged imaged in the in 700the 700 nm channel.nm channel. The The concentration concentration of NT*MNV of NT*MNV VPg TMVPg was TM increasedwas increased from 0.5fromµM 0.5 to μ 8Mµ Mto in8 μ 2-foldM in increments.2-fold increments. NT*CAT NT*CAT (8 µM) was(8 μM) included was included as a negative as a negative control andcontrol FOX-1 and (8FOX-1µM) as(8 aμ positiveM) as a positive control. control.

TheCell conserved cycle analysis N-terminal showed region that atof 12norovirus h post-transfection, VPg proteins MNV is involved VPg induced in binding an ofincrease, NTPs, tonucleotidylation, approximately 70%and ofcell cells cycle in G0/G1manipulation phase with [11,17,25]. a corresponding We were decreaseinterested in in S determiningphase cells. (Figureif mutations9b,d). that MNV eliminate VPg TM RNA induced binding an accumulationhave any effect of on cells the in cell G0/G1, cycle, toas previously63%, significantly this has different only been from investigated both mock in transfected relation to cells single and point MNV mutations VPg, indicating or trunca- that tionswhile of there the motif was still [17]. a To change determine in the the G0/G1 importance population of positively it was decreased charged amino (Figure acids9b). for In acontrast, G0/G1 cell transfection cycle arrest, of MNV mRNA VPg transcripts K2-12 showed encoding no significant MNV VPg changes TM and in MNV the percentage VPg K2- 12of cellswere intransfected each phase into of theasynchronous cell cycle compared RAW-Blue to mockcells and transfected the percentage cells (Figure of cells9d). in Thateach phaseMNV VPgof the TM cell binds cycle RNA measured and induces by flow a partialcytometry cell cycle(Figure arrest 9). Expression while MNV of VPg VPg K2-12 con- structsdoes not was possess confirmed either by of thesewestern functions blot and corroborates each construct previous was shown findings to onexpress the importance to similar levelsof the as N-terminal wild-type region MNV forVPg cell (Figure cycle 9c,e). arrest and that this correlates with the basic patch. However,Cell cycle further analysis research showed will need that toat be12 performedh post-transfection, to confirm MNV whether VPg thereinduced is a an direct in- crease,mechanistic to approximately relationship between70% of cells RNA in bindingG0/G1 phase and cell with cycle a corresponding manipulation. decrease in S phase cells. (Figure 9b,d). MNV VPg TM induced an accumulation of cells in G0/G1, to 63%, significantly different from both mock transfected cells and MNV VPg, indicating

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Figure 9. Manipulation of the cell cycle by MNV VPg with N-terminal mutations mutations.. RAW-Blue cells were transfected with 4–5 µμg of transcript RNA corresponding to MNV VPg, MNV VPg TM, or MNVMNV VPgVPg K2-12.K2-12. Mock transfected (MT) cells were seededseeded atat thethe timetime of of transfection transfection as as a negativea negative control. contro Atl. At 12 12 h posth post transfection, transfection, cells cells were were harvested harvested for westernfor western blot blotand flowand flow cytometry cytometry analysis anal ofysis the of cell the cycle. cell cycle. (a) Representative (a) Representative histograms histograms from onefrom of on threee of experiments.three experiments. The positions The posi- of tionsthe G0/G1 of the andG0/G1 G2/M and phaseG2/M populationsphase populations of cells of are cells labelled are labelled on the ton= 0the histogram. = 0 histogram. The S phase The S lies phase between lies between the G0/G1 the G0/G1 and G2/M populations and is indicated by hatched lines. (c,e) Analysis of total cell lysate by western blot to confirm and G2/M populations and is indicated by hatched lines. (c,e) Analysis of total cell lysate by western blot to confirm expression of MNV VPg, MNV VPg TM, and MNV VPg K2-12, with actin as a loading control. (b,d) The histograms were expression of MNV VPg, MNV VPg TM, and MNV VPg K2-12, with actin as a loading control. (b,d) The histograms were analysed using MODfit LT 3.0 and the percentage of cells in each phase of the cell cycle are shown. Red indicates percent- ageanalysed of cells using in G0/G1 MODfit phase, LT 3.0 green and theindica percentagetes the S ofphase, cells inand each blue phase indicates of the the cell G2 cycle/M phase. are shown. The results Red indicates present percentage the mean andof cells SD infrom G0/G1 three phase, independent green indicates experime thents. S phase,Statistical and significance blue indicates was the dete G2/Mrmined phase. using The a one-way results present ANOVA the with mean Dun- and nett’sSD from post-test. three independent ; not significant, experiments. * p ≤ 0.05, Statistical ** p ≤ 0.01 significance and *** p was≤ 0.001. determined using a one-way ANOVA with Dunnett’s post-test. ns; not significant, * p ≤ 0.05, ** p ≤ 0.01 and *** p ≤ 0.001. 4. Discussion 4.1.4. Discussion MNV VPg Is an RNA Binding Protein 4.1. MNVWe used VPg EMSA Is an RNA gels Bindingto show Proteinthat MNV VPg binds to RNA as predicted by bioinfor- maticWe analysis used EMSAand consistent gels to show with observed that MNV ph VPgosphocellulose binds to RNA binding as predicted of this protein by bioinfor- [20]. maticThe analysis VPg proteins and consistent used in with this study observed were phosphocellulose expressed as a fusion binding with of the this NT* protein protein. [20]. Here Thewe show VPg proteins that the usedaddition in this of the study NT* were fusion expressed proteinas to anorovirus fusion with VPg the enabled NT* protein. good expressionHere we show in T.ni that cells. the addition MNV VPg of the was NT* capable fusion of protein binding to norovirusto RNA both VPg with enabled the goodNT* fusionexpression or without in T.ni cells.(Figure MNV 3 and VPg Figure was capable S1), indicating of binding that to the RNA presence both with of the NT*fusion fusion did notor without affect this (Figure specific3 and function. Figure S1), indicating that the presence of the fusion did not affect this specificThe binding function. of NT*MNV VPg was specific for RNA even with an excess of DNA in the experiment,The binding suggesting of NT*MNV that VPg RNA was is specific the favoured for RNA binding even with partner. an excess RNA of binding DNA in theoc- curredexperiment, in a non-specific suggesting manner, that RNA with is the binding favoured of MNV binding VPg partner. to all 12 RNA pentaprobe binding sequences occurred tested,in a non-specific representing manner, all possible with binding five nucleo of MNVtide combinations, VPg to all 12 pentaprobe the most common sequences length tested, of RNArepresenting sequences all targeted possible fiveby RNA nucleotide binding combinations, proteins. This the result most is commonconsistent length with the of RNA VPg proteinssequences from targeted the Potyviridae by RNA binding and Picornaviridae proteins. This viral result families is consistent that also with bind the RNA VPg in proteins a non- from the Potyviridae and Picornaviridae viral families that also bind RNA in a non-specific specific manner [22,23,46]. We have also shown that this RNA binding is not unique to manner [22,23,46]. We have also shown that this RNA binding is not unique to MNV with

Viruses 2021, 13, 1282 12 of 16

the VPg protein of the GII.4 Sydney 2012 norovirus also showing a non-specific interaction with RNA. A wide range of proteins show non-specific RNA binding, with an estimated half of RNA binding proteins falling into this category [47]. Examples of non-specific RNA binding protein include translation initiation factors such as eIF4E [48] and proteins involved in RNA splicing, including the SR protein family [26,49,50]. Non-specific RNA binding proteins typically interact with sites that appear to be devoid of specific sequence or structural motifs. However, the non-specific interaction of VPg with RNA pentaprobes does not preclude a specific target(s) for VPg. There are multiple ways in which specific RNA binding can be achieved, including the specific recognition of RNA secondary structure. For example, the FUS protein binds specifically to AU-rich stem-loops but without a specific sequence motif [51]. The ends of the norovirus genome contain several evolutionarily conserved RNA structures [52,53]. We were unable to identify any enhanced binding to RNA probes corresponding to the 50 and 30 ends of the viral genome using the assays employed in this study. RNA binding proteins can also have specificity conferred by interaction with other proteins. The VPg protein from shows specific RNA binding activity in conjunc- tion with the viral polymerase for RNA genome structures [54–56]. The MNV polymerase protein has RNA binding activity [57] and there is interaction between the VPg protein and the RNA polymerase for nucleotidylylation of the VPg and for initiation of RNA synthesis [10,11,58], and it is possible that this interaction confers specificity upon the VPg interaction.

4.2. Identification of a Motif/Region of MNV VPg Required for Binding to RNA The N-terminus of MNV VPg was predicted to interact with RNA but does not contain any sequences traditionally associated with RNA binding motifs. Instead, there is a correlation between the potential for RNA binding and propensity for disorder by the N-terminal amino acids (Figure2). Many RNA binding proteins contain regions of intrinsic disorder [59]. One of these non-canonical RNA binding regions has been termed the K/R basic patch. Basic patches are normally composed of 4–8 lysine amino acids and occasionally arginine amino acids, which form a highly positive and exposed surface for binding to RNA [26,60]. The conserved N-terminal region of MNV VPg contains five lysine and two arginine residues within the first 12 amino acids, consistent with a disordered basic patch (Figure7). Given the resemblance to a K/R basic patch, it seemed likely that the N-terminal region of MNV VPg, which is enriched in lysine and arginine amino acids would be required for RNA binding activity. In support of this prediction, PVA VPg has been shown to bind RNA in a sequence independent manner, which is dependent on a stretch of amino acids with a high local positive charge [23,46]. Alanine substitutions to MNV VPg at positions K5, K7, and R10 did not inhibit RNA binding while mutation of all lysine and arginine residues within the first 12 amino acids inhibited binding consistent with non-canonical RNA binding. These results indicate that K5, K7, and R10 residues are not, by themselves, essential for RNA binding, and the interaction appears to involve a larger region of the N-terminus of VPg. Future work should look to identify the minimum requirements for charged residues to enable RNA binding by MNV VPg. In Jembrana disease virus, RNA binding is mediated by arginine residues, which form electrostatic interactions with RNA that is stabilized by the surrounding amino acids [61]. Similarly, with PVA VPg, it has been proposed that the protein has a loosely folded and positively charged contact surface at the edge of the protein [23]. Mutation of lysine residues in this area is predicted to drop the surface charge of PVA VPg, resulting in decreased attraction between the VPg and either RNA or individual NTPs [23]. The conserved N- terminal motif of MNV VPg shares similarities with the RNA binding motif of PVA VPg, particularly the lysine residues (Figure2). Given the decreased RNA binding when charged amino acids are replaced with alanine, it seems likely that the N- terminal residues of MNV VPg allow binding to RNA in a similar manner via electrostatic interactions. Viruses 2021, 13, 1282 13 of 16

4.3. Biological Significance of RNA Binding by MNV VPg RNA binding appears to be a conserved function of norovirus, potyvirus, and pi- cornavirus VPg proteins. The biological significance of RNA binding by norovirus and potyvirus VPg proteins is not clear. The charged residues in the N-terminal region of MNV VPg have been linked with nucleotidylylation, NTP binding, and induction of a cell cycle arrest. We have investigated a possible link with induction of a G0/G1 phase cell cycle arrest. Mutation of all K and R residues in the first 12 amino acids of MNV VPg results in no cell cycle arrest and no RNA binding. The MNV VPg TM with only three charged amino acids replaced, induced a partial cell cycle arrest and still bound to RNA. Future experiments should address the affinity for RNA binding by mutant and wild type VPg proteins to further elucidate a potential relationship between these functions. RNA binding proteins (RBPs) play a crucial role in the regulation of cell cycle progres- sion [62]. These proteins participate in a variety of post-transcriptional RNA processing steps, including translation, RNA stability, localisation, and splicing for both mRNA and non-coding . This study has shown that the N-terminal K/R basic patch of MNV VPg is important for induction of a cell cycle arrest and binding to RNA; perturbation of the patch affects both of these biological activities. However, a causal relationship between RNA binding and the other functions associated with the K/R patch is yet to be established. The sequence conservation in noroviruses and the ability of VPg proteins from a range of RNA viruses to bind RNA non-specifically points towards an important role for VPg RNA binding in the replication of these RNA viruses.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/v13071282/s1, Figure S1, MNV VPg binds RNA. Author Contributions: Conceptualization, A.M.M., V.L.Y., and V.K..; methodology, A.M.M. and V.L.Y.; writing—original draft preparation, A.M.M. and V.L.Y.; writing—review and editing, A.M.M., V.L.Y., and V.K.W.; visualization, A.M.M.; supervision, V.K.W.; All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the New Zealand Ministry for Business Innovation and Employment, grant number UOOX1904. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available in the manuscript and Supplementary Figure S1. Acknowledgments: The authors thank Joel Mackay for the gift of the plasmids containing 12 pen- taprobes (Addgene plasmid # 83326–83337). Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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