Article Temporal Regulation of Distinct Internal Ribosome Entry Sites of the Paralysis

Anthony Khong 1, Jennifer M. Bonderoff 1, Ruth V. Spriggs 2, Erik Tammpere 1, Craig H. Kerr 1, Thomas J. Jackson 2, Anne E. Willis 2 and Eric Jan 1,*

Received: 3 December 2015; Accepted: 11 January 2016; Published: 19 January 2016 Academic Editor: Craig McCormick

1 Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC V6T 1Z3, Canada; [email protected] (A.K.); [email protected] (J.M.B.); [email protected] (E.T.); [email protected] (C.H.K.) 2 Medical Research Council Toxicology Unit, Leicester LE1 9HN, UK; [email protected] (R.V.S.); [email protected] (T.J.J.); [email protected] (A.E.W.) * Correspondence: [email protected]; Tel.: +1-604-827-4226

Abstract: Internal ribosome entry is a key mechanism for viral protein synthesis in a subset of RNA viruses. (CrPV), a member of Dicistroviridae, has a positive-sense single strand RNA genome that contains two internal ribosome entry sites (IRES), a 51untranslated region (51UTR) and intergenic region (IGR) IRES, that direct translation of open reading frames (ORF) encoding the viral non-structural and structural proteins, respectively. The regulation of and the significance of the CrPV IRESs during infection are not fully understood. In this study, using a series of biochemical assays including radioactive-pulse labelling, reporter RNA assays and ribosome profiling, we demonstrate that while 51UTR IRES translational activity is constant throughout infection, IGR IRES translation is delayed and then stimulated two to three hours post infection. The delay in IGR IRES translation is not affected by inhibiting global translation prematurely via treatment with Pateamine A. Using a CrPV replicon that uncouples viral translation and replication, we show that the increase in IGR IRES translation is dependent on expression of non-structural proteins and is greatly stimulated when replication is active. Temporal regulation by distinct IRESs within the CrPV genome is an effective viral strategy to ensure optimal timing and expression of viral proteins to facilitate infection.

Keywords: virus; translation; IRES; ribosome; dicistrovirus

1. Introduction Viral protein synthesis is an essential process in all viral life cycles. As such, viruses have adapted diverse strategies to recruit the host ribosome and the translational machinery. Numerous positive strand RNA viruses use a strategy whereby infection leads to an inhibition of host translation concomitant with an increase in viral protein synthesis [1]. One of the best-studied examples of this switch from host to viral protein synthesis is during poliovirus infection: the translation factors eIF4G and poly A binding protein are cleaved by virally-encoded proteases thereby shutting off cap-dependent translation [2–4]. By contrast, the poliovirus internal ribosome entry site, IRES, recruits the ribosome using a subset of translation factors and IRES trans-acting factors (ITAFs) to direct cap-independent viral protein synthesis [5]. Therefore, the strategies that underlie the switch from host to viral protein synthesis such as the signalling pathways that target translation factors and the mechanisms that direct viral protein expression are fundamental to virus infection.

Viruses 2016, 8, 25; doi:10.3390/v8010025 www.mdpi.com/journal/viruses Viruses 2016, 8, 25 2 of 20

Viruses 2016, 8, x 2 of 19 The Dicistroviridae family uses an interesting strategy for viral protein synthesis: the monopartite plus strandThe ~9Dicistroviridae kb RNA genome family containsuses an twointeresting IRESs, strate the 5gy1untranslated for viral protein region synthesis: (51UTR) andthe the intergenicmonopartite (IGR) plus IRES strand that ~9 direct kb RNA translation genome contains of two two open IRESs, reading the 5'untranslated frames (ORFs) region encoding (5'UTR) the non-structuraland the intergenic and structural (IGR) IRES proteins, that direct respectively translation (Figure of two 1openA) [ reading6]. Infection frames of (ORFs) Drosophila encoding S2 cells by thethe dicistrovirus non-structuralCricket and structural paralysis virusproteins,(CrPV) respecti resultsvely in(Figure the rapid 1A) [6]. shut Infection off of host of Drosophila protein synthesis S2 cells by the dicistrovirus Cricket paralysis virus (CrPV) results in the rapid shut off of host protein concomitant with preferential viral protein synthesis involving supramolar expression of the structural synthesis concomitant with preferential viral protein synthesis involving supramolar expression of proteins compared to the non-structural proteins [7–9]. The significance and the regulation of the CrPV the structural proteins compared to the non-structural proteins [7–9]. The significance and the IRESregulation translation of duringthe CrPV infection IRES translation have not during been infection examined have in detail.not been examined in detail.

Figure 1. Viral non-structural and structural protein expression in CrPV-infected S2 cells. (A) Figure 1. Viral non-structural and structural protein expression in CrPV-infected S2 cells. (A) Schematic Schematic of the genome arrangement of CrPV; (B) Autoradiography of pulse-labelled protein of thelysates genome from arrangement S2 cells either of mock- CrPV; or (CrPV-infecteB) Autoradiographyd (MOI 10) ofresolved pulse-labelled on a 12% proteinSDS-PAGE lysates gel. The from S2 cells eitherprotein mock- lysates or were CrPV-infected collected from (MOI S2 10)cells resolved at the indicated on a 12% times SDS-PAGE hours post-infection gel. The protein (h.p.i.) lysates and were collectedmetabolically from S2 labelled cells at with the indicated[35S]-Met/Cys times for hourstwenty post-infectionminutes at the end (h.p.i.) of each and time metabolically point (top). The labelled withidentities [35S]-Met/Cys of specific for CrPV twenty viral minutes proteins, at as the shown end ofon eachthe right time of pointthe gel, (top). were Thepreviously identities described of specific CrPV[7,8] viral and proteins, determined as shown by immunoblotting on the right of with the α gel,-RdRp were and previously α-VP2 peptide described antibodies [7,8] or and predicted determined by by immunoblottingmolecular weight with α(below);-RdRp and(C) αRaw-VP2 densitometric peptide antibodies quantitation or predicted of [35S]-Met/Cys by molecular pulse-labelled weight (below); (C) RawRdRp*, densitometric RdRp**, RdRp***, quantitation 2C, VP0, of and [35 S]-Met/CysVP1, 2, and 3 pulse-labelledduring CrPV infection RdRp*, from RdRp**, three RdRp***, independent 2C, VP0, and VP1,experiments 2, and 3(±s.d.). during RdRp*, CrPV infectionRdRp**, and from RdRp*** three independent denote polyproteins experiments containing (˘s.d.). RdRp RdRp*, at the RdRp**, approximate sizes of 120 kDa, 105 kDa, and 100 kDa respectively. and RdRp*** denote polyproteins containing RdRp at the approximate sizes of 120 kDa, 105 kDa, and 100 kDa respectively. The 5'UTR- and IGR IRES can support translation in a number of translation systems both in vitro and in vivo, including in yeast, insect and mammalian extracts and cells [10–13]. The IGR IRES Thehas been 51UTR- studied and extensively IGR IRES can [14–19]; support the IGR translation IRES directly in a number recruits the of translation ribosome without systems the both aid ofin vitro and intranslation vivo, including initiation in factors yeast, and insect initiates and mammaliantranslation from extracts a non-AUG and cells codon [10 [14].–13 ].Biochemical The IGR IRESand has beenstructural studied extensivelystudies have revealed [14–19]; that the the IGR IGR IRES IRES directly adopts distinct recruits structural the ribosome domains without that enable the the aid of translationIRES to initiationoccupy the factors intersubunit and initiatescore of the translation ribosome in from order a to non-AUG gain access codon to the [ribosomal14]. Biochemical A and P and sites [15,16,20–23]. Indeed, the CrPV pseudoknot I domain of the IGR IRES structurally mimics the structural studies have revealed that the IGR IRES adopts distinct structural domains that enable the anticodon of a tRNA[15,22,23]. Recent cryo-EM studies have demonstrated that the PKI domain first IRES to occupy the intersubunit core of the ribosome in order to gain access to the ribosomal A and P sites [15,16,20–23]. Indeed, the CrPV pseudoknot I domain of the IGR IRES structurally mimics the anticodon of a tRNA [15,22,23]. Recent cryo-EM studies have demonstrated that the PKI domain first Viruses 2016, 8, 25 3 of 20 occupies the A site followed by translocation to the P site and thereby leaving the A site unoccupied for delivery of the initial aminoacyl-tRNA [22,23]. By contrast, studies on the mechanism of the 51UTR IRES have been limited [13,24]; using a reconstitution approach, Terenin et al. (2005) showed that eIF1, eIF2, and eIF3 are required for 51UTR IRES dependent translation of a related dicistrovirus Rhapdopsilum padi virus (RhPV) [24]. Differences in the mechanism of the CrPV 51UTR and IGR IRESs likely contribute to distinct expression levels of non-structural and structural proteins. Supporting this, 51UTR IRES translation is weaker than IGR IRES translation [9] and that IGR IRES translation is stimulated during CrPV infection using transfection approaches of reporter RNAs [25]. However, the significance of this strategy by dicistroviruses is not well understood. Many RNA viruses control the timing of viral protein expression in order to facilitate viral replication early in infection and viral packaging later in infection [26]. For example, in infections, non-structural proteins are expressed prior to structural protein synthesis in order to coordinate replication and viral assembly temporally [26]. In this study, using a series of approaches including radioactive pulse-labelling, reporter construct assays and ribosome profiling, we demonstrate that the 51UTR and IGR IRES activities are temporally regulated during CrPV infection. Specifically, IGR IRES translation is delayed until later times in infection when compared to 51UTR IRES translation. The delay is not due to a general decrease in host translation, an increase in eIF2α phosphorylation during infection or a decrease in host transcription. Using a novel CrPV replicon that uncouples the effects of replication and translation, our data suggest that non-structural viral proteins may be partly responsible for the increase in IGR IRES dependent translation during infection. In summary, CrPV uses an IRES-dependent temporal regulation strategy in order to ensure optimal expression of structural and non-structural proteins and to facilitate the timing of events during the viral life cycle.

2. Materials and Methods

2.1. Cell Culture, Virus Infection, and Viral Titers Drosophila S2 cells were maintained in Shields and Sang M3 insect media (Sigma-Aldrich, Oakville, ON, Canada) supplemented with 10% fetal bovine serum (SSM3) at 25 ˝C as described previously [7]. For virus infections, S2 cells were washed with PBS, then incubated with CrPV for the indicated MOI for 30 min and subsequently, washed with PBS and resuspended with SSM3. Viral titers were determined by a fluorescence-focusing assay as described [7].

2.2. Reporter Construct and CrPV2 Replicon The construction of the CrPV minigenome reporter was described in Kerr et al. (2015) [27]. For the CrPV2(Fluc), CrPV2(Fluc)-ORF1stop, and CrPV2(Fluc)-mutRdRp replicons, standard recombinant DNA technology was used to construct these plasmids. Unique restriction sites that facilitated insertion of the firefly luciferase gene into the CrPV-2 clone [27] were introduced by site-directed mutagenesis. Specifically, an XhoI site was introduced by substituting two nucleotides within ORF2 that encodes VP2 and a SacII site was introduced by adding the restriction sequence, 51-CCGCGG-31, after ORF2 in CrPV2. The primer pairs used for introducing XhoI are 51-ATGAAGATAAA AGACTCGAGTCAGAACAGAAAGAAATTGTACATT-31 and 51-CTCGAGTCTTTTATCTTCATA AACAAGAAAATTCACACATTGAAA-31 and the primer pairs used for introducing SacI are 51-TAGCACGCGCCTAACCGCGGCTAACTATTTGCTTTGTATTTTAAG-31 and 51-CCGCGGTT AGGCGCGTGCTACATTTACTAAAGGTGGAACTCCAAG-31. Mutations were sequenced to verify. To generate CrPV2(Fluc), firefly luciferase gene was amplified from the CrPV minigenome reporter with primers containing XhoI and SacII flanking the luciferase gene (51-AAACTCGAGATGGAAGA CGCCAAAAACATA-31 and 51-AAACCGCGGTTACACGGCGATCTTTCCGCC-31). The CrPV-2 plasmid containing XhoI and SacII and the amplified firefly luciferase gene were digested with XhoI and SacII restriction enzymes. The backbone of the CrPV-2 plasmid was then gel purified (Qiagen, Hilden, Viruses 2016, 8, 25 4 of 20

Germany). Subsequently, the PCR-purified (Qiagen) digested firefly luciferase gene was combined in a ligation reaction with the CrPV-2 backbone template as described in the manufacturer’s protocol (Thermo Fisher Scientific, Waltham, MA, USA). After transforming the ligation reaction, DH5α bacteria colonies were picked and insertion of the firefly luciferase gene into the CrPV-2 clone was sequenced verified. To generate CrPV2(Fluc)-ORF1stop and CrPV2(Fluc)-mutRdRp replicons, site directed mutagenesis was performed. For CrPV2(Fluc)-ORF1stop, the primers used for site-directed mutagenesis were 51-ATTCTTACAATGTGATCATGTCTTTTTAATAAACAAACAACAACGCAACCAACAAC-31 and 51-GTTGTTGGTTGCGTTGTTGTTTGTTTATTAAAAAGACATGATCACATTGTAAGAAT-31. For CrPV2(Fluc)-mutRdRp, the primers used for site directed mutagenesis were 51-GATGATAAGCTACG GAGCTGCCAATTGCCTAAATATTT-31 and 51-AAATATTTAGGCAATTGGCAGCTCCGTAGCTTATC ATC-31. Clones were fully sequenced for verification.

2.3. RNA Transfection and Luciferase Assay In vitro transcription reactions using T7 RNA polymerase was performed as described previously [25]. RNA was polyadenylated (CellScript, Madison, WI, USA), purified (RNeasy kit, Qiagen) and the integrity of the RNA was determined by visualizing it on an agarose gel. Transfection of 1 µg RNA (lipofectamine 2000, Invitrogen) into S2 cells (1.5 ˆ 106 cells per mL; 12-well plates) was performed as described by the manufacturer (Invitrogen). Cells were harvested with passive lysis buffer (Promega, Madison, WI, USA) and assayed for luciferase activity (Promega) using a microplate luminometer (Berthold Technologies, Centro LB 960, Bad Wildbad, Germany).

2.4. Western Blotting As described previously [7], cells were washed once with 1ˆ PBS and harvested in lysis buffer (20 mM HEPES, 150 mM NaCl, 1% Triton X-100, 10% glycerol, 1 mM EDTA, 10 mM tetrapyrophosphate, 100 mM NaF, 17.5 mM β-glycerophosphate, and protease inhibitors (Roche, Basel, Switzerland)). Equal amounts of lysates, as determined by Bradford assay, were resolved on a 10%–15% acrylamide SDS-PAGE gel. For western blotting, the following antibodies were used: 1:10,000 CrPV VP2 antibody (PL Laboratories, Port Moody, BC, Canada), 1:10,000 CrPV RdRP antibody (PL Laboratories), or 1:500 phospho-eIF2α antibody (Cell Signaling, Whitby, ON, Canada).

2.5. [35S]-Met/Cys Pulse-Labelling As described previously [7], 5 ˆ 106 S2 cells were pulse labelled with 250 µCi [35S]-Met/Cys (Perkin-Elmer, Waltham, MA, USA) for twenty minutes, washed with 0.5 mL cold PBS twice and harvested with 50 µL lysis buffer. Equal amounts of lysates were resolved with SDS-PAGE. The gel was dried and radioactive bands were analyzed by a phosphorimager (Amersham Pharmacia Biotech, Amersham, UK). Densitometric analysis on the pulse-labelled bands was performed using ImageJ software [28].

2.6. Immunoprecipitation S2 lysates were incubated with a fixed amount of CrPV RdRp or VP2 rabbit polyclonal primary antibodies respectively in immunoprecipitation lysis buffer (50 mM Tris-HCL, pH 7.5, 150 mM NaCl, 1% Nonidet P40, 0.5% sodium deoxycholate, and protease inhibitors (Roche)) for one hour at 4 ˝C with gentle agitation. Fifty microliters of protein G-agarose (Roche) were incubated with the reaction overnight at 4 ˝C. Mixtures were centrifuged at 2000 rpm for one minute and the precipitate was washed twice with the immunoprecipitation lysis buffer, twice with wash buffer 1 (50 mM Tris-HCL, pH 7.5, 500 mM NaCl, 0.1% Nonidet P40, and 0.05% sodium deoxycholate), and once with wash buffer 2 (50 mM Tris-HCL, 0.1% Nonidet P40, and 0.05% sodium deoxycholate). The precipitate was resuspended in 50 µL SDS loading dye and removed from the beads by heating at 95 ˝C for ten minutes and centrifugation at 13,200 rpm. The samples were resolved by SDS-PAGE. The gel was Viruses 2016, 8, 25 5 of 20 dried and imaged with a phosphoimager (Amersham Pharmacia Biotech). Densitometric analysis on the pulse-labelled bands was performed using ImageJ software [28].

2.7. dsRNA-Mediated Knockdown The protocol for dsRNA-mediated knockdown in Drosophila S2 cells was adapted from Cherry et al. (2005) [29]. Snapdragon [30], a web-based tool, was used to identify appropriate primers for amplifying fragments of genes that are intended for knockdown while minimizing off target effects. Specifically, the PCR products of dPERK (437–853 nucleotides, NM_141281) and dGCN2 (492–1034 nucleotides, NM_057882) was RT-PCR amplified from total RNA of Drosophila S2 cells using primers that contain the T7 promoter at both ends. dPERK was PCR amplified using: 51-ACTGACTAATACGACTCACTATAGGGCTGGGCACCCAACTACTGAT-31 and 51-ACTGACTAATACGACTCACTATAGGGAACGGAAACACCGTATGAGC-31. dGCN2 was PCR amplified using: 51-ACTGACTAATACGACTCACTATAGGGGAGACTCCTCGAACGGACTG-31 and 51-ACTGACTAATACGACTCACTATAGGGGGAAGTAGAGCGTCTCCGTG-31. The PCR products were used as templates in in vitro transcription reactions using T7 RNA polymerase. dsRNA was purified and the integrity was verified by gel analysis. dsRNA (13 µg/million cells) was incubated with S2 cells in serum-free SSM3 media for one hour, followed by the addition of complete SSM3 media.

2.8. S2 Translation Extracts and in Vitro Translation Assay The protocol for S2 translation extracts was adapted from Brasey et al. (2003) and Roy et al. (2004) [31,32]. Specifically, 2.0 ˆ 109 mock-infected or 6 h.p.i. CrPV-infected (MOI 10) S2 cells were pelleted at 1000 g for eight minutes, washed once with PBS and resuspended in 2 mL hypotonic buffer (10 mM HEPES-KOH (pH 7.4), 10 mM KOAc, 0.5 mM MgOAc, 1 mM DTT). After incubation in hypotonic buffer on ice for five minutes, the cells were lysed by extrusion 25 times through a 23-gauge needle. The extracts were then adjusted to 50 mM KOAc. The supernatant (S2 translation extract) was collected upon clearing all cell debris by centrifugation at 16,000 g for five minutes at 4 ˝C, aliquoted, and stored at ´80 ˝C. Prior to use, the S2 translation extract was supplemented with 0.2 U creatine phosphokinase/µL. Each translation assay was carried out in a final volume of 10 µL containing 6.5 µL S2 translation extract, 2 µL 5ˆ master mix (10 µM complete amino acids, 40 mM creatine phosphate, 100 mM HEPES-KOH (pH 7.6), 5 mM ATP, 1 mM GTP, 2.5 mM spermidine, 500 mM KOAc, and 5 mM MgOAc), 0.5 µL Ribolock RNase Inhibitor (40 U/µL, Thermo Fisher Scientific), and 9 nM template RNA for thirty minutes at 30 ˝C. After incubation, half of the reaction was assayed for luciferase activity (Promega).

2.9. Ribosome Profiling The ribosome profiling experiment was adapted from Ingolia et al. (2009) [33]. Specifically, Drosophila S2 cells were infected with CrPV (MOI 10) or mock-infected. At the indicated time points, samples were subject to total RNA extraction using Trizol (Thermo Fisher Scientific) for RNAseq analysis or were subject to RNA isolation from ribosomes. For the latter, cells were first treated with cycloheximide (100 µg/mL), pelleted, washed with PBS containing cycloheximide (100 µg/mL), pelleted again, and lysed with 1ˆ RPF buffer (300 mM NaCl, 15 mM MgCl2, 15 mM Tris-HCl pH 7.5, 2 mM DTT, and 100 µg/mL cycloheximide) containing 1% Triton ˆ100 and 500 U/mL RNaseIn on ice for ten minutes. Afterwards, the lysates were spun at 10,000 rpm for ten minutes at 4 ˝C. The supernatant was collected and is now referred to as S10 lysates. S10 lysates were treated with 2 U/µL RNase I (Thermo Fisher Scientific) for thirty minutes at room temperature. The S10 lysate was loaded onto a 10%–50% sucrose gradient containing 15 mM Tris-HCl pH 7.5, 300 mM NaCl, 15 mM MgCl2, 2 mM dithiothreitol, 100 µg/mL cycloheximide, and 0.1 U/µL SUPERase*in (Ambion) and ultracentrifuged for three hours at 35,000 rpm. Gradients were fractionated using a Brandel syringe pump and ISCO UV detector; the top two monosome peak fractions were extracted twice with acid phenol and ethanol precipitated. The RNA collected from these monosome peak fractions Viruses 2016, 8, 25 6 of 20 are now referred to as ribosome protected fragments (RPFs). The RPFs were depleted of 2S rRNA by subtractive hybridization [34]. Specifically, RPFs were incubated with 100 pmol of biotinylated DNA oligo (TACAACCCTCAACCATATGTAGTCCAAGCA, Integrated DNA Technologies, Coralville, IA, USA) at 65 ˝C for two minutes and then at 37 ˝C for fifteen minutes in 2ˆ SSC (300 mM NaCl, 30 mM sodium citrate). An excess of streptavidin-coated beads was added and incubation continued on a rotator for fifteen minutes at 25 ˝C. The supernatant was recovered and precipitated by ethanol. Finally, the precipitated RPF RNA was gel-excised using 27 and 33 nucleotides RNA oligonucleotide markers. Total RNA was prepared for RNAseq using the SOLiD Total RNAseq kit (Thermo Fisher Scientific). The gel-excised RPF RNA was prepared for RNAseq using the Small RNA Library Procotol from the SOLiD Total RNAseq Kit (Applied Biosystems). Libraries were sequenced on the SOLiD 4 platform.

2.10. Calculations of Translational Efficiency The raw csfasta and qual files produced by each run of SOLiD 4 were converted to xsq files using the XSQ Tools package from life technologies™ (Thermo Fisher Scientific). Reads within these xsq files were mapped to the CrPV genome using “genomic resequencing” mapping within LifeScope™ from life technologies™. Reference files for mapping were created using the sequence and annotations provided in NC_003924.1 (Wilson et al. (2009)) [35]. A mapping quality threshold of 8 was used. Each mapped read was labelled depending on its position with relation to the two ORFs of the CrPV genome. The raw numbers of reads were converted to rpM (reads per million) to normalize for the different library sizes between SOLiD runs. Translational efficiency was calculated using rpM values for the ribosome protected fragments (RPF) and for the Whole Transcriptome set (WT), and is RPF divided by WT. To find any translational efficiency changes across the two ORFs, the CrPV genome was split into five sections, namely 51UTR, ORF1, IGR, ORF2, and 31UTR. A t-test statistical analysis was performed to determine if there were significant changes in translational efficiency values.

2.11. Uridine Labelling of RNA S2 cells (5 ˆ 106) were pretreated with actinomycin D for fifteen minutes, followed by the addition of 5 µCi of [3H]-uridine (Perkin Elmer) for fifteen minutes. Total RNA was extracted (Trizol, Thermo Fisher Scientific). Equal amounts of RNA were separated in a denaturing agarose gel and transferred to nylon membrane. Levels of radioactivity were detected by phosphorimaging and quantified using ImageQuant software.

3. Results

3.1. Translation of CrPV ORF1 and ORF2 during CrPV Infection Although it is well established that viral structural proteins are expressed in molar excess over the viral non-structural proteins during CrPV infection [7–9], we carefully re-examined viral protein synthesis in CrPV-infected S2 cells by metabolic [35S]-Met/Cys pulse-labelling at different times after infection. As observed previously [7–9], host translation decreases dramatically two to three hours post infection (h.p.i) concomitant with the synthesis of CrPV proteins (Figure1B). The processed and unprocessed viral structural and non-structural proteins were annotated based on previous studies [7–9] and from immunoblots using antibodies that recognize RNA-dependent RNA polymerase (RdRp) and VP2 structural protein (Figure1B) [ 7]. Using an RdRp antibody for Western blot analysis, we detected unprocessed RdRp non-structural proteins at 120 (RdRp*), 105 (RdRp**), and 100 kDA (RdRp***) and the mature 3D at 60 kDa. A close inspection of the pulse-labelled proteins showed that the non-structural proteins such as the unprocessed 120 kDa RdRp and 2C are detected first at two h.p.i. whereas the structural proteins are not detected until after three h.p.i. (Figure1B), which is similar to that observed previously [ 7,9]. Quantitation of the viral proteins confirmed that the non-structural proteins are first expressed by two h.p.i. and increase steadily at later times of infection and that the viral structural proteins are not detected until three to four Viruses 2016, 8, 25 7 of 20 h.p.i. (Figure1C). We note that quantitation of the pulse-labelled non-structural proteins at two h.p.i. may be masked due to ongoing host protein synthesis at this time of infection (Figure1B). To detect theViruses non-structural 2016, 8, x proteins more accurately, we monitored synthesis by immunoprecipitation7 of 19 of pulse-labelleddue to ongoing structural host andprotein non-structural synthesis at this proteins time fromof infection CrPV-infected (Figure 1B). cell To lysates detect (Figurethe S1). Specifically,non-structural we pulse-labelled proteins more cells accurately, for one hourwe monitored followed bysynthe lysissis andby immunoprecipitation immunoprecipitation of using α-RdRPpulse-labelled and α-VP2 antibodies.structural and We non- initiallystructural optimized proteins thefrom immunoprecipitation CrPV-infected cell lysates conditions (Figure S1). to ensure that theSpecifically, antibody is we saturating pulse-labelled by incubating cells for one increasing hour followed amounts by lysis of and [35S]-Met/Cys immunoprecipitation labelled using lysates with a constantα-RdRP amount and ofα-VP2 antibody antibodies. (Figure We initially S1A,B). optimized Immunoprecipitation the immunoprecipitation with the conditionsα-RdRp antibodyto ensure pulled that the antibody is saturating by incubating increasing amounts of [35S]-Met/Cys labelled lysates α down unprocessedwith a constant and amount mature of antibody RdRp from(Figure infected S1A,B). Immunoprecipitation lysates whereas the with-VP2 the α-RdRp immunoprecipitated antibody all fourpulled structural down proteins, unprocessed likely and as a packagedmature RdRp virion from (Figure infected S1A,B). lysates At whereas two h.p.i., the non-structural α-VP2 proteinsimmunoprecipitated were readily detected all four by structuralα-RdRp proteins, pulldowns. likely as In a contrast,packaged virion the structural (Figure S1A,B). proteins At two were not detectedh.p.i., until non-structural three h.p.i. (Figure proteins S1C,D). were readily Furthermore, detected whereasby α-RdRp the pulldowns. pulse-labelled In contrast, RdRp is the relatively constantstructural throughout proteins infection, were not the detected structural until protein three h.p.i. immunoprecipates (Figure S1C,D). Furthermore, increase significantly whereas the at later pulse-labelled RdRp is relatively constant throughout infection, the structural protein times of infection, supporting the notion that translation of the two ORFs are distinctly and temporally immunoprecipates increase significantly at later times of infection, supporting the notion that regulatedtranslation during of infection the two ORFs (Figure are distinctly S1C,D). Inand summary, temporally theregulated pulse-labeling during infection experiments (Figure S1C,D). suggest that translationIn summary, of the downstream the pulse-labeling ORF experiments driven by suggest the IGR that IRES translation is delayed of the until downstream three to ORF four driven h.p.i. by the IGR IRES is delayed until three to four h.p.i. 3.2. Ribosome Profiling of Viral RNA in CrPV-Infected S2 Cells 3.2. Ribosome Profiling of Viral RNA in CrPV-Infected S2 Cells

A IGR IRES ORF1 ORF2 5’ IRES 1A 2B 2C 3A 3C RdRp VP2 VP3 VP1 3’(A)n VPg VP4 h.p.i. 105 104 2 103 102 105 104 4 103 102 105 104 6 103 102 1 1000 2000 3000 4000 5000 6000 7000 8000 9000 Nucleotide position B 0.8

0.7 ORF1 ORF2 0.6

0.5

0.4

0.3

Translational efficiency Translational 0.2

0.1

0 246 Time (h.p.i) Figure 2. Ribosome profiling of the CrPV RNA in infected S2 cells. (A) CrPV RNA sequence coverage Figure 2.(x-axis)Ribosome in rpM from profiling the ribosome-associated of the CrPV RNA fractions in infected(y-axis) at S2two, cells. four and (A )six CrPV h.p.i (MOI RNA 10). sequence coverageThe (x coverage,-axis) in as rpM indicated, from is the aligne ribosome-associatedd to the viral genome fractionsshown above; (y-axis) (B) Translational at two, four efficiency and six h.p.i (MOI 10).was The calculated coverage, by dividing as indicated, reads per is million aligned valu toes the for viral the ribosome genome protected shown fragments above; (B (RPF)) Translational by efficiencythe was Whole calculated Transcriptome by dividing set (WT). reads The TE per was million determined values from for the the average ribosome of two protected independent fragments (RPF) byexperiments. the Whole Transcriptome set (WT). The TE was determined from the average of two independent experiments. Viruses 2016, 8, 25 8 of 20

The distinct temporal expression of non-structural and structural proteins may be explained by one of two broad hypotheses. (1) The increase in structural protein synthesis can be described by some form of passive regulation during infection—a combination of ribosome availability upon host translational repression, different intrinsic affinities for ribosomes and requirements for translation initiation factors between the 51IRES and the IGR IRES and/or an increase in viral RNAs; (2) alternatively, the increase in viral structural protein synthesis may be driven by some active form of regulation during infection; in other words, IGR IRES translation may be activated or inhibited, possibly by protein factors binding to the IGR IRES and/or by changes in the structure of IGR IRES during infection. To address these two broad hypotheses, we examined IRES activities indirectly by ribosome profiling. Ribosome profiling is a next-generation sequencing-based approach that identifies ribosome occupancy across mRNAs at high nucleotide resolution [33]. RNAseq analysis, which is performed in parallel, takes into account for mRNA abundance, thus the translational efficiency of each mRNA can be calculated. We measured ribosome occupancy on the CrPV RNA at 2, 4 and 6 h.p.i (Figure2A). Ribosome protected fragments (RPF) were found predominantly within the two main ORFs, and as expected very little RPFs were found within the IGR and 31UTR. To determine the TE of each ORF, we divided the number of ribosome-protected footprints (RPFs) for each ORF to the average read frequency across the viral RNA genome. At two h.p.i, the translational efficiencies of ORF1 and ORF2 are approximately equal (Figure2B), which suggests that both ORFs are translated similarly. While the translational efficiency of ORF1 remains relatively constant as infection progresses, the translational efficiency of ORF2 increases by approximately 2.5 fold over the course of infection (Figure2B). These results suggest that the increased ribosome occupancy on ORF2 is a reflection of increased translation by the IGR IRES at later times of infection.

3.3. IRES Translation Using CrPV Minigenome Reporter Analysis The ribosome profiling and pulse-labelling experimental results suggest that translation of the CrPV ORFs is regulated. However, it is possible that the increase in ORF2 expression at 3–4 h.p.i. may be reflected in the rapid increase in viral RNA levels due to replication [7]. To uncouple the effects of viral translation from replication, we directly examined 51UTR and IGR IRES translation using a CrPV reporter minigenome construct whereby the viral ORF1 and ORF2 are replaced with Renilla and firefly luciferase gene, respectively (Figure3A) [ 27]. Therefore, Renilla and firefly luciferase activities are used to monitor the 51UTR and IGR IRES-mediated translation, respectively. In vitro transcribed minigenome RNA was transfected into S2 cells, which were then infected with CrPV an hour later. Previously, we showed that this approach resulted in luciferase activity that increases linearly in the first six hours, indicating that the reporter RNA is engaged in translation during this time [25]. Cells were harvested at 1–5 h after infection and assayed for luciferase activity (Figure3B,C). As expected, in mock-infected cells transfected with the minigenome RNA, Renilla luciferase activity increased over time in the first five hours after transfection. In CrPV-infected cells, Renilla luciferase activity increased over time at a similar rate as in mock-infected cells, suggesting that 51UTR IRES translation is relatively constant during infection (Figure3B). In contrast, firefly luciferase activity accumulated gradually early in infection and sharply increased after two h.p.i., suggesting that IGR IRES translation is delayed at early times of infection and is stimulated at later time points (Figure3C). To confirm that IGR IRES translation is being monitored, we used a mutant minigenome reporter (∆PK1) [19] that contains mutations in pseudoknot I that are known to ablate IGR IRES activity. Firefly luciferase activity was severely inhibited in cells transfected with the ∆PK1 minigenome reporter (Figure3C). Viruses 2016, 8, 25 9 of 20 Viruses 2016, 8, x 9 of 19 A

minigenome IGR IRES 5’ IRES Renilla 5’

Firefly AAAn 3’

B 5’IRES C IGR IRES 6 14 ) ) 6 5 5 12 Mock Mock CrPV 10 CrPV 4 Mock (∆PKI) 8 CrPV (∆PKI) 3 6 Relative 2 Relative 4 1 2 Firefly luciferase (X10 Renilla luciferase (X10 0 0 0 1 2345 012345 Time (h.p.i) Time (h.p.i) D E minigenome 160 2.5 140 Mock Mock 2.0 CrPV 120 CrPV

100 1.5 80

60 1.0

40 Normalized luciferase 0.5

Normalized Firefly luciferase 20

0 0 7 7 FL m G-FL m G-FL-Poly(A) 5’IRES IGR IRES

FigureFigure 3. 3.CrPVCrPV 5'UTR 51UTR and and IGR IGR IRES IRES translation. translation. ( A) SchematicSchematic ofof the the CrPV CrPV minigenome; minigenome; (B (,CB,)C S2) S2 cells cellswere were transfected transfected with with the the CrPV CrPV minigenome minigenome RNA RNA one one hour hour prior prior to mockto mock or CrPVor CrPV infection. infection. Cells Cellswere were harvested harvested at the at indicatedthe indicated time time points points and and assayed assayed for ( Bfor) Renilla (B) Renilla or (C or) firefly (C) firefly luciferase luciferase activity. activity.A mutation A mutation within wi thethin IGR the IRES IGR (∆ IRESPKI), ( whichΔPKI), disruptswhich disrupts pseudoknot pseudoknot I of the IRES,I of the was IRES, used was to validateused to thatvalidate IGR IRESthat IGR activity IRES was activity monitored; was monitored; (D) Monocistronic (D) Monocistronic firefly RNA firefly (FL), 5 1RNA-capped (FL), monocistronic 5'-capped monocistronicfirefly RNA (mfirefly7G-FL), RNA and (m 571G-FL),-capped and and 5'-capped polyadenylated and polyaden monocistronicylated monocistronic firefly RNA were firefly incubated RNA werein S2incubated translation in S2 extracts translation for thirty extracts minutes for thirty and assayedminutes forand firefly assaye luciferased for firefly activity. luciferase The activity. results are Thenormalized results are tonormalized FL incubated to FL in incubated mock extracts; in mock (E) Minigenomeextracts; (E) Minigenome RNA was incubated RNA was in incubated S2 translation in S2 extractstranslation from extracts mock orfrom infected mock cells or infected for thirty ce minuteslls for thirty and assayedminutes forand Renilla assayed and for firefly Renilla luciferase and fireflyactivities. luciferase The resultsactivities. are The normalized results are to mocknormalized extracts. to (mockD,E) Averagesextracts. ( areD,E shown) Averages from are at least shown three fromindependent at least three experiments independent (˘s.d.). experiments (±s.d.).

ToTo further further confirm confirm that that these these effects effects are areat the at thetranslational translational level, level, we compared we compared IRES IRES activity activity in S2in translation S2 translation extracts extracts from fromcells cellsthat were that were mock mock infected infected or CrPV-infected or CrPV-infected at 6 h.p.i. at 6 h.p.i. Incubation Incubation of 7 monocistronicof monocistronic firefly firefly luciferase luciferase reporter reporter RNA RNA that that is 5'-capped is 51-capped (m (mG-FLuc)7G-FLuc) or or5'-capped 51-capped and and polyadenylated (m7G-FLuc-Poly(A)) in S2 extracts resulted in a 38 fold and a 133 fold increase in translation, respectively, as compared to an uncapped, nonpolyadenylated reporter RNA (FL)

Viruses 2016, 8, 25 10 of 20 polyadenylated (m7G-FLuc-Poly(A)) in S2 extracts resulted in a 38 fold and a 133 fold increase in translation, respectively, as compared to an uncapped, nonpolyadenylated reporter RNA (FL) (Figure3D), thus showing that the S2 extract supports cap- and poly (A)-dependent translation. In CrPV-infected extracts, a relatively lower levels in luciferase activities were observed with m7G-FL and m7G-FL-poly(A) RNA, respectively (Figure3D), consistent with CrPV infection leading to inhibition of host translation [7–9]. To address if IGR IRES activity differs in extracts from mock infected or infected cells, we incubated the minigenome RNA in these extracts (Figure3A) and assayed for luciferase activities. Similar to the in vivo results, the CrPV-infected extracts displayed higher IGR IRES activity (Figure3E). On the other hand, the 5 1-UTR IRES activity is similar in mock and infected extracts (Figure3E). In summary, these results demonstrate that the CrPV 5 1UTR and IGR IRESs are differentially and temporally regulated during CrPV infection.

3.4. Role of Transcriptional Shutoff in Viral Protein Synthesis under CrPV Infection A number of viruses inhibit transcription and/or translation during infection. We hypothesize that these widespread disruptions in cellular homeostasis may have an effect on IGR IRES translation. To examine this, we first tested if cellular transcription is affected during CrPV infection, which may be responsible for changes in IGR IRES-dependent translation. Several studies have examined the changes in transcriptomes during dicistrovirus infection in Drosophila S2 cells and in [36–39]. In general, dicistroviruses repress the transcription of host genes [39]. We verified that CrPV infection repressed cellular transcription by pulse labelling mock and CrPV-infected S2 cells with [3H]-uridine for 15 min at specific time points after infection (Figure4A). RNA was extracted, resolved on an agarose gel, and transferred to a nylon membrane. Methylene blue staining readily detected the 18S/28S rRNA, which migrated at a size of ~2 kb, confirming that the RNA is intact (Figure4B). In Drosophila, the mature 28S rRNA undergoes cleavage producing two products of approximate size to each other that coincides with the size of the 18S rRNA [40]. Labeling with [3H]-uridine in mock-infected cells produced two predominant radiolabelled bands that migrated at ~8 kb and ~5 kb (Figure4A). Because we only labelled with [ 3H]-uridine for a short period, these labelled RNAs likely represent immature, unprocessed rRNA precursors. As expected, treatment of mock-infected cells with actinomycin D completely inhibited host transcription (Figure4A). CrPV infection at an MOI of 10 resulted in labeling of a ~9 kb RNA, which increased during the course of infection demonstrating viral replication (Figure4A). To distinguish between host and viral transcription, host transcription was inhibited by treating cells with actinomycin D (5 µg/mL) prior to [3H]-uridine-labeling. As predicted, radiolabelled viral RNA was only detected in actinomycin D-treated infected cells (Figure4A,C). Viral RNA synthesis increased during the first 4 h of infection before levelling off and correlated with the increase in viral RNA as measured by Northern blot analysis (Figure4D). In contrast, host transcription was rapidly inhibited within one hour of infection (~80% inhibition) and remained shut off during infection (Figure4A,C). Host transcription was calculated by subtracting the amount of [3H]-uridine labelled RNA in cells treated with actinomycin D from that in cells without actinomycin D. The quantitations of both viral and host transcription is shown in Figure4C. Since CrPV infection inhibits host transcription, we tested if this effect impacts the temporal regulation of CrPV protein synthesis. We pretreated cells in the absence or presence of actinomycin D for three hours prior to infection and compared the rate of viral protein synthesis in S2 cells by [35S]-Met/Cys pulse labelling. If shutoff of transcription is necessary for the enhanced structural protein synthesis at three h.p.i, IGR IRES translation may be prematurely stimulated. No significant changes in viral non-structural and structural protein synthesis were observed between cells pretreated with actinomycin D, suggesting that host transcriptional shutoff does not contribute significantly to the temporal regulation of viral ORF protein synthesis (Figure4F,G). Viruses 2016, 8, 25 11 of 20 Viruses 2016, 8, x 11 of 19

A B C CrPV CrPV CrPV infection (normalizedmock) to Viral incorporation M 1 2 4 6 h.p.i M 1246h.p.i 70 100 - +-+ - + - + - + Act D - +-+ - + - + - + Act D 60 kb kb 80 50 40 6 60 6 3 40 30 3 20 20 1 1 10

]-uridine pulse 0 0 3

% Host incorporation % Host M1246h.p.i [H Methylene Blue Staining Host Virus [H3]-uridine pulse D E 2

0 1 23456 h.p.i 1.5

CrPV RNA 1

GAPDH RNA 0.5 CrPV/GAPDH RNA CrPV/GAPDH 78910 0 0 1 234567 h.p.i FGDMSO Actinomycin D 123456 h.p.i 123456h.p.i kDa - +-+ - + - + - + - + CrPV kDa - +-+ -+-+-+-+ CrPV 170 RdRp* 170 RdRp* 130 RdRp** 130 RdRp** 100 RdRp*** 100 RdRp*** 70 70 RdRp RdRp 55 55 2C 2C 40 VP0 40 VP0 VP1 VP1 35 VP2 35 VP2 S]-Met labeling S]-Met labeling S]-Met

35 VP3 35 VP3 [ [ 25 25

Figure 4. (A) Mock- and CrPV-infected S2S2 cellscells werewere pulse-labelledpulse-labelled withwith [[33H]-uridine for ten minutes at thethe indicatedindicated hourshours postpost infectioninfection (h.p.i.)(h.p.i.) before harvesting.harvesting. Where indicated, the cells were left untreated or treated with actinomycin D (Act(Act D,D, 55 µµg/mL)g/mL) for for fifteen fifteen minutes minutes prior prior to to pulse-labeling. pulse-labeling. RNA was extracted, loaded on a denaturing agaroseagarose gel, transferred to a nylonnylon membrane,membrane, and analyzed byby phosphorimager phosphorimager analysis. analysis. Representative Representative autoradiogram autoradiogram and a corresponding and a corresponding methylene bluemethylene stain areblue shown stain in are (A ,Bshown), respectively; in (A,B), (C )respectively; Quantitation (C of) hostQuantitation and viral of transcription host and ratesviral intranscription CrPV-infected rates cells. in CrPV The-infected rate of host cells. transcription The rate of at host each tr timeanscription point was at each calculated time point using was the formulacalculated [(Total using radioactivitythe formula [(Total in lane radioactivity´ Act D) ´in (Totallane − radioactivityAct D) − (Total in radioactivity lane + Act in D)] lane / (Total+ Act radioactivityD)] / (Total radioactivity in lane ´ Act in D). lane The − rateAct ofD). host The transcription rate of host wastranscription normalized was to normalized that in mock-infected to that in cellsmock-infected given as 100%. cells Thegiven rate as of100%. viral The transcription rate of viral was transcription calculated at was each calculat h.p.i. byed using at each the h.p.i. formula by [(Totalusing the radioactivity formula [(Total in lane +radioactivity Act D) / (Total in radioactivitylane + Act D) in / lane(Total – Act radioactivity D)]. The rate in oflane viral – transcriptionAct D)]. The wasrate normalizedof viral transcription to that in mock-infected was normalized cells to given that as in 0%. mock-infected Shown are representative cells given as gels 0%. and Shown averages are fromrepresentative at least 2 gels independent and averages experiments; from at least (D 2) independent Viral RNA levelsexperiments; were detected (D) Viral by RNA Northern levels were blot analysis;detected (byE) ViralNorthernRNA blot levels analysis; were quantified (E) Viral usingRNA ImageQuantlevels were quantified and plotted using to show ImageQuant levels of viraland RNA/GAPDHplotted to show RNA levels at differentof viral RNA/GAPDH time points post RNA infection; at different (F,G) Autoradiography time points post of infection; pulse-labelled (F,G) proteinAutoradiography lysates resolved of pulse-labelled on a 12% SDS-PAGE protein lysates gel from resolved S2 cells on mock- a 12% or SDS-PAGE CrPV-infected gel (MOIfrom 10)S2 cells and treatedmock- or with CrPV-infected DMSO (F) or (MOI 50 nM 10)Act and D treated (G) three with hours DMSO prior (F to) or infection. 50 nM Act RdRp*, D (G RdRp**,) three hours and RdRp***prior to denoteinfection. polyproteins RdRp*, RdRp**, containing and RdRp*** RdRp at denote the approximate polyproteins sizes containing of 120 kDa, RdRp 105 at kDa, the andapproximate 100 kDa, respectively.sizes of 120 AverageskDa, 105 arekDa, shown and from100 kDa, at least respecti three independentvely. Averages experiments are shown (˘ s.d.).from at least three independent experiments (±s.d.). 3.5. Role of eIF2α Phosphorylation in Viral Protein Synthesis under CrPV Infection 3.5. Role of eIF2α Phosphorylation in Viral Protein Synthesis under CrPV Infection Virus-mediated translational shutoff is a strategy used by many RNA viruses to increase the cellularVirus-mediated pool of ribosomes translational for viral shutoff protein is synthesis a strategy and used to inhibit by many antiviral RNA innate viruses responses to increase during the infectioncellular pool [1,41 of]. CrPVribosomes infection for viral leads protein to a significant synthesi shutoffs and to of inhibit translation antiviral at two innate to three responses h.p.i. which during is theinfection approximate [1,41]. CrPV time wheninfection IGR leads IRES to translation a significant is stimulated shutoff of [ 7translation]. We previously at two showedto three thath.p.i. eIF2 whichα is phosphorylatedis the approximate approximately time whenat IGR this IRES time posttranslation infection, is stimulated however, forced [7]. We dephosphorylation previously showed of eIF2 thatα dideIF2 notα impactis phosphorylated host translational approximately shut off or viral at protein this synthesistime post [7]. Asinfection, several studieshowever, [14,25 ,forced42,43] dephosphorylation of eIF2α did not impact host translational shut off or viral protein synthesis [7]. As several studies [14,25,42,43] have shown that eIF2α phosphorylation stimulates IGR IRES

Viruses 2016, 8, 25 12 of 20 haveViruses shown 2016 that, 8, x eIF2α phosphorylation stimulates IGR IRES translation, we re-examined12 of whether 19 phosphorylation of eIF2α during CrPV infection has an effect on the regulation of 51UTR and IGR IRES translation, we re-examined whether phosphorylation of eIF2α during CrPV infection has an effect translation. To this end, we inhibited eIF2α phosphorylation by treating S2 cells with double stranded on the regulation of 5'UTR and IGR IRES translation. To this end, we inhibited eIF2α RNAs, dsRNAs, directed against the two known eIF2α kinases in Drosophila, dPERK and dGCN2. phosphorylation by treating S2 cells with double stranded RNAs, dsRNAs, directed against the two Sinceknown both dPERKeIF2α kinases and dGCN2 in Drosophila, are required dPERK for and eIF2 dGα CN2.phosphorylation Since both dPERK in arsenite-treated and dGCN2 are S2 cells, dsRNArequired knockdown for eIF2 efficienciesα phosphorylation were assessed in arsenite-treated indirectly S2 by cells, immunoblotting dsRNA knockdown for eIF2 efficienciesα phosphorylation were in arsenite-treatedassessed indirectly dPERK by immunoblotting and dGCN2-depleted for eIF2αS2 phosphorylation cells [44]. Treating in arsenite-treated cells with both dPERK dPERK and and dGCN2dGCN2-depleted dsRNAs but S2 not cells singly [44]. inhibitedTreating cells arsenite-induced with both dPER eIF2K andα phosphorylation,dGCN2 dsRNAs but which not singly is similar to thatinhibited observed arseni previouslyte-induced (Figure eIF2α phosphorylation,5A) [ 44], thus validating which is similar our knockdown to that observed conditions. previously We next determined(Figure 5A) which [44], kinase thus validating is responsible our knockdown for eIF2α cophosphorylationnditions. We next indeterm CrPV-infectedined which kinase S2 cells. is We incubatedresponsible GFP dsRNAsfor eIF2α as phosphorylation a control or dPERK in CrPV-infected or dGCN2 S2 dsRNAs cells. We individually incubated GFP or together dsRNAs to as S2 a cells control or dPERK or dGCN2 dsRNAs individually or together to S2 cells prior to mock- or prior to mock- or CrPV-infection. While CrPV-infected S2 cells treated with control (GFP), GCN2 or CrPV-infection. While CrPV-infected S2 cells treated with control (GFP), GCN2 or PERK dsRNAs PERK dsRNAs resulted in phosphorylation of eIF2α, treating infected cells with both PERK and GCN2 resulted in phosphorylation of eIF2α, treating infected cells with both PERK and GCN2 dsRNAs dsRNAsabolished abolished eIF2α eIF2phosphorylationα phosphorylation (Figure 5A). (Figure Thus,5 A).our Thus,result suggests our result that suggests both GCN2 that and both PERK GCN2 and PERKare activated are activated in CrPV-infected in CrPV-infected S2 cells to induce S2 cells eIF2 toαinduce phosphorylation. eIF2α phosphorylation. To determine whether To determine host whetheror viral host orprotein viral proteinsynthesis synthesis is perturbed, is perturbed, we monitored we monitored protein proteinsynthesis synthesis by pulse-labelling by pulse-labelling in in CrPV-infectedCrPV-infected S2 S2 cells cells treatedtreated with control dsRN dsRNAsAs (Figure (Figure 5B)5B) or orwith with PERK PERK and and GCN2 GCN2 dsRNAs dsRNAs (Figure(Figure5C). Despite5C). Despite inhibiting inhibiting eIF2 eIF2ααphosphorylation, phosphorylation, host host translational translational shutoff shutoff andand viral viral protein protein synthesissynthesis occurred occurred to the to samethe same extent extent and and at roughly at roughly the the same same time time as inas cellsin cells treated treated with with the the control dsRNAscontrol (Figure dsRNAs5B,C). (Figure Thus, in5B,C). agreement Thus, with in previousagreement findings with previous [ 7], eIF2 αfindingsphosphorylation [7], eIF2α is not phosphorylation is not responsible for host translation shutoff or viral protein synthesis and does responsible for host translation shutoff or viral protein synthesis and does not appear to contribute to not appear to contribute to the temporal regulation of IGR IRES-mediated translation of CrPV ORF2 the temporal regulation of IGR IRES-mediated translation of CrPV ORF2 in CrPV-infected S2 cells. in CrPV-infected S2 cells.

A control PERK dsRNA (GFP) PERK GCN2 GCN2 CrPV 6 h.p.i - + --+ --+ --+ - Arsenite - --+ --+ --+ - +

P-eIF2α

Tubulin

B control (GFP) dsRNA PERK and GCN2 dsRNAs 123456h.p.i 123456h.p.i kDa - +-+ - + - + - + - + CrPV kDa - +-+ - + - + - + - + CrPV 130 RdRp* RdRp* 100 RdRp** 130 RdRp** RdRp*** 100 RdRp*** 70 70 RdRp (3D) RdRp (3D) 55 55 2C 2C 40 VP0 40 VP0

35 VP1,VP2, 35 VP1,VP2, VP3 VP3 S]-Met labeling S]-Met labeling 35 35 [ [

Figure 5. Role of dPERK and dGCN2 eIF2α kinases in CrPV-infected S2 cells. (A) Immunoblots Figure 5. Role of dPERK and dGCN2 eIF2α kinases in CrPV-infected S2 cells. (A) Immunoblots (anti-phospho-eIF2α (P-eIF2α) and anti-Tubulin antibodies) of lysates from cells treated with control (anti-phospho-eIF2α (P-eIF2α) and anti-Tubulin antibodies) of lysates from cells treated with control (GFP) dsRNAs or singly dPERK or dGCN2 dsRNAs or both dPERK/dGCN2 dsRNAs for 48 h (GFP) dsRNAs or singly dPERK or dGCN2 dsRNAs or both dPERK/dGCN2 dsRNAs for 48 h followed followed by arsenite (500 µM) treatment or infection with CrPV (MOI 10); (B) [35S]-Met/Cys 35 by arsenitepulse-labelled (500 µ proteinM) treatment lysates from or infection control or with dPERK CrPV and (MOI dGCN2 10); dsRNA-treated (B)[ S]-Met/Cys S2 cells pulse-labelledthat were proteineither lysates mock from or CrPV-infected control or dPERK (MOI and10) dGCN2for the in dsRNA-treateddicted times (h.p.i.) S2 cells were that resolved were eitheron a 12% mock or CrPV-infectedSDS-PAGE(MOI gel. 10)Shown for theare indictedrepresentative times (h.p.i.)autoradiographs were resolved from onat aleast 12% SDS-PAGEthree independent gel. Shown are representativeexperiments. RdRp*, autoradiographs RdRp**, and fromRdRp*** at least denote three polyproteins independent contai experiments.ning RdRp at RdRp*,the approximate RdRp**, and RdRp***sizes denoteof 120 kDa, polyproteins 105 kDa, and containing 100 kDa, respectively. RdRp at the approximate sizes of 120 kDa, 105 kDa, and 100 kDa, respectively. Viruses 2016, 8, 25 13 of 20

3.6. PrematureViruses 2016, 8 Translational, x Shutoff and CrPV IRES Translation 13 of 19

3.6.We Premature next asked Translational whether prematurelyShutoff and CrPV repressing IRES Translation host translation can alter the temporal regulation of ORF1 and ORF2 expression in CrPV infected cells. We reasoned that premature inhibition of host translationWe maynext stimulateasked whether IGR IRESprematurely translation repressing earlier host in infected translation cells. can Treatment alter the oftemporal mammalian regulation of ORF1 and ORF2 expression in CrPV infected cells. We reasoned that premature cells with the chemical compound, Pateamine A (PatA), which deregulates eIF4A activity, results in inhibition of host translation may stimulate IGR IRES translation earlier in infected cells. Treatment a rapidof mammalian decrease in cells cap-dependent with the chemical translation compound, and stimulatesPateamine A IGR (PatA), IRES-mediated which deregulatestranslation eIF4A [ 25,45]. We treatedactivity, mock-results orin a CrPV-infected rapid decrease S2in cap-dependent cells with PatA translation at one h.p.i. and stimulates and monitored IGR IRES-mediated protein synthesis by pulse-labeling.translation [25,45]. As We expected, treated mock- PatA or inhibited CrPV-infected overall S2 cells host with protein PatA at synthesis one h.p.i.after and monitored only one hour of treatmentprotein synthesis (Figure by6B, pulse-labeling. compare lane As 1 expected, to 3). The PatA effects inhibited of PatA overall continued host protein to abolishsynthesis translation after afteronly 5 h one of treatment hour of treatment (Figure (Figure6B, lanes 6B, 5, compare 7, 9 and lane 11). 1 Comparedto 3). The effects to DMSO- of PatA treatedcontinued cells to abolish (Figure 6A), PatAtranslation treatment after of 5 CrPV-infected h of treatment (Figure cells resulted 6B, lanes in 5, a7, premature9 and 11). Compared significant to DMSO- shut off treated of host cells protein synthesis(Figure by 6A), 2 h.p.i.PatA treatment (Figure6 B,of CrPV lane-infected 3). In contrast cells resulted and interestingly, in a premature only significant the CrPV shut non-structuraloff of host protein synthesis by 2 h.p.i. (Figure 6B, lane 3). In contrast and interestingly, only the CrPV proteins could be visibly detected at this time point (Figure6B, lane 4) and it is not until 3 h.p.i. non-structural proteins could be visibly detected at this time point (Figure 6B, lane 4) and it is not that synthesis of the structural proteins is detected, which is similar to the time that is observed in until 3 h.p.i. that synthesis of the structural proteins is detected, which is similar to the time that is DMSO-treatedobserved in infectedDMSO-treated cells (Figureinfected6 A,B).cells (Figure Moreover, 6A,B). the Moreover, extent of the viral extent non-structural of viral non-structural and structural proteinand synthesisstructural andprotein RNA synthesis synthesis and were RNA similar synthesis between were DMSO- similar and between PatA-treatments DMSO- and over the coursePatA-treatments of infection over (Figure the6 C).course Finally, of infection PatA treatment (Figure 6C). did notFinally, significantly PatA treatment affect did intracellular not CrPVsignificantly yield, thus affect suggesting intracellular that CrPV premature yield, thus translational suggesting that shut premature off in S2 translational cells does shut not affectoff in viral replicationS2 cells does (Figure not6 affectC). To viral ensure replication these (Figure results 6C). are To not ensure specific these to results PatA, are we not prematurely specific to PatA, inhibited cellularwe translationprematurely withinhibited DTT, cellular a treatment translation that induces with DTT, eIF2 αaphosphorylation treatment that induces and also eIF2 knownα to stimulatephosphorylation IGR IRES translationand also known (Figure to6 D,E).stimulate Like IGR PatA IRES treatment, translation we observed(Figure 6D,E). no differences Like PatA in the treatment, we observed no differences in the timing or extent of viral non-structural and structural timing or extent of viral non-structural and structural protein synthesis in untreated and DTT-treated protein synthesis in untreated and DTT-treated infected cells (Figure 6D,E). Thus, forced premature infected cells (Figure6D,E). Thus, forced premature inhibition of protein synthesis does not disrupt inhibition of protein synthesis does not disrupt the temporal regulation of IGR IRES-mediated the temporaltranslation. regulation of IGR IRES-mediated translation.

ABDMSO Pat A C 123456h.p.i 123456h.p.i kDa - +-+ - + - + - + - + CrPV kDa - +-+ - + - + - + - + CrPV 170 170 RdRp* RdRp* 8 130 130 10 RdRp** 100 RdRp** 100 RdRp*** RdRp*** 70 70 107 RdRp (3D) 55 55 RdRp (3D)

6 2C 2C FFU/uL 10 40 VP0 40 VP0 S]-Met labeling S]-Met labeling

35 VP1 35 VP1 [ 35 [ 35 5 VP2 VP2 10 VP3 VP3 Pat A lanes 123456789101112 lanes 123456789101112 DMSO

D No drug E DTT 246h.p.i 246h.p.i kDa - +-+ - + CrPV kDa - +-+ - + CrPV 170 170 RdRp* 130 RdRp* 130 RdRp** RdRp** 100 100 RdRp*** 70 RdRp*** 70 RdRp (3D) 55 RdRp (3D) 55 2C 40 2C 40 VP0 VP0 VP1 VP1 35 VP2 35 VP2 VP3 VP3 S]-Met labeling S]-Met labeling S]-Met 35 35 [ 25 [ 25

lanes 1 2 3 4 5 6 lanes 1 2 3 4 5 6

FigureFigure 6. Effects 6. Effects of of pateamine pateamine A A (PatA) (PatA) and DTT on on Cr CrPVPV protein protein synthesis. synthesis. Pulse-labelled Pulse-labelled protein protein lysateslysates from from mock- mock- or or CrPV-infected CrPV-infected (MOI(MOI 10) 10) S2 S2 cells cells treated treated with with either either (A) (DMSO;A) DMSO; (B) 50 ( BnM) 50 nM pateaminepateamine A; A; (E )(E 50) 50 nM nM DTT; DTT; ( D(D)) or or leftleft untreated were were resolved resolved on ona 12% a 12% SDS-PAGE SDS-PAGE gel. The gel. cells The cells were metabolically labelled with [35S]-Met/Cys for twenty minutes at the end of each time point. were metabolically labelled with [35S]-Met/Cys for twenty minutes at the end of each time point. Shown are representative autoradiographs from at least three independent experiments; (C) Viral Shown are representative autoradiographs from at least three independent experiments; (C) Viral titers titers from infected lysates treated with DMSO or PatA after 10 h.p.i. RdRp*, RdRp**, and RdRp*** from infected lysates treated with DMSO or PatA after 10 h.p.i. RdRp*, RdRp**, and RdRp*** denote denote polyproteins containing RdRp at the approximate sizes of 120 kDa, 105 kDa, and 100 kDa polyproteinsrespectively. containing RdRp at the approximate sizes of 120 kDa, 105 kDa, and 100 kDa respectively.

Viruses 2016, 8, 25 14 of 20 Viruses 2016, 8, x 14 of 19

3.7.3.7. Non-StructuralNon-Structural ViralViral ProteinsProteins PromotePromote ExpressionExpressionof ofStructural Structural Viral Viral Proteins Proteins BecauseBecause IGRIGR IRES-mediatedIRES-mediated translationtranslation isis notnot perturbedperturbed byby prematurepremature inhibitioninhibition ofof cellularcellular translationtranslation oror transcription,transcription, wewe nextnext askedasked ifif oneone oror moremore non-structuralnon-structural viralviral proteinsproteins maymay bebe responsibleresponsible forfor thethe regulationregulation ofof IGRIGR IRESIRES activity.activity. To testtest this,this, wewe createdcreated aa CrPVCrPV repliconreplicon (CrPV2(Fluc))(CrPV2(Fluc)) byby replacingreplacing thethe ORF2ORF2 structuralstructural proteinsproteins ofof thethe CrPV-2CrPV-2 infectiousinfectious cloneclone withwith fireflyfirefly luciferaseluciferase [[27].27]. Thus, Thus, firefly firefly luciferase luciferase expression expression in inthis this replicon replicon is under is under the thecontrol control of the of IGR the IRES IGR IRES(Figure (Figure 7A).7 A).To Toaddress address whether whether expression expression of of the the non-structural non-structural proteins affectsaffects IGRIGR IRESIRES translation,translation, wewe generatedgenerated another another replicon replicon (CrPV2(Fluc)-ORF1 (CrPV2(Fluc)-ORF1stopstop),), whichwhich containscontains twotwo stopstop codonscodons withinwithin the the most most N-terminal N-terminal protein protein of of ORF1, ORF1, CrPV1A, CrPV1A, and and thus thus prevents prevents ORF1 ORF1 expression expression (Figure (Figure7A). The7A).in The vitro intranscribed vitro transcribed replicons replicons (Figure (Figure7B) were 7B) transfected were transfected into S2 cellsinto andS2 cells luciferase and luciferase activity wasactivity measured was measured at different at timedifferent points time after points transfection after transfection (hours post (hours transfection post transfection (h.p.t)). Whereas (h.p.t)). (CrPV2(Fluc)-ORF1Whereas (CrPV2(Fluc)-ORF1stop) RNA transfectionsstop) RNA transfections led to relatively led low to luciferaserelatively activity, low luciferase CrPV2(Fluc) activity, RNA transfectionsCrPV2(Fluc) resultedRNA transfections in a significant resulted increase in a insignificant luciferase increase activity, in especially luciferase between activity, 12 especially and 20 h afterbetween transfection 12 and 20 (Figure h after7C). transfection These results (Figure suggest 7C). that These expression results ofsuggest the non-structural that expression proteins of the contributesnon-structural to the proteins increase contributes in IGR IRES to the translation. increase in IGR IRES translation.

A

IGR VPg 5’UTR IRES 3’UTR CrPV-2(Fluc) 5` 1A 2B 2C 3A 3C 3D Fluc 3`

C T/C T A C/A C 971 974 5763 5766 CrPV-2(Fluc)-ORF1stop CrPV-2(Fluc)-mutRdRp

B in vitro transcribed RNAs

CrPV-2(Fluc) CrPV-2(Fluc)- CrPV-2(Fluc) mutRdRp -ORF1stop CD

14 2.5 CrPV-2(Fluc) CrPV-2(Fluc)-ORF1stop stop 12 CrPV-2(Fluc)-ORF1 CrPV-2(Fluc)-mutRdRp CrPV-2(Fluc)-mutRdRp 2 10 8 1.5

6 1 4 Relative luciferase Relative Relative luciferase Relative 0.5 2 0 0 0 5 10 15 20 0 5 10 15 20 h.p.t h.p.t

FigureFigure 7.7. Non-structural viral proteinsproteins promotepromote IGRIGR IRESIRES translation.translation. (A) SchematicsSchematics ofof thethe stop CrPV2(Fluc),CrPV2(Fluc), CrPV2(Fluc)-ORF1CrPV2(Fluc)-ORF1stop andand CrPV2(Fluc)-mutRdRp replicons; ( B)) In vitrovitro transcribedtranscribed stop CrPV2(Fluc),CrPV2(Fluc), CrPV2(Fluc)-ORF1CrPV2(Fluc)-ORF1stop andand CrPV2(Fluc)-mutRdRp RNAs RNAs that were usedused inin thethe transfectionstransfections asas shownshown inin ((CC,D,D);); ((CC)) FireflyFirefly luciferaseluciferase activitiesactivities fromfrom S2S2 cellscells transfectedtransfected withwith CrPV2(Fluc),CrPV2(Fluc), CrPV2(Fluc)-ORF1CrPV2(Fluc)-ORF1stopstop andand CrPV2(Fluc)-mutRdRp RNAs at differentdifferent timetime pointspoints transfection.transfection. The The results results are are normalized normalized to its to respective its respective three-hour three-hour post transfection post transfection (h.p.t); (D(h.p.t);) Results (D) areResults from are (C) from but only (C) but showing only showing CrPV-2(Fluc)-ORF1 CrPV-2(Fluc)-ORF1stop and CrPV(FLuc)-mutRdRP.stop and CrPV(FLuc)-mutRdRP. Shown areShown results are fromresults an from average an average of three of independent three independent experiments experiments (˘s.d.). (±s.d.).

The increase in luciferase activity observed from CrPV2(Fluc) transfections is likely due to The increase in luciferase activity observed from CrPV2(Fluc) transfections is likely due to contributions to an increase in IGR IRES translation and replication. To uncouple these effects, we contributions to an increase in IGR IRES translation and replication. To uncouple these effects, mutated two conserved residues (D1620A and D1621A) within the RdRp that have been shown to be we mutated two conserved residues (D1620A and D1621A) within the RdRp that have been important for replicase activity (CrPV2(Fluc)-mutRdRp) [46]. Transfection with shown to be important for replicase activity (CrPV2(Fluc)-mutRdRp) [46]. Transfection with (CrPV2(Fluc)-mutRdRp) RNA did not lead to an increase in luciferase activity as compared to the (CrPV2(Fluc)-mutRdRp) RNA did not lead to an increase in luciferase activity as compared to the CrPV2(Fluc) transfection (Figure 7B), suggesting that replication of the CrPV replicon contributes CrPV2(Fluc) transfection (Figure7B), suggesting that replication of the CrPV replicon contributes significantly to the increase in luciferase activity from CrPV2(Fluc) transfections. However, a careful comparison of luciferase activities between (CrPV2(Fluc)-mutRdRp) and (CrPV2(Fluc)-ORF1stop)

Viruses 2016, 8, 25 15 of 20 significantly to the increase in luciferase activity from CrPV2(Fluc) transfections. However, a careful comparison of luciferase activities between (CrPV2(Fluc)-mutRdRp) and (CrPV2(Fluc)-ORF1stop) transfections (Figure7D) showed that there is a reproducible two-fold increase in luciferase activity of (CrPV2(Fluc)-mutRdRp) over (CrPV2(Fluc)-ORF1stop) transfections, indicating that IGR IRES activity is stimulated similar to that observed with the reporter construct assays and ribosome profiling (Figures2,3A,B and7D). These results further suggest that expression of a non-structural protein(s) contribute to the increase in IGR IRES activity.

4. Discussion Dicistroviruses utilize a strategy whereby distinct IRESs regulate translation of non-structural and structural protein ORFs. While it has been well established that the viral structural proteins are produced in supramolar excess over non-structural viral proteins [7–9], the significance and the mechanisms underlying this phenomenon have not been investigated in detail. Here, we demonstrate that translation of the two ORFs via the 51UTR and IGR IRESs are temporally regulated (Figure8). Using a series of biochemical and molecular assays, we show conclusively that there is a reproducible delay, albeit slight, in IGR IRES translation compared to 51UTR IRES translation during CrPV infection (Figures1–3). We also showed that this temporal regulation is not dependent on the repression of host transcriptional or translational inhibition, cellular processes that are perturbed during CrPV infection (Figures4–6). Using CrPV(Fluc) replicons to decouple viral translation from viral replication, we showed that the increase in IGR IRES translation is partly dependent on viral non-structural proteins (Figure7). We propose that CrPV utilizes a dual IRES translational control strategy to ensure that optimal expression of viral non-structural proteins occurs prior to synthesis of viral structural proteins (Figure8), and as a result, this leads to the temporal regulation of viral replication and virus assembly during infection. Regulation of IRES translation is key for some virus infections. For instance, poliovirus uses a strategy via temporal cleavage of host factors such as the translation factors eIF4G and poly (A) binding protein and IRES-trans-acting factors, Poly(rC) binding protein 2 and polypyrimidine tract binding protein 1, to regulate the switch from host translation to IRES-mediated protein synthesis as well as the switch from viral translation to replication [3,47–49]. In this study, we examined whether the distinct temporal regulation of non-structural and structural protein synthesis during CrPV infection may be driven by the shutoff of host translation and the different factor requirements between IGR IRES and 51UTR dependent translation [13,14,24]. Surprisingly, attempts to disrupt overall host translation by either inhibiting eIF2α phosphorylation or affecting eIF4A activity during infection did not affect the timing of 51UTR and IGR IRES translation (Figures5 and6). Although it has been shown that CrPV IGR IRES translation is stimulated when eIF2α is phosphorylated or eIF4A activity is disrupted [14,25,42], it is clear that indirect effects through translational shutoff are not a prerequisite for IGR IRES stimulation during CrPV infection. One possible explanation is that there may be a more direct effect on IGR IRES translation during infection: (i) IGR IRES translation may be inhibited at early time points during infection or (ii) IGR IRES translation may be activated at later times. Our data suggest that besides RdRp, one or more non-structural proteins contribute to the increased structural protein synthesis during CrPV infection (Figure7). Specifically, a reproducible two-fold increase in luciferase activity was noted when comparing a mutant replicon that is defective in replicating (CrPV2(Fluc)-mutRdRp) to a mutant replicon that expresses no non-structural viral proteins (CrPV2(Fluc)-ORF1stop) (Figure7). These results suggest that non-structural proteins may act directly or indirectly to regulate IGR IRES translation during CrPV infection, potentially via structural conformations of the IGR IRES and/or affecting ribosome recruitment (Figure7). A possible target is the ribosome. It has been shown that depletion of a subset of ribosomal proteins results in inhibition of IRES-dependent but not cap-dependent translation [29,50–52], suggesting that heterogeneity of ribosomes may have an effect on IRES translation. For example, depletion of a subset of ribosomal proteins inhibits IGR IRES Viruses 2016, 8, 25 16 of 20 translation and infectivity by a related dicistrovirus Drosophila C virus [29,50,51]. Interestingly, cells depleted of RACK1, a component of the 40S subunit, leads to inhibition of 51UTR IRES but not IGR IRES translation during infection, thus further supporting that the two CrPV IRESs are differentially Viruses 2016, 8, x 16 of 19 regulated at the ribosome level [53]. Thus, it remains to be examined whether specific post-translational modifications or depletion of key ribosomal proteins during CrPV infection may contribute to the post-translational modifications or depletion of key ribosomal proteins during CrPV infection may temporal regulation of IRES translation. contribute to the temporal regulation of IRES translation.

Figure 8. Model of CrPV 5'UTR IRES and IGR IRES-dependent translation during infection. Early in Figure 8. Model of CrPV 51UTR IRES and IGR IRES-dependent translation during infection. Early in infection (0–2 hpi), 5'UTR IRES-dependent translation predominates to produce viral non-structural infection (0–2 hpi), 51UTR IRES-dependent translation predominates to produce viral non-structural proteins such as the 3D RNA-dependent RNA polymerase, the 3C-like protease and helicase. (1) IGR proteins such as the 3D RNA-dependent RNA polymerase, the 3C-like protease and helicase. (1) IGR IRES translation may be inhibited directly or indirectly by a yet unknown factor; (2) At 3–4 hpi, IRES translation may be inhibited directly or indirectly by a yet unknown factor; (2) At 3–4 hpi, non-structural viral proteins may directly or indirectly stimulate IGR IRES translation (dashed non-structural viral proteins may directly or indirectly stimulate IGR IRES translation (dashed arrow), arrow), leading to viral structural protein expression; (3) Replication of the viral genome enhances leading to viral structural protein expression; (3) Replication of the viral genome enhances structural structural protein expression by providing more translatable CrPV genomes and/or stimulating IGR protein expression by providing more translatable CrPV genomes and/or stimulating IGR IRES activity. IRES activity. In effect, the temporal regulation of viral protein synthesis via IRES control allows viral In effect, the temporal regulation of viral protein synthesis via IRES control allows viral replication to replication to occur prior to structural protein synthesis. occur prior to structural protein synthesis.

As described earlier, a number of viruses, including poliovirus, recruit ITAFs to facilitate viral translationAs described [54,55]. earlier, It is possible a number that of viruses,a similar including phenomena poliovirus, is occurring recruit ITAFsunder toCrPV facilitate infection. viral translationHowever, there [54,55 have]. It is yet possible to be any that reports a similar of phenomenahost (or viral) is occurringfactors other under than CrPV the infection.ribosome However,that bind to the IGR IRES. Moreover, given the compact structure of the IRES, it is difficult to imagine a trans-acting factor binding to the IGR IRES [16,20–23]. Nevertheless, if an ITAF is involved, our data suggest it is not likely a newly transcribed cellular RNA or translated host protein that mediates IGR IRES-dependent translation during infection (Figures 4 and 6). A key finding from our studies is that replication of the CrPV genome is necessary for the dramatic increase in firefly luciferase, which monitors IGR IRES translation in the replicon system

Viruses 2016, 8, 25 17 of 20 there have yet to be any reports of host (or viral) factors other than the ribosome that bind to the IGR IRES. Moreover, given the compact structure of the IRES, it is difficult to imagine a trans-acting factor binding to the IGR IRES [16,20–23]. Nevertheless, if an ITAF is involved, our data suggest it is not likely a newly transcribed cellular RNA or translated host protein that mediates IGR IRES-dependent translation during infection (Figures4 and6). A key finding from our studies is that replication of the CrPV genome is necessary for the dramatic increase in firefly luciferase, which monitors IGR IRES translation in the replicon system (Figure7). In contrast, despite the significant increase in viral RNA during CrPV infection, 51UTR IRES-mediated translation is not stimulated (Figure1), thus underscoring the differential regulation of the distinct IRESs during CrPV infection. Because IGR IRES-mediated translation is stimulated during infection by approximately 3–4 fold (Figure3)[ 25], the supramolar expression of structural proteins is likely due to a sum of both the effects of the increase in replicating CrPV viral RNA genome and the stimulation of IGR IRES translation. Alternatively, IGR IRES translation may be stimulated even more within viral progeny genomes. It has been reported that newly transcribed vaccinia RNAs during infection are preferentially translated during infection [56]. Thus, a similar scenario may be occurring in CrPV replicating genomes that results in preferential IGR IRES translation, a hypothesis that warrants further examination. In a recent report, Wu et al. (2014) demonstrated that Pelo is required for enhanced structural protein synthesis during dicistrovirus infection but does not affect IGR IRES translation [57]. Pelo is the Drosophila homolog for Dom34, which is responsible for the recycling of stalled 80S ribosomes on mRNAs [58]. Wu et al. (2014) speculated that the action of Pelo during infection provides dicistrovirus genomes greater access to ribosomes for high level synthesis of viral structural proteins [58]. Nevertheless, in cells depleted of Pelo, dicistrovirus structural proteins are still expressed in supramolar excess over non-structural proteins [57], thus ribosome recycling is likely not the sole reason for structural protein synthesis that is observed late in infection.

5. Conclusions Many viruses use a strategy to coordinate temporal expression of non-structural and structural proteins [26]. We propose CrPV uses an IRES-based strategy to initiate expression of non-structural proteins for viral translation and replication and delay of expression of the structural proteins during infection, which may be a strategy generalized within the Dicistroviridae family.

Supplementary Materials: The following is available online at www.mdpi.com/1999-4915/8/1/25/s1, Figure S1: CrPV protein synthesis by immunoprecipitation analysis. Acknowledgments: We thank George Mackie for insightful discussions. This work was supported by a Canadian Institute of Health Research operating grant (MOP-81244) to Eric Jan, MRC Programme grant funding to Anne Willis and an NSERC Graduate Scholarship to Anthony Khong. Author Contributions: Anthony Khong, Jennifer M. Bonderoff, Ruth V. Spriggs, Craig H. Kerr, Thomas J. Jackson, Anne E. Willis and Eric Jan conceived and designed the experiments; Anthony Khong, Jennifer M. Bonderoff, Ruth V. Spriggs, Erik Tammpere, Craig H. Kerr and Thomas J. Jackson performed the experiments; Anthony Khong, Jennifer M. Bonderoff, Ruth V. Spriggs, Craig H. Kerr, Thomas J. Jackson, Anne E. Willis and Eric Jan analyzed the data; Anthony Khong, Jennifer M. Bonderoff, Ruth V. Spriggs, Erik Tammpere, Craig H. Kerr and Thomas J. Jackson contributed reagents/materials/analysis tools; Anthony Khong, Jennifer M. Bonderoff, Anne E. Willis and Eric Jan wrote the paper. Conflicts of Interest: The authors declare no conflict of interest.

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