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Ribosomal Protein P0 Promotes Potato Virus A Infection and Functions in Viral Translation Together with VPg and eIF(iso)4E

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that PVA RNA can be targeted by VPg to a specific gene expression pathway that protects the viral RNA from degradation and facilitates its translation. Here, we show that P0 is essential for this activity of VPg, similar to eIF4E/eIF(iso)4E. We also demon-strate that VPg, P0, and eIF(iso)4E synergistically enhance viral translation. Interestingly, the positive effects of VPg and P0 on viral translation were negatively correlated with the cell-to-cell spread of infection, suggesting that these processes may compete for viral RNA.

V

iruses have acquired mechanisms by which they efficiently recruit the host translational machinery. Viral RNA transla-tion is sometimes accompanied by the repression of host mRNA translation (1–4) by strategies that frequently involve the virus-induced modification of translation initiation factors. An alterna-tive strategy is illustrated by aCauliflower mosaic virus(CaMV) protein, which associates directly with the ribosome and regulates its function (5). Many host proteins that function in conventional translation also participate in viral RNA replication (6). For ex-ample, the replicase of bacteriophage Q␤contains four host pro-teins connected to translation, i.e., ribosome-associated HF-1, ri-bosomal S1, and elongation factors EF-Tu and EF-Ts (7). The eukaryotic homolog of EF-Tu is eEF1A, a protein frequently asso-ciated with viral replicases (8). Different subunits of eIF3 have also been shown to be functional components of both the Brome mosaic virus(BMV) andTobacco mosaic virus(TMV) replicases (9–11).

Studies showing that only replicated viral RNAs are efficiently translated have indicated that positive-stranded RNA virus trans-lation and replication are functionally coupled (12–14). The pro-cess of RNA replication is associated with virus-induced cellular membrane structures called viral replication complexes (RCs) (15). The model virus of this study,Potato virus A(PVA), is a positive-stranded RNA virus belonging to the genus Potyvirus. Several host proteins that function in translation have been asso-ciated with RCs ofTurnip mosaic virus(TuMV) (genusPotyvirus), including eIF(iso)4E, PABP, and eEF1A (16–18). Since both PABP and eEF1A interact with the TuMV RNA-dependent RNA polymerase (RdRp), PABP and eEF1A may function in RNA rep-lication (18, 19). Most recessive genes conferring resistance to infection by potyviruses are alleles of eIF4E and eIF(iso)4E, and successful infection commonly requires compatible interactions between viral VPg and eIF4E and eIF(iso)4E (20).

Ribosomes are essential for translation. In plants, they contain a structure referred to as a ribosomal stalk (21), which forms a lateral protrusion from the ribosome and is composed of the acidic proteins P0, P1, and P2 (22) and the plant-specific protein P3 (23,24). P proteins are also present in non-ribosome-associ-ated pools (25–27). P0 functions as a scaffold for the stalk

struc-ture by interacting with 28S rRNA, and the remaining P proteins assemble to form the extended stalk via interactions with P0. P0 is a protein conserved across kingdoms, as shown by the ability of P0 genes from worm, mammals, and protozoa to functionally com-plementSaccharomyces cerevisiaelacking endogenous P0 (28–30). P0 is the only essential P protein for bothin vitrotranslational activity of yeast ribosomes and cell survival (31,32). Ribosomal stalk proteins can affect several aspects of ribosome function, including transla-tional capacity, polysome pattern, and ribosomal subunit joining (33,34). P proteins are also regarded as having different effects on the translation of distinct mRNAs in yeast (31). In this study, we show that ribosomal P proteins are important for PVA infection of Nicotiana benthamiana. We found that P0, a viral RNP compo-nent, has functions distinct from those of the other P proteins in regulating PVA RNA expression.

MATERIALS AND METHODS

Plants.Nicotiana benthamianaplants were grown in a greenhouse at 22°C for an 18-h day period and at 18°C for a 6-h night period and used for experiments at the 4- to 6-leaf stage.

Protein analysis.Viral RNP complexes were purified from infected plants, and P0 was identified by proteomic tools as described previously (35). Ribosomes were isolated as described previously (36), except that the phosphatase inhibitors were omitted. P proteins were detected by West-ern blot analysis using a human autoimmune disease serum against ribo-somal P antigen (catalog no. HP0-0100; Immunovision).

Viruses, plant overexpression, and gene silencing constructs.PVA and firefly luciferase (FLUC) constructs were described previously (37). A P0 plant expression vector was constructed by generating a Gateway Clon-ing Technology (Invitrogen)-compatible cDNA ofArabidopsis thaliana

60S acidic ribosomal protein P0 (RPP0C) (GenBank accession no. NM_111960) by PCR. The cDNA was inserted into pMDC32 (38),

Received19 November 2012Accepted25 January 2013

Published ahead of print30 January 2013

Address correspondence to Kristiina Mäkinen, kristiina.makinen@helsinki.fi.

Copyright © 2013, American Society for Microbiology. All Rights Reserved.

doi:10.1128/JVI.03198-12

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pGWB17, and pGWB18 (39) via pDONR/Zeo (Invitrogen), using stan-dard Gateway cloning. An eIF(iso)4E Gateway-compatible PCR product was recombined via pDONR/Zeo (Invitrogen) into pGWB18. The GUS and VPg plant expression constructs were described previously (40). P-protein-silencing vectors were constructed by generating Gateway-com-patibleN. benthamianaP-protein cDNA fragments, which were inserted into pHELLSGATE8 (pHG8) (41) via pDONR/Zeo; empty pHG8 was used as a control. The silencing constructs for eIF4E and eIF(iso)4E were described previously (40). All plant expression vectors were used to trans-formAgrobacterium tumefaciensstrain C58C1/pGV2260.

Gene silencing by transient expression of hairpin RNA.The method used for transientAgrobacterium-mediated silencing was described previ-ously (42).Agrobacteriumcells carrying hairpin vectors (pHG8) with gene-specific inserts were infiltrated into leaves. Hairpins were either cotransformed with wild-type (wt) PVA or pretransformed 4 days before inoculation of mutant PVAs. Gene silencing was verified by reverse tran-scription (RT)-PCR. Here, total RNA was extracted from plant leaves 4 days after infiltration ofAgrobacteriumcells carrying hairpin constructs. Total RNA was treated with DNase I, and cDNA was synthesized by using Moloney murine leukemia virus (M-MLV) reverse transcriptase and oligo(dT) primers. The same primers that were used to generate the se-quences for cloning of cDNAs into pHG8 were used for PCR amplifica-tion.

Analysis of infection foci by fluorescence microscopy. Agrobacte-riumcells carrying green fluorescent protein (GFP)-tagged PVA were in-filtrated into plant leaves at an optical density at 600 nm (OD600) of 0.0005. Exogenous proteins or RNA hairpins were coexpressed by coin-filtratingAgrobacteriumcells carrying the respective expression cassettes. Infection foci were examined by using a Zeiss Axio Scope.A1 microscope and a 2.5⫻or 10⫻objective. The area of individual infection foci and the percentage of infected tissue were determined by using microscope soft-ware. For each condition, data on infection foci were collected from a minimum of three individual plants and 50 foci.

RLUC- and qPCR-based PVA infection assay.Luciferase quantita-tion of virus-derivedRenillaluciferase (RLUC) and control FLUC and quantitative RT-PCR (qPCR) were described previously(37,40). Details ofAgrobacteriumOD600s used to transform PVA and other expression cassettes, time points of data collection, and other relevant information are given in the respective figure legends and the text. Error bars represent the standard deviations.

Nucleotide sequence accession numbers.Data have been deposited in the GenBank database under accession no.NM111960(A. thaliana

RPP0C); FN666434 [Nicotiana tabacumeIF(iso4)E]; and JN227614 (P0), JN227615 (P1), JN227616 (P2), and JN227617 (P3) (N. benthamiana si-lencing fragments).

RESULTS

Ribosomal P0 copurifies with viral replication proteins from infected plants.To identify host proteins participating in PVA infection, two viral replication proteins, VPg and RdRp, were ex-pressed as Strep III-tagged fusion proteins from their native posi-tions in the viral genome and used to affinity purify membrane-associated viral protein complexes from infected Nicotiana benthamianaplants. The composition of these complexes was an-alyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), which showed that HSP70, viral RdRp, NIa, and VPg were all found in both the VPg- and RdRp-tagged samples (35). In this study, we report the identification of a protein homol-ogous toArabidopsis thaliana 60S acidic ribosomal protein P0 (P0) (GenBank accession no.AAL07229). Western blot analysis using human anti-P autoimmune disease serum showed that P0 was specifically copurified with both Strep III-tagged VPg and RdRp but was not visible in the control sample derived from leaves infected with nontagged PVA (Fig. 1A).

The serum used to detect P0 also recognizes the ribosomal stalk proteins P1, P2, and P3 via their homologous C termini (23,43). These proteins are small (10 to 15 kDa) and may correspond to the weakly detected lower-molecular-weight proteins (Fig. 1A). The patterns of viral RNP- and ribosome-associated P proteins dif-fered. A Western blot analysis with the human anti-P autoim-mune disease serum of a ribosome-enriched fraction derived from noninfected N. benthamiana leaves yielded several proteins (Fig. 1B) with a pattern corresponding to that of maize ribosomal P proteins (44). In contrast, the viral RNP complex contained more P0 than the other P proteins. None of the other P proteins was detected by LC-MS/MS analysis, providing further evidence that they were not as abundant as P0 in the purified viral RNP.

Exogenous P0 promotes PVA infection.We inserted the Re-nillaluciferase (RLUC) reporter gene into PVA infectious cDNA to enable sensitive quantification of viral infection (Fig. 2) (37). To analyze the role of P0 in PVA infection, we constructed silenc-ing and overexpression constructs for P0. P0 was transiently si-lenced by expressing a P0-targeting RNA hairpin in plant leaves usingAgrobacteriuminfiltration, with the control being the empty hairpin vector. Western blot analysis using anti-P serum showed that silencing reduced the expression level of P0 (Fig. 3A). In con-trast, when P0 was overexpressed, no obvious P0 accumulation could be detected (Fig. 3B), similar to findings in yeast cells, which do not accumulate P0 protein despite increased levels of P0 mRNA (45). We therefore verified ectopic P0 expression using N-and C-terminally fused myc tags N-and found that these proteins could be detected by using anti-myc IgGs (Fig. 3C). To address the role of P0 in infection, exogenous P0, with and without myc tags, was expressed along with RLUC-tagged wt PVA, and the effect on RLUC activity at 7 days after infection (DAI) was analyzed (Fig. 3D). GUS was expressed as a control for P0. Exogenous ex-pression of both tagged and nontagged P0 increased viral RLUC activity by 3-fold, whereas silencing of P0 reduced viral RLUC activity by 3-fold (Fig. 3D).

We also estimated the average area of individual infection foci formed by wt GFP-tagged PVA in the presence of exogenous P0 or GUS at 3 DAI (Fig. 3E). GFP fluorescence was detected in numer-ous cells within separate foci at this time, showing that infection had already spread by cell-to-cell movement (Fig. 3F). Expression of exogenous P0 increased the average area of infection foci com-pared with the control. It is important to emphasize that very low FIG 1Ribosomal P0 is a component of membrane-associated viral RNPs. (A) Western blotting showing the presence of P proteins in the RNP complexes obtained by affinity purification, using Strep III-tagged PVA RdRp (RdRpSIII)

and Strep III-tagged VPg (VPgSIII), from membrane fractions of infectedN. benthamianaleaves. A sample purified from plants infected with wt PVA was used as a control. (B) Western blotting of P proteins associated with isolated ribosomes fromN. benthamianaleaves. Human anti-P autoimmune disease serum was used to detect P proteins in panels A and B.

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concentrations ofAgrobacterium(OD600⫽0.0005) were used to

transform RLUC/GFP-tagged wt PVA (Fig. 3DtoF), resulting in the initiation of 1 infection focus per mm2(e.g., seeFig. 4B). This

low density reduced any possible effects due to the Agrobacterium-based inoculation method and left space for the infection to spread by cell-to-cell movement. However, it was necessary to measure the diameter of the infection foci at 3 DAI, as at 4 DAI, many foci will have coalesced (data not shown). Separate infection foci in which cell-to-cell movement had occurred by 3 DAI were also achieved with TuMV under similar inoculation conditions (46).

Ribosomal stalk proteins are important for PVA infection. The results described above showed that P0 is important for PVA infection. Since P0 is essential for cellular translation as part of the ribosomal stalk structure, altered P0 homeostasis may affect PVA infection through effects on either the ribosomal stalk or other P0 functions. To analyze the possible role of the ribosomal stalk in PVA infection, RNA hairpins were expressed to silence the other stalk P proteins (P1, P2, and P3) as well as P0, and wt PVA RLUC activities were analyzed at 3, 6, and 9 DAI (Fig. 4A). Although silencing of any P protein reduced viral RLUC activity, the latter was higher during P0 silencing than during P1, P2, or P3 silencing at 9 DAI.

The infection foci of GFP-tagged PVA were also analyzed by

fluorescence microscopy. First, the number of GFP foci was cal-culated at 3 DAI (Fig. 4B), a time point when most foci were isolated clusters of cells showing PVA-derived GFP fluorescence (e.g., seeFig. 3F). Silencing of P proteins did not alter the amount of initially infected cells compared with the control, indicating that the reduction in viral RLUC activity during P-protein silenc-ing was not due to reduced initiation but to reduced progression of infection. Moreover, silencing of the P proteins resulted in a slight reduction in the average areas of infection foci at 3 DAI (Fig. 4C). At 6 DAI, however, the leaves of both the control and P-pro-tein-silenced plants were fully infected, as GFP fluorescence was uniform throughout the leaves (Fig. 4D). Fluorescence intensity, as well as viral RLUC activity, was strongest in the control (Fig. 4A). Together, these results show that the silencing of ribo-somal stalk proteins affects PVA infection by reducing the cel-lular level of infection but has little effect on the cell-to-cell spread of infection. RT-PCR verified silencing of the target transcripts (Fig. 4E).

P0 has functions that are distinct from those of other P pro-teins in PVA infection.Coordination of the potyvirus genome between translation, replication, and other cellular functions is complex, as these stages are coupled, and inhibition of virus infec-tion due to the silencing of P proteins may result from disrupinfec-tion of any of those processes. We therefore utilized different RLUC-FIG 2Schematic representation of the constructs used in this study. The transcription CaMV 35S promoter and the nopaline synthase (NOS) and octopine synthase (OCS) terminators are in gray, and cDNAs are in white. Viral constructs lacking either the PVA 5=- or 3=-UTR were derived from the PVA⌬GDD

construct. Both monocistronic RLUC and monocistronic GUS were expressed from similar constructs to FLUC and VPg. eIF(iso)4E was expressed from a similar construct as P0 in the pGWB18 vector backbone.

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tagged PVA mutants to determine which viral process is affected under each experimental condition. PVACPmutis a virus with a

coat protein (CP) mutation that undergoes replication in inocu-lated cells but is incapable of virus particle assembly and cell-to-cell and systemic movement (37). PVA⌬GDDcarries a mutation in the RdRp that inhibits replication (37); it therefore does not carry VPg at its 5=end, differentiating it from authentic viral RNAs. The validity of the use of this construct for studying PVA translation is addressed in Discussion.

P proteins were silenced by transforming RNA hairpins 4 days prior to inoculation of mutant PVAs, along with firefly luciferase (FLUC) as an internal control forAgrobacterium-mediated non-viral expression. PVA RLUC activity and RNA accumulation were assayed at 3 DAI (Fig. 5AandB). Importantly, the RNA level was 10-fold higher for PVACPmut than for PVA⌬GDDin the control plants, indicating that most of the PVACPmutRNA was derived

from replication. PVACPmutRNA levels were markedly reduced by silencing of P1, P2, and P3, suggesting reduced replication. This may have been due to reduced translation, as both PVA⌬GDDand FLUC RNA levels were unaffected, although the corresponding luciferase activities decreased (Fig. 5BandC). In contrast to the other P proteins, elevated PVACPmut RNA levels were detected

after P0 silencing, suggesting that P0 can affect PVA RNA

accu-mulation differently from the other stalk proteins and thus ex-plaining why P0 was identified from membrane-associated viral RNPs (Fig. 1). The ratio of RNA to RLUC activity reflects the translation of accumulated RNA. The 4-fold increase in the ratio of PVACPmutRNA to RLUC during P0 silencing (Fig. 5D) suggests that despite the accumulation of PVACPmutRNA, it was not

effi-ciently translated.

P0 can increase viral translation.Since the expression of exog-enous P0 increased wt PVA infection (Fig. 3D), we analyzed whether exogenous P0 expression affected RLUC-tagged PVA⌬GDD and

FLUC activities and RNA accumulation. Exogenous P0 increased RLUC activity but did not alter PVA⌬GDDRNA levels (Fig. 6A),

sug-gesting that P0 enhanced viral translation. FLUC activity and RNA levels did not respond to exogenous P0. The positive effect of exoge-nous P0 expression on RLUC accumulation was similar when de-rived from PVA⌬GDDand wt PVA infection (Fig. 3D), indicating that

P0 could promote wt infection through increased viral translation. The 5=-UTR is an important part of the viral RNA response to P0.The viral untranslated regions (UTRs) appear to promote PVA RNA translation, as the removal of either UTR reduced viral protein accumulation (40). We analyzed the importance of viral UTRs in the P0 promotion of viral translation by expressing exogenous P0 with PVA⌬GDD lacking either the 5=-UTR FIG 3Ribosomal P0 promotes PVA infection. (A) P0 was silenced by expressing an RNA hairpin (hp) that targets P0 transcripts (hpP0), followed by Western blotting using human anti-P autoimmune disease serum directed against the ribosomal P antigen. The empty hairpin vector was expressed as a control (hp⫺). (B) Exogenous P0 was expressed, and P0 levels were analyzed by Western blotting as described above for panel A. GUS was expressed as a control. (C) Exogenous expression of myc-tagged P0 followed by Western blotting. P0Nand P0Chave N- and C-terminal myc fusions, respectively. The membrane stained for total

protein by Ponceau S is shown at the position of the RUBISCO large subunit as a loading control in panels A to C. (D) Exogenous P0 with or without myc tags or GUS (control) was coexpressed with wt PVA. In parallel, P0 was silenced, and the level of luciferase activity derived from RLUC-tagged wt PVA was determined 7 days after infection (DAI). TheAgrobacteriumOD600values used for infiltration were as follows: 0.0005 for wt PVA and 0.5 for P0, GUS, hp⫺, and hpP0. (E)

Fluorescence microscopy of the average area of infection foci formed by GFP-tagged wt PVA at 3 DAI. (F) Representative infection foci from leaves expressing GUS and P0. Scale bar, 200␮m. The number of analyzed foci was⬎100 for both GUS and P0.ⴱ,P⬍0.05;ⴱⴱ,P⬍0.01 (determined by Student’sttests).

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(PVA⌬GDD⌬5=UTR

) or the 3=-UTR (PVA⌬GDD⌬3=UTR

) and deter-mined viral RLUC activity (Fig. 7A). RLUC activity hardly in-creased for PVA⌬GDD⌬5=UTR

, suggesting that the 5=-UTR is impor-tant for exogenous P0 to enhance viral translation. Exogenous P0 increased RLUC activity derived from PVA⌬GDD⌬3=UTR

more than that from PVA⌬GDD. Hence, the 5=- and 3=-UTRs exerted opposite

effects on viral RNA translation in the presence of exogenous P0. We further analyzed if monocistronic RLUC transcripts carrying the viral 5=-UTR could respond to exogenous P0 expression (Fig. 7B). Its RLUC activity was unaltered by exogenous P0 expression, suggesting that the viral RNA has additional properties required for P0 to increase its translation.

Next, we correlated RLUC activities with PVA⌬GDD, PVA⌬GDD⌬5=UTR, and PVA⌬GDD⌬3=UTRRNA levels (Fig. 7C).

De-letion of the 5=-UTR reduced RLUC activity more than RNA levels compared with PVA⌬GDD, whereas an absence of the 3=-UTR

re-duced RLUC activity but not RNA levels, showing that both UTRs are important, especially for viral RNA translation. We also com-pared RLUC activities and RNA levels of monocistronic RLUC transcripts fused with two different 5=-UTRs, a cloning vector-derived random leader sequence (5=-UTRRef) and the PVA 5= -UTR (Fig. 7D). The viral 5=-UTR did not enhance either the ac-cumulation or translation of the RLUC transcript. These findings

indicate that the viral 5=-UTR is essential for efficient viral trans-lation and is required for a transtrans-lational response of PVA⌬GDDto

exogenous P0.

P0 is required for VPg-enhanced viral translation.We re-ported previously that VPg requires the PVA 5=-UTR to increase viral translation when expressed intransbut that the PVA 5=-UTR alone is not sufficient to transfer this response to a monocistronic transcript (40), similarly to our results for P0. We therefore ana-lyzed whether P0 and VPg were connected in promoting viral translation. As the expressions of exogenous VPg (40) and P0 increase RLUC activity from both wt PVA and PVA⌬GDD, we used PVA⌬GDDin this expression analysis to exclude any effects of virus

replication and movement on viral protein accumulation. Either VPg or GUS (control) was coexpressed with PVA⌬GDDduring

P-protein silencing (Fig. 8). VPg increased viral RLUC activity 3-fold in control and P1-, P2-, and P3-silenced leaves but not during P0 silencing, showing that of these P proteins, only P0 was required to increase viral translation.

PVA translation is increased further by P0 and VPg together. To further analyze the connection between P0 and VPg in viral translation, PVA⌬GDDwas coexpressed with P0 and/or VPg, and RLUC activity and RNA levels were determined at 3 DAI (Fig. 9A). As before, P0 increased RLUC activity but not the PVA⌬GDDRNA FIG 4Ribosomal P proteins are important for PVA infection. (A) RLUC activity of wt PVA was determined at 3, 6, and 9 DAI during silencing of P proteins by using RNA hairpins (hp) targeting each P protein (hpP0, hpP1, hpP2, and hpP3). An empty hairpin vector was expressed as a control (hp⫺). TheAgrobacterium

OD600values used for infiltration are described in the legend ofFig. 3D. (B) Average numbers of infection foci per mm23 days later. Twelve areas of 4 mm2were

analyzed under each condition by using fluorescence microscopy. (C) The average size of infection foci was determined. Over 100 foci were analyzed under each condition.ⴱ,P⬍0.05;ⴱⴱ,P⬍0.01 (determined by Student’sttests). (D) Representative leaf tissue images of PVA-derived GFP fluorescence in infection foci during P-protein silencing at 3 and 6 DAI. Scale bar, 1 mm. (E) RT-PCR was used to verify silencing of P0 to P3 transcripts achieved by expression of RNA hairpins. The expected PCR products are indicated by arrows, and the size marker (M) is on the left.

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level (Fig. 6A), whereas VPg increased both (40). Further increases in RLUC activity and viral RNA levels were observed when P0 and VPg were coexpressed. The levels of monocistronic FLUC RNA and FLUC activity remained essentially unaltered (Fig. 9B), show-ing that the response was specific for PVA⌬GDDRNA.

To determine whether P0 and VPg together could affect natu-ral infection, lower leaves of plants were inoculated with wt PVA, and after 7 days, P0 and VPg were expressed by Agrobacterium-mediated transformation in systemically infected upper leaves. Viral RLUC activity was determined 3 days after infiltrating P0 and VPg expression constructs into systemically infected leaves, equal to 10 days after inoculation of the plants with PVA (Fig. 9C). Expression of either exogenous VPg or P0 increased viral RLUC activity. Exogenous P0 increased systemic infection by wt PVA (Fig. 3D) and PVA⌬GDD(Fig. 6Aand7A), further indicating that

the P0 response functions in bona fide PVA infection. Expression of exogenous VPg increased viral RLUC activity in systemically infected leaves but less than that observed for PVA⌬GDD(Fig. 9A).

The synergistic effect of VPg and P0 on PVA gene expression could not be detected here.

Exogenous VPg increases viral translation together with eIF(iso)4E and P0 but reduces the spread of infection. We showed previously that exogenously expressed VPg is unable to enhance viral translation when both eIF4E and eIF(iso)4E are si-lenced (40). To extend this finding, we cosilenced eIF4E and eIF(iso)4E and analyzed RLUC activities and RNA levels of RLUC-tagged wt PVA at 9 DAI (Fig. 10A). Both RLUC activity and viral RNA levels were reduced during eIF4E/eIF(iso)4E silencing and during P0 silencing (Fig. 10A). As both P0 and eIF4E/eIF(iso)4E are required for normal PVA infection and for a VPg-mediated boost in translation, the effects of different combi-nations of eIF(iso)4E, VPg, and P0 on wt PVA RLUC activity were determined at 3 DAI (Fig. 10B). To our surprise, RLUC activity was not increased by VPg or eIF(iso)4E and was increased only slightly by the combination of VPg, P0, and eIF(iso)4E but was markedly increased by VPg plus eIF(iso)4E. We therefore ana-lyzed the effects of VPg, P0, and eIF(iso)4E expression on the development of infection foci by GFP-tagged wt PVA by deter-mining the average area of infection foci at 3 DAI (Fig. 10C), at which time cell-to-cell movement had already occurred (see also Fig. 3Fand4D). Exogenous VPg expression reduced the intercel-lular spread of infection (Fig. 10C), an effect even more pro-nounced when the proportion of infected to noninfected tissue was determined at 4 DAI (Fig. 10DandF). Interestingly, coexpres-sion of P0 with VPg reduced cell-to-cell infection spread even more than VPg alone (Fig. 10D), despite an increase when P0 was expressed alone (Fig. 3F). Also, the positive effect of VPg and eIF(iso)4E coexpression on PVA RLUC activity was overpowered when P0 was coexpressed. Importantly, the total number of infec-tion foci remained similar under these condiinfec-tions, showing that altering the PVA infection initiation rate was not responsible for the observed effects (Fig. 10E).

FIG 5P0 has functions distinct from those of other P proteins in PVA infec-tion. (A to C) RLUC activity and RNA accumulation were determined for PVACPmut(A), PVA⌬GDD(B), and FLUC (C) during P-protein silencing at 3

DAI. Hairpins were applied 4 days before PVAs. TheAgrobacteriumOD600

values used for infiltration were as follows: 0.05 for PVACPmutand PVA⌬GDD,

0.005 for FLUC, and 0.4 for hairpins. Luciferase activities and RNA amounts were analyzed by using the same samples. (D) Ratio of RNA amount to viral RLUC activity for PVACPmut, calculated by dividing the average relative values

calculated from data presented in panel A.ⴱ,P⬍0.05;ⴱⴱ,P⬍0.01 (deter-mined by Student’sttests).

FIG 6P0 can increase viral translation. (A) Exogenous P0 was coexpressed with PVA⌬GDDand FLUC, and luciferase activity and RNA amounts were

assessed at 3 DAI. GUS was coexpressed as a control. TheAgrobacteriumOD600

values used for infiltration were as follows: 0.05 for PVA⌬GDD, 0.005 for FLUC,

and 0.5 for GUS and P0.ⴱⴱ,P0.01 (determined by Student’sttests).

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When wt PVA is infiltrated at a high OD600, VPg increases viral

RLUC activity (40). At an OD600of 0.05, PVA RNA expression is

often initiated from adjacent cells, leaving less space for cell-to-cell spread of infection (37). At a 100-fold-lower OD, the size of infec-tion foci was severely reduced by exogenous VPg expression (Fig. 10D), with the lack of increased wt PVA RLUC activities (Fig. 10B) likely being due to impaired cell-to-cell spread of infection. Exog-enous VPg increased viral RLUC activity when expressed in sys-temically infected leaves (Fig. 9C), showing that this mechanism operates during authentic infection. As the VPg-mediated in-crease of RLUC activity was less than that for PVA⌬GDD,

intercel-lular spread was likely hindered. The synergistic effect of VPg and

P0 coexpression was not detected, which may also be explained by restricted cell-to-cell movement. Finally, we analyzed how the same combinatory expressions affected PVA⌬GDDRLUC activity

(Fig. 10G). Viral RLUC activity was higher when VPg was

coex-FIG 7The 5=-UTR is important for PVA RNA translation and the P0 re-sponse. (A) Exogenous P0 was expressed with PVA⌬GDDor with PVA⌬GDD

lacking either its 5=-UTR (PVA⌬GDD⌬5=UTR) or its 3=-UTR (PVA⌬GDD⌬3=UTR),

and RLUC activities were determined at 3 DAI. GUS was expressed as a con-trol. Results are expressed as relative luciferase activity. TheAgrobacterium

OD600values used for infiltration were as follows: 0.05 for PVAs and 0.5 for

GUS and P0. (B) Exogenous P0 or GUS was expressed with a monocistronic RLUC transcript carrying the PVA 5=-UTR (5=UTRPVA), and RLUC

activ-ities were determined. TheAgrobacteriumOD600values used for

infiltra-tion were as follows: 0.05 for the PVA 5=-UTR and 0.5 for GUS and P0. (C) Actual RLUC activities and RNA levels for PVA⌬GDD, PVA⌬GDD⌬5=UTR, and

PVA⌬GDD⌬3=UTR. (D) Monocistronic RLUC constructs with a reference

leader (5=UTRRef) or the PVA 5=-UTR were transformed using an OD 600of

0.01, and the RLUC activity and RNA amounts were quantified.ⴱ,P⬍0.05;

ⴱⴱ,P0.01 (determined by Student’sttests).

FIG 8VPg requires P0 to enhance viral translation. VPg was coexpressed with PVA⌬GDDduring P-protein silencing, and the RLUC activities were

quantitated at 3 DAI. Results are expressed as relative values. The Agrobac-teriumOD600values used for infiltration were as follows: 0.05 for PVA⌬GDD

and 0.5 for GUS and VPg.ⴱ,P⬍0.05; ⴱⴱ,P⬍0.01 (determined by Student’sttests).

FIG 9P0 and VPg act synergistically to increase PVA translation. (A and B) VPg and/or P0 was coexpressed with PVA⌬GDD(A) and control FLUC (B), and

luciferase activity and RNA levels were quantified at 3 DAI. GUS was expressed as a control. TheAgrobacteriumOD600values used for infiltration were as

follows: 0.05 for PVA⌬GDD, 0.005 for FLUC, and 0.25 for P0 and VPg. Agro-bacteriumcarrying theuidAgene (GUS) was used to adjust all infiltrated sam-ples to the same final OD600. (C) GUS/P0/VPg coexpressions, as in panels A

and B, in leaves systemically infected with RLUC-tagged wt PVA. The lower leaves were inoculated with PVA, and 7 days later, the systematically infected upper leaves were infiltrated withAgrobacteriumcarrying GUS/P0/VPg ex-pression cassettes. Viral RLUC activity was determined 3 days later, corre-sponding to 10 days after PVA inoculation.ⴱ,P⬍0.05;ⴱⴱ,P⬍0.01 (deter-mined by Student’sttests).

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pressed with eIF(iso)4E than when either was expressed alone, indicating that eIF(iso)4E and VPg increased PVA⌬GDDRLUC

activity synergistically. The highest RLUC activity was achieved by coexpressing VPg with both eIF(iso)4E and P0. Control FLUC activity was unaffected under these conditions (Fig. 10H), indicat-ing that viral RNA has specific properties by which it responds to ectopic expression of VPg, P0, and eIF(iso)4E.

DISCUSSION

Virus infection relies on mechanisms that protect viral RNA from being degraded and guarantee efficient production of viral pro-teins in a cellular environment. We have shown here that ribo-somal P0 is important for PVA infection by promoting viral trans-lation along with eIF(iso)4E and the viral protein VPg. P0 functions as part of the ribosomal stalk, with all the acidic P pro-teins that constitute the ribosomal stalk being important for PVA infection. Our findings also suggest that P0 functions as an

extrari-bosomal protein in PVA infection and regulates viral RNA func-tions.

Ribosomal stalk proteins are important for PVA infection. Few previous reports have linked ribosomal stalk proteins to virus infection.Lassa virusZ protein interacts with the nuclear fraction of P0 (47). Assembly of the 60S ribosomal subunit occurs in the nucleus, and upon export to the cytoplasm, the subunit matures through processes including association of the stalk (48). This suggests that Z protein interactions probably did not involve ri-bosomes. L-A virus has been found to propagate to higher levels in yeast strains lacking P1 and P2, with the viral Gag protein being associated with P0 (49). We found that silencing of all P proteins reduced PVA infection substantially. Because translation and rep-lication correlate positively for some animal positive-stranded RNA viruses (50,51), the reduction in PVA infection following P-protein silencing may be due to reduced translation, resulting in reduced replication. Genome-wide RNA interference (RNAi) FIG 10Exogenous VPg increases viral translation with eIF(iso)4E and P0 but reduces spread of infection. (A) P0 and both eIF4E and eIF(iso)4E were silenced by expression of the corresponding RNA hairpins (hpP0 and hp4Es), and the levels of luciferase activity and RNA derived from RLUC-tagged wt PVA were determined at 9 DAI. The empty hairpin vector was expressed as a control (hp⫺). TheAgrobacteriumOD600values used for infiltration were as follows: 0.0005

for wt PVA, 0.4 for hp⫺and hpP0, and 0.2 each for hp4Es [hpeIF4E and hpeIF(iso)4E]. (B to E) eIF(iso)4E was coexpressed with RLUC-tagged wt PVA in different combinations with VPg and/or P0. RLUC activity (B) and the area of infection foci (C) were determined at 3 DAI, and the percentage of GFP-tagged wt PVA-infected tissue at 4 DAI (D) and the number of established GFP-tagged PVA infection foci at 3 DAI (E) were determined. (F) Representative pictures of GFP-tagged wt PVA-infected tissue at 4 DAI during GUS and VPg coexpression. Scale bar, 1 mm. (G and H) Effects of the same coexpressions on PVA⌬GDD(G)

and FLUC (H) luciferase activities. eIF(iso)4E, P0, and VPg each used anAgrobacteriumOD600of 0.25 for infiltration, and GUS was used to adjust all infiltrated

samples to the same final bacterial density. The otherAgrobacteriumOD600values used for infiltration were as follows: 0.0005 for RLUC/GFP-tagged wt PVA, 0.05

for PVA⌬GDD, and 0.005 for FLUC.,P0.05;ⴱⴱ,P0.01 (determined by Student’sttests).

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(53–55). TuMV and TMV (genusTobamovirus) infections were highly reduced by knockdown of several ribosomal proteins (56). The knockdown of RPS2, RPS6, RPL7, RPL13, and RPL19 re-duced the number of TuMV infection foci, regardless of whether the plants were inoculated with virus particles orAgrobacterium. In contrast, P-protein silencing did not reduce the number of infection foci and affected the spread of PVA only slightly. The other ribosomal proteins may therefore have roles distinct from those of the stalk proteins in potyvirus infection. Clearly, ribo-somal protein homeostasis is important for infection by potyvirus as well as other viruses.

Extraribosomal regulation of PVA RNA by P0.Our findings suggest that compared with other P proteins, P0 has additional functions. For example, P0 was a component of viral RNP com-plexes purified via both VPg and RdRp, whereas the other P pro-teins were not, suggesting that the latter were not prominent com-ponents of purified viral RNPs (Fig. 1). These viral RNP complexes may have been derived from RCs, as they were purified from membrane fractions containing potyviral RNA synthesis ac-tivity (57,58). HSP70 is associated with potyviral RCs and the RdRp (19) and was present in our purified samples, together with viral RNA, RdRp, NIa, and VPg (35). Therefore, P0 may associate with viral RNP complexes at the sites of viral RCs, possibly as an extraribosomal protein.

The abundant accumulation of PVACPmutRNA during P0 si-lencing (Fig. 5A) suggests a functional role for P0 in PVA RNA regulation. This accumulation occurred only during silencing of P0 and not during silencing of the other P proteins, suggesting that, independent of stalk function, P0 can affect PVA RNA accu-mulation. Although replicating PVACPmutRNA accumulated in

single cells in the absence of P0, this RNA was not translated effi-ciently (Fig. 5). P0 was required for wt PVA to reach normal levels of infection (Fig. 3D), most likely due to the function of P0 in viral translation. In particular, we found that P0 was the only P protein required for a specific viral translation mechanism involving viral VPg and eIF(iso)4E (Fig. 8to10). Ribosomal proteins have been shown to have extraribosomal functions, e.g., plant L10 in gemi-nivirus infection (59) and S1 associated with bacteriophage Q␤ replicase (7). Extraribosomal P0 is part of the mRNA-regulating human IMP1 and STAU1 mRNP complexes (60, 61) and may function in nucleus-associated DNA repair (62). Taken together with our findings, P0 likely regulates viral RNA via extraribosomal mechanisms.

P0 and eIF(iso)4E are essential for VPg to promote PVA RNA translation.Several potential translational components are asso-ciated with RCs of TuMV, including eIF(iso)4E, PABP, eEF1A, and AtRH8 (16–18, 63), but their roles have remained elusive. They may function to regulate viral translation (64), as translation is coupled with replication (35) and RCs (65) during potyvirus infection. P0 is found in membrane-associated viral RNP com-plexes (Fig. 1) and promotes PVA translation, suggesting that it

(Fig. 8) and that PVA⌬GDDtranslation (Fig. 5B) was less sensitive to reduced P0 amounts than wt PVA infection (Fig. 3D). Higher RNA and VPg levels of replicating viruses may require more P0 and eIF4E/eIF(iso)4E, making their translation more sensitive to silencing of these proteins. P0 transcription is induced early in PVA infection (66), and TuMV infection upregulates eIF4E (67), suggesting that the demand for these proteins is increased during natural potyvirus infection. This was supported by results show-ing that the silencshow-ing of both of these host factors reduced PVA infection similarly (Fig. 10A).

During infection, VPg-linked viral RNA is produced within membrane-associated RCs. Our results using PVA⌬GDDshow that

viral RNA does not have to be produced via replication in authen-tic RCs for VPg, P0, and eIF(iso)4E to efficiently promote its translation. The viral 5=-UTR is an important RNA element by which P0 and VPg increase PVA translation (Fig. 7A) (40). Poty-virus TEV has an IRES element in the 5=-UTR that can recruit ribosomes independently of eIF4Es via direct interactions with eIF4G (54). TEV VPg has been shown to be associated with the eIF4F protein complex consisting of eIF4E and eIF4G, reducing the affinity of eIF4F for the conventional cap structure found in the 5=-UTR of cellular mRNAs and facilitating the translation of transcripts carrying the TEV 5=-UTR (68). The presence or ab-sence of a 5=cap did not alter the ability of VPg to facilitate the translation of TEV 5=-UTR transcripts. By analogy, the capped 5=-UTR of PVA⌬GDDRNA may qualify as a substrate for IRES

translation. Furthermore, as wt PVA RNA responds to exogenous VPg by increased translation in systematically infected leaves (Fig. 9C), it can be concluded that 5=VPg-linked PVA RNA also qual-ifies for VPg-mediated enhancement of PVA RNA translation, suggesting that this mechanism can operate during natural infec-tion. Therefore, the mechanism of the VPg response may involve PVA 5=-UTR-driven IRES translation. Its significance may be re-flected by the reduced infection observed during eIF4E/eIF(iso)4E and P0 silencing. eIF4E and eIF(iso)4E are critical host compo-nents by which potyviruses establish infection, by interacting with VPg (20). Although the mechanisms by which eIF4E and eIF(iso)4E act during potyvirus infection have been hard to deter-mine, our results show that they are essential for VPg to increase PVA translation (40).

Coordination of viral RNA functions. Although VPg en-hanced wt PVA gene expression (Fig. 9C) (40), it restricted the spread of infection (Fig. 10D). Reduced viral spread likely masked the increase in viral gene expression levels during active cell-to-cell movement. A comparison of areas of infection foci (Fig. 10D) with RLUC activity (Fig. 10B) indicated that VPg also enhanced translation under these conditions. Our experiments assessing the VPg-mediated effect on viral translation disturbed the natural stoichiometry of VPg production. Thus, the more PVA RNA is allocated to translation via VPg, the less is available for assembly and movement. Obviously, the correct stoichiometry of viral

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tein products is a prerequisite for a balanced natural infection. P0 and VPg coexpression increased the steady-state amount of PVA⌬GDDRNA (Fig. 9A), indicating that replication was not

nec-essary for RNA accumulation. Because the transcriptions of con-trol FLUC and PVA⌬GDDare both driven by the 35S promoter, the

specific effects of P0 and VPg on PVA⌬GDDRNA translation and accumulation are likely posttranscriptional. The 20- to 100-fold increase in the PVA⌬GDDexpression level is an intriguing example of how extensively posttranscriptional mRNA regulation can af-fect gene expression. VPg may reroute PVA⌬GDDRNA from deg-radation pathways to translation. Although there have been other examples of the interdependence between mRNA translation and degradation (69,70), it needs to be considered whether our results with PVA⌬GDDreflect the mechanisms operating during natural PVA infection. This RNA is derived by nuclear transcription and carries a 5=-cap structure instead of 5=-linked VPg present in the authentic viral RNA. The specific effects observed for PVA⌬GDD

compared with our nonviral FLUC control (Fig. 10GandH) sug-gest that PVA⌬GDDRNA has virus-specific features that mediate

gene expression responses that are at least somewhat similar to those observed with authentic viral RNA (Fig. 9Cand10B) (40). Interestingly, we observed that PVA RNA induces the formation of P0-containing granule structures (A. Hafrén and K. Mäkinen, unpublished results). Similar to wt PVA, PVA⌬GDDRNA also in-duced P0- but not P1- or P2-containing granule-like structures in plant cells, whereas the nonviral control FLUC did not. These granules may represent structures associated with viral RNA me-tabolism, similar to the P bodies and stress granules described, for example, for virus-infected animal cells (71).

Finally, it is noteworthy that silencing of P0 caused abundant accumulation of PVACPmutRNA. We are presently unable to

dis-tinguish between the contributions of RNA synthesis via replica-tion and RNA degradareplica-tion to the steady-state PVA RNA level. Moreover, mutant CP may contribute to the different outcomes of P0 silencing for PVACPmutand wt PVA. For example, P0 may be

an essential host factor required for coordinating PVA RNA func-tions among translation, replication, degradation, and assembly/ movement pathways. In summary, we hypothesize that (i) P0 is an essential host component required to achieve a normal strength of infection and (ii) P0 regulates PVA RNA translation together with VPg and eIF(iso)4E.

ACKNOWLEDGMENTS

We thank the CSIRO for kindly providing the pHELLSGATE8 silencing plasmid.

We gratefully acknowledge financial support from the Academy of Finland (grant no. 115922 and 1138329 to K.M. and grant no. 127969 to K.E.).

REFERENCES

1.Bushell M, Sarnow P.2002. Hijacking the translation apparatus by RNA viruses. J. Cell Biol.158:395–399.

2.Dreher TW, Miller WA.2006. Translational control in positive strand RNA plant viruses. Virology344:185–197.

3.Lloyd RE.2006. Translational control by viral proteinases. Virus Res. 119:76 – 88.

4.Thompson SR, Sarnow P.2003. Enterovirus 71 contains a type I IRES element that functions when eukaryotic initiation factor eIF4G is cleaved. Virology315:259 –266.

5.Park HS, Himmelbach A, Browning KS, Hohn T, Ryabova LA.2001. A plant viral “reinitiation” factor interacts with the host translational ma-chinery. Cell106:723–733.

6.Ahlquist P, Noueiry AO, Lee WM, Kushner DB, Dye BT.2003. Host factors in positive-strand RNA virus genome replication. J. Virol.77: 8181– 8186.

7.Blumenthal T, Carmichael GG.1979. RNA replication: function and structure of Qbeta-replicase. Annu. Rev. Biochem.48:525–548. 8.Li Z, Pogany J, Tupman S, Esposito AM, Kinzy TG, Nagy PD.2010.

Translation elongation factor 1A facilitates the assembly of the tombusvi-rus replicase and stimulates minus-strand synthesis. PLoS Pathog. 6:e1001175. doi:10.1371/journal.ppat.1001175.

9.Osman TA, Buck KW.1997. The tobacco mosaic virus RNA polymerase complex contains a plant protein related to the RNA-binding subunit of yeast eIF-3. J. Virol.71:6075– 6082.

10. Quadt R, Kao CC, Browning KS, Hershberger RP, Ahlquist P.1993. Characterization of a host protein associated with brome mosaic virus RNA-dependent RNA polymerase. Proc. Natl. Acad. Sci. U. S. A.90:1498 – 1502.

11. Taylor DN, Carr JP.2000. The GCD10 subunit of yeast eIF-3 binds the methyltransferase-like domain of the 126 and 183 kDa replicase proteins of tobacco mosaic virus in the yeast two-hybrid system. J. Gen. Virol. 81:1587–1591.

12. Mizumoto H, Iwakawa HO, Kaido M, Mise K, Okuno T.2006. Cap-independent translation mechanism of red clover necrotic mosaic virus RNA2 differs from that of RNA1 and is linked to RNA replication. J. Virol. 80:3781–3791.

13. Sanz MA, Castello A, Carrasco L.2007. Viral translation is coupled to transcription in Sindbis virus-infected cells. J. Virol.81:7061–7068. 14. Sanz MA, Castello A, Ventoso I, Berlanga JJ, Carrasco L.2009. Dual

mechanism for the translation of subgenomic mRNA from Sindbis virus in infected and uninfected cells. PLoS One4:e4772. doi:10.1371/journal .pone.0004772.

15. Miller S, Krijnse-Locker J.2008. Modification of intracellular membrane structures for virus replication. Nat. Rev. Microbiol.6:363–374. 16. Beauchemin C, Boutet N, Laliberte JF.2007. Visualization of the

inter-action between the precursors of VPg, the viral protein linked to the ge-nome of turnip mosaic virus, and the translation eukaryotic initiation factor iso 4E in planta. J. Virol.81:775–782.

17. Beauchemin C, Laliberte JF.2007. The poly(A) binding protein is inter-nalized in virus-induced vesicles or redistributed to the nucleolus during turnip mosaic virus infection. J. Virol.81:10905–10913.

18. Thivierge K, Cotton S, Dufresne PJ, Mathieu I, Beauchemin C, Ide C, Fortin MG, Laliberte JF.2008. Eukaryotic elongation factor 1A interacts with turnip mosaic virus RNA-dependent RNA polymerase and VPg-Pro in virus-induced vesicles. Virology377:216 –225.

19. Dufresne PJ, Thivierge K, Cotton S, Beauchemin C, Ide C, Ubalijoro E, Laliberte JF, Fortin MG.2008. Heat shock 70 protein interaction with turnip mosaic virus RNA-dependent RNA polymerase within virus-induced membrane vesicles. Virology374:217–227.

20. Wang A, Krishnaswamy S.2012. Eukaryotic translation initiation factor 4E-mediated recessive resistance to plant viruses and its utility in crop improvement. Mol. Plant Pathol.13:795– 803.

21. Gonzalo P, Reboud JP.2003. The puzzling lateral flexible stalk of the ribosome. Biol. Cell95:179 –193.

22. Wool IG, Chan YL, Gluck A, Suzuki K.1991. The primary structure of rat ribosomal proteins P0, P1, and P2 and a proposal for a uniform no-menclature for mammalian and yeast ribosomal proteins. Biochimie73: 861– 870.

23. Bailey-Serres J, Vangala S, Szick K, Lee CH.1997. Acidic phosphopro-tein complex of the 60S ribosomal subunit of maize seedling roots. Com-ponents and changes in response to flooding. Plant Physiol.114:1293– 1305.

24. Szick K, Springer M, Bailey-Serres J.1998. Evolutionary analyses of the 12-kDa acidic ribosomal P-proteins reveal a distinct protein of higher plant ribosomes. Proc. Natl. Acad. Sci. U. S. A.95:2378 –2383.

25. Sanchez-Madrid F, Vidales FJ, Ballesta JP.1981. Effect of phosphoryla-tion on the affinity of acidic proteins from Saccharomyces cerevisiae for the ribosomes. Eur. J. Biochem.114:609 – 613.

26. Tsurugi K, Ogata K.1985. Evidence for the exchangeability of acidic ribosomal proteins on cytoplasmic ribosomes in regenerating rat liver. J. Biochem.98:1427–1431.

27. Zinker S, Warner JR.1976. The ribosomal proteins of Saccharomyces cerevisiae. Phosphorylated and exchangeable proteins. J. Biol. Chem.251: 1799 –1807.

28. Aruna K, Chakraborty T, Rao PN, Santos C, Ballesta JP, Sharma S.

on November 7, 2019 by guest

http://jvi.asm.org/

(11)

are not required for cell viability but regulate the pattern of protein ex-pression in Saccharomyces cerevisiae. Mol. Cell. Biol.15:4754 – 4762. 32. Remacha M, Jimenez-Diaz A, Santos C, Briones E, Zambrano R,

Ro-driguez Gabriel MA, Guarinos E, Ballesta JP.1995. Proteins P1, P2, and P0, components of the eukaryotic ribosome stalk. New structural and functional aspects. Biochem. Cell Biol.73:959 –968.

33. Huang C, Mandava CS, Sanyal S.2010. The ribosomal stalk plays a key role in IF2-mediated association of the ribosomal subunits. J. Mol. Biol. 399:145–153.

34. Martinez-Azorin F, Remacha M, Ballesta JP.2008. Functional charac-terization of ribosomal P1/P2 proteins in human cells. Biochem. J.413: 527–534.

35. Hafren A, Hofius D, Ronnholm G, Sonnewald U, Makinen K.2010. HSP70 and its cochaperone CPIP promote potyvirus infection in Nicoti-ana benthamiNicoti-ana by regulating viral coat protein functions. Plant Cell 22:523–535.

36. Williams AJ, Werner-Fraczek J, Chang IF, Bailey-Serres J.2003. Regu-lated phosphorylation of 40S ribosomal protein S6 in root tips of maize. Plant Physiol.132:2086 –2097.

37. Eskelin K, Suntio T, Hyvarinen S, Hafren A, Makinen K.2010. Renilla luciferase-based quantitation of potato virus A infection initiated with Agrobacterium infiltration of N. benthamiana leaves. J. Virol. Methods 164:101–110.

38. Curtis MD, Grossniklaus U.2003. A gateway cloning vector set for high-throughput functional analysis of genes in planta. Plant Physiol.133:462– 469.

39. Nakagawa T, Kurose T, Hino T, Tanaka K, Kawamukai M, Niwa Y, Toyooka K, Matsuoka K, Jinbo T, Kimura T.2007. Development of series of gateway binary vectors, pGWBs, for realizing efficient con-struction of fusion genes for plant transformation. J. Biosci. Bioeng. 104:34 – 41.

40. Eskelin K, Hafren A, Rantalainen KI, Makinen K.2011. Potyviral VPg enhances viral RNA translation and inhibits reporter mRNA translation in planta. J. Virol.85:9210 –9221.

41. Helliwell C, Waterhouse P.2003. Constructs and methods for high-throughput gene silencing in plants. Methods30:289 –295.

42. Johansen LK, Carrington JC.2001. Silencing on the spot. Induction and suppression of RNA silencing in the Agrobacterium-mediated transient expression system. Plant Physiol.126:930 –938.

43. Elkon K, Skelly S, Parnassa A, Moller W, Danho W, Weissbach H, Brot N.1986. Identification and chemical synthesis of a ribosomal protein antigenic determinant in systemic lupus erythematosus. Proc. Natl. Acad. Sci. U. S. A.83:7419 –7423.

44. Szick-Miranda K, Bailey-Serres J.2001. Regulated heterogeneity in 12-kDa P-protein phosphorylation and composition of ribosomes in maize (Zea mays L.). J. Biol. Chem.276:10921–10928.

45. Santos C, Ballesta JP.1994. Ribosomal protein P0, contrary to phospho-proteins P1 and P2, is required for ribosome activity and Saccharomyces cerevisiae viability. J. Biol. Chem.269:15689 –15696.

46. Dunoyer P, Thomas C, Harrison S, Revers F, Maule A.2004. A cysteine-rich plant protein potentiates potyvirus movement through an interaction with the virus genome-linked protein VPg. J. Virol.78:2301–2309. 47. Borden KL, CampbellDwyer EJ, Carlile GW, Djavani M, Salvato MS.

1998. Two RING finger proteins, the oncoprotein PML and the arenavirus Z protein, colocalize with the nuclear fraction of the ribosomal P proteins. J. Virol.72:3819 –3826.

48. Lo KY, Li Z, Bussiere C, Bresson S, Marcotte EM, Johnson AW.2010. Defining the pathway of cytoplasmic maturation of the 60S ribosomal subunit. Mol. Cell39:196 –208.

49. Krokowski D, Tchorzewski M, Boguszewska A, McKay AR, Maslen SL, Robinson CV, Grankowski N.2007. Elevated copy number of L-A virus

Dev.19:445– 452.

53. Basso J, Dallaire P, Charest PJ, Devantier Y, Laliberte JF.1994. Evidence for an internal ribosome entry site within the 5=non-translated region of turnip mosaic potyvirus RNA. J. Gen. Virol.75(Part 11):3157–3165. 54. Gallie DR.2001. Cap-independent translation conferred by the 5=leader

of tobacco etch virus is eukaryotic initiation factor 4G dependent. J. Virol. 75:12141–12152.

55. Niepel M, Gallie DR.1999. Identification and characterization of the functional elements within the tobacco etch virus 5=leader required for cap-independent translation. J. Virol.73:9080 –9088.

56. Yang C, Zhang C, Dittman JD, Whitham SA.2009. Differential require-ment of ribosomal protein S6 by plant RNA viruses with different trans-lation initiation strategies. Virology390:163–173.

57. Martin MT, Garcia JA.1991. Plum pox potyvirus RNA replication in a crude membrane fraction from infected Nicotiana clevelandii leaves. J. Gen. Virol.72(Part 4):785–790.

58. Schaad MC, Jensen PE, Carrington JC.1997. Formation of plant RNA virus replication complexes on membranes: role of an endoplasmic retic-ulum-targeted viral protein. EMBO J.16:4049 – 4059.

59. Carvalho CM, Santos AA, Pires SR, Rocha CS, Saraiva DI, Machado JP, Mattos EC, Fietto LG, Fontes EP.2008. Regulated nuclear trafficking of rpL10A mediated by NIK1 represents a defense strategy of plant cells against virus. PLoS Pathog.4:e1000247. doi:10.1371/journal.ppat.1000247. 60. Brendel C, Rehbein M, Kreienkamp HJ, Buck F, Richter D, Kindler S.

2004. Characterization of Staufen 1 ribonucleoprotein complexes. Biochem. J.384:239 –246.

61. Jonson L, Vikesaa J, Krogh A, Nielsen LK, Hansen T, Borup R, Johnsen AH, Christiansen J, Nielsen FC.2007. Molecular composition of IMP1 ribonucleoprotein granules. Mol. Cell. Proteomics6:798 – 811. 62. Yacoub A, Kelley MR, Deutsch WA.1996. Drosophila ribosomal protein

PO contains apurinic/apyrimidinic endonuclease activity. Nucleic Acids Res.24:4298 – 4303.

63. Huang TS, Wei T, Laliberte JF, Wang A.2010. A host RNA helicase-like protein, AtRH8, interacts with the potyviral genome-linked protein, VPg, associates with the virus accumulation complex, and is essential for infec-tion. Plant Physiol.152:255–266.

64. Grangeon R, Cotton S, Laliberte JF.2010. A model for the biogenesis of turnip mosaic virus replication factories. Commun. Integr. Biol.3:363–365. 65. Cotton S, Grangeon R, Thivierge K, Mathieu I, Ide C, Wei T, Wang A,

Laliberte JF.2009. Turnip mosaic virus RNA replication complex vesicles are mobile, align with microfilaments, and are each derived from a single viral genome. J. Virol.83:10460 –10471.

66. Vuorinen AL, Gammelgard E, Auvinen P, Somervuo P, Dere S, Valkonen JPT.2010. Factors underpinning the responsiveness and higher levels of virus resistance realised in potato genotypes carrying virus-specific R genes. Ann. Appl. Biol.157:229 –241.

67. Leonard S, Viel C, Beauchemin C, Daigneault N, Fortin MG, Laliberte JF.2004. Interaction of VPg-Pro of turnip mosaic virus with the transla-tion initiatransla-tion factor 4E and the poly(A)-binding protein in planta. J. Gen. Virol.85:1055–1063.

68. Khan MA, Miyoshi H, Gallie DR, Goss DJ.2008. Potyvirus genome-linked protein, VPg, directly affects wheat germ in vitro translation: inter-actions with translation initiation factors eIF4F and eIFiso4F. J. Biol. Chem.283:1340 –1349.

69. Coller J, Parker R. 2004. Eukaryotic mRNA decapping. Annu. Rev. Biochem.73:861– 890.

70. Jacobson A, Peltz SW.1996. Interrelationships of the pathways of mRNA decay and translation in eukaryotic cells. Annu. Rev. Biochem.65:693– 739.

71. Beckham CJ, Parker R.2008. P bodies, stress granules, and viral life cycles. Cell Host Microbe3:206 –212.

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Figure

FIG 1 Ribosomal P0 is a component of membrane-associated viral RNPs. (A)Western blotting showing the presence of P proteins in the RNP complexesobtained by affinity purification, using Strep III-tagged PVA RdRp (RdRpSIII)and Strep III-tagged VPg (VPgSIII), f
FIG 3 Ribosomal P0 promotes PVA infection. (A) P0 was silenced by expressing an RNA hairpin (hp) that targets P0 transcripts (hpP0), followed by Western(B) Exogenous P0 was expressed, and P0 levels were analyzed by Western blotting as described above for p
FIG 4 Ribosomal P proteins are important for PVA infection. (A) RLUC activity of wt PVA was determined at 3, 6, and 9 DAI during silencing of P proteins by
FIG 6 P0 can increase viral translation. (A) Exogenous P0 was coexpressedwith PVAand 0.5 for GUS and P0.�GDD and FLUC, and luciferase activity and RNA amounts wereassessed at 3 DAI
+3

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A content analysis of the five most popular scripted broadcast television programs for each year was conducted in order to assess the frequency of nerd characters, as well as

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The steganalytic algorithm using reversible texture synthesis Mechanism is used to extract the source texture from original image .this also extracts the