Genome Replication and Regulation of Viral Gene Expression

Differential Roles of Hsp70 and Hsp90 in the Assembly of the Replicase Complex of a Positive-Strand RNA Plant Virus

Akira Mine, Kiwamu Hyodo, Yuri Tajima, Kusumawaty Kusumanegara, Takako Taniguchi, Masanori Kaido, Kazuyuki Mise, Hisaaki Taniguchi, Tetsuro Okuno
DOI: 10.1128/JVI.01659-12

ABSTRACT

Assembly of viral replicase complexes of eukaryotic positive-strand RNA viruses is a regulated process: multiple viral and host components must be assembled on intracellular membranes and ordered into quaternary complexes capable of synthesizing viral RNAs. However, the molecular mechanisms underlying this process are poorly understood. In this study, we used a model virus, Red clover necrotic mosaic virus (RCNMV), whose replicase complex can be detected readily as the 480-kDa functional protein complex. We found that host heat shock proteins Hsp70 and Hsp90 are required for RCNMV RNA replication and that they interact with p27, a virus-encoded component of the 480-kDa replicase complex, on the endoplasmic reticulum membrane. Using a cell-free viral translation/replication system in combination with specific inhibitors of Hsp70 and Hsp90, we found that inhibition of p27-Hsp70 interaction inhibits the formation of the 480-kDa complex but instead induces the accumulation of large complexes that are nonfunctional in viral RNA synthesis. In contrast, inhibition of p27-Hsp90 interaction did not induce such large complexes but rendered p27 incapable of binding to a specific viral RNA element, which is a critical step for the assembly of the 480-kDa replicase complex and viral RNA replication. Together, our results suggest that Hsp70 and Hsp90 regulate different steps in the assembly of the RCNMV replicase complex.

INTRODUCTION

Most plant and animal viruses are positive-strand RNA viruses, which have single-stranded messenger-sense genomic RNAs. These viruses often induce host membrane rearrangements to form organelle-like compartments in which viral genomic RNAs are replicated via negative-strand RNA intermediates by the viral replicase complexes (10). Viral replicase complexes comprise multiple proteins, including viral auxiliary proteins, viral RNA-dependent RNA polymerase (RdRP), and host proteins (61). Viral replicase complexes have been studied extensively by characterizing their RdRP activities and the functions of the viral and host components of the complexes. These studies have provided important information about the mechanisms regulating genome replication (15, 19, 47, 89), viral pathogenicity (68, 69), and virus-host interactions (24, 25, 32, 33). However, an important question remains: how do multiple viral and host components assemble properly into the replicase complex?

Molecular chaperones are essential for cell viability by ensuring folding of newly synthesized proteins, refolding of misfolded or aggregated proteins, protein complex assembly and disassembly, membrane translocation of organellar and secretory proteins, protein degradation, and activities of regulatory proteins in signal transduction pathways (12, 18, 51). In eukaryotic cells, the abundant and highly conserved molecular chaperones heat shock proteins Hsp70 and Hsp90 play central roles in the biological processes mentioned above, and the activities of Hsp70 and Hsp90 are modulated by a variety of cochaperones (37, 80). Considering their pivotal roles in cells, it is not surprising that Hsp70 and Hsp90 are involved together with their cochaperones in virus infection (62). For instance, Hsp70 facilitates the assembly and disassembly of viral capsids (7, 26, 46), promotes the subcellular transport of tombusvirus replicase proteins and affects the activity or assembly of tombusvirus replicase complexes (71, 90), controls potyvirus gene expression in cooperation with its cochaperone CPIP (17), and positively and negatively affects the genome replication of various viruses (5, 45, 91). Hsp90 affects the early stages of Bamboo mosaic virus (BaMV) infection by binding to the genomic RNA (20), increases the synthesis or stability of viral proteins (4, 8), supports the assembly and nuclear import of influenza A virus RNA polymerase complex (59, 63), and tightly regulates hepatitis C virus replication in cooperation with FKBP8 and hB-ind1 cochaperones (67, 79, 88).

Hsp70 and Hsp90 sometimes work together in the activation or maturation of viral and cellular proteins. For example, Hsp90 together with Hsp70 and a variety of cochaperones regulate the actions of steroid receptors and the responses to ligands (16). It has been reported recently that Hsp70, Hsp90, and their cochaperones facilitate the incorporation of small RNAs into Argonaute proteins, which play central roles in posttranscriptional gene silencing (22, 23, 31, 55). In the case of hepadnavirus reverse transcriptase, Hsp70 and Hsp40 cochaperones are essential for the specific binding of the reverse transcriptase to pregenomic RNA templates, and Hsp90 facilitates this step (77, 78). However, the coordinate functions of these molecular chaperones in other biological processes such as multicomponent complex assembly are poorly understood.

To elucidate the molecular mechanisms of the replication of positive-strand RNA viruses, we used Red clover necrotic mosaic virus (RCNMV) as a model. RCNMV is a positive-strand RNA plant virus and a member of the genus Dianthovirus in the family Tombusviridae. The bipartite genomic RNAs, RNA1 and RNA2, possess neither a cap structure at the 5′ end (58) nor a poly(A) tail at the 3′ end (48, 95). Instead, RNA1 and RNA2 use distinct cap/poly(A)-independent mechanisms to produce all viral proteins (30, 57, 58, 74). RNA1 encodes N-terminally overlapping replicase component proteins, a 27-kDa auxiliary protein (p27), and an 88-kDa protein with an RdRP motif (p88). p88 is produced via programmed −1 position ribosomal frameshifting (81, 94). RNA1 also encodes a coat protein (CP), which is translated from CP subgenomic RNA (76, 86). A small noncoding RNA (SR1f) with a potential function in regulating RCNMV infection is generated from RNA1. RNA2 encodes a movement protein that is required for viral movement in plants (34, 36, 93).

We have developed a model to study the assembly processes of viral replicase complexes by detecting the RCNMV replicase complex as a functional protein complex with an apparent molecular mass of 480 kDa using blue native polyacrylamide gel electrophoresis (BN-PAGE) (53). The 480-kDa replicase complexes are tightly associated with the membrane of the endoplasmic reticulum (ER) and retain the ability to synthesize complementary RNAs by specifically recognizing RCNMV RNAs (41, 53). The assembly of the 480-kDa replicase complex requires both p27-p27 and p27-p88 interactions (52). In addition to p27 and p88, the 480-kDa complexes contain several host proteins, whereas p27 and p88 seem to interact with both the host components of the 480-kDa complex and many other host proteins, including chaperones, ribosomal proteins, and cytoskeletal proteins, which are absent in the complex (53).

The functions of p27 and p88 in viral RNA replication have been investigated. Both p27 and p88 interact with RNA1 in a translation-coupled manner (27), and these interactions are important for the cis-preferential replication of RNA1 (66). p27 but not p88 binds specifically to the Y-shaped RNA element (YRE) of RNA2 in trans (27), and this interaction is essential for the recruitment of RNA2 into replication (1, 21). In contrast, the functions of host proteins in RCNMV RNA replication are currently unknown.

In this study, we investigated the functions of two host molecular chaperones, Hsp70 and Hsp90, in RCNMV RNA replication. Gene silencing and pharmacological inhibition of Hsp70 and Hsp90 revealed that these molecular chaperones are required for RCNMV RNA replication. A series of in vivo and in vitro protein interaction experiments showed that both Hsp70 and Hsp90 interact with p27 via protein-protein contacts on the ER membrane. Further studies using a cell-free viral translation/replication system showed that when p27-Hsp70 interaction is blocked, p27 forms large complexes that are nonfunctional in viral RNA synthesis. In contrast, in the absence of p27-Hsp90 interaction, p27 was unable to bind to a viral RNA element, such as YRE, which is a critical step for the assembly of the 480-kDa replicase complexes. These results provide strong evidence that Hsp70 and Hsp90 have different functions in regulating the assembly of the RCNMV replicase complex.

MATERIALS AND METHODS

Molecular cloning and plasmid construction.pUCR1 (84) and pRC2|G (96) are full-length cDNA clones of RNA1 and RNA2 of the RCNMV Australian strain, respectively. pBI_C-CY, pBI_C-NY, pBI_N-NY, and pBI_N-CY were kind gifts from Takashi Araki (Kyoto University, Kyoto, Japan). Constructs described previously that were used in this study include pBICp35 (83), pBICp19 (83), pBICB3aGFP (35), pBICRMsG (36), pBICER:mCherry (34), pBICRC1 (84), pUBRC1 (84), pBICRC2 (84), pUBRC2 (84), pUBp35 (83), pUBp88 (84), pBICp27 (84), pBICp88 (84), pBICp27-FLAG (53), pBICp27-HA (53), pUCp27-FLAG (52), pUCp88-T7 (52), pBYL2 (53), pBINTRA6 (73), pTV00 (73), pPVX.NbHsp70c-1 (38), and pBE2113-GUS (54). pET42a and pUC118 were purchased from TaKaRa Bio Inc. (Shiga, Japan). Escherichia coli DH5α was used for the construction of all plasmids, except that E. coli Top10 (Invitrogen, Carlsbad, CA) was used for the construction of pBYLHsp70. All plasmids constructed in this study were verified by sequencing.

(i) pBYLHsp70 and pBYLHsp90.To isolate tobacco BY-2 Hsp70 and Hsp90 cDNA fragments by reverse transcription-PCR (RT-PCR), we used two sets of degenerate primers, Hsp70deg-F (5′-AARAAYCARGTNGCNATGAA-3′) plus Hsp70deg-R (5′-CATNCGYTCDATYTCYTCYTT-3′) and Hsp90deg-F (5′-AAGGCGCGCCATGGCGGABRCAGAGACGTTT-3′) plus Hsp90deg-R (5′-AAGGCGCGCCTTAGTCAACYTCCTCCATCTT-3′), respectively. Both 5′ rapid amplification of cDNA ends (5′-RACE) and 3′-RACE techniques were carried out to identify both ends. Full-length Hsp70 and Hsp90 cDNAs were amplified with RT-PCR using the primer pair sets AscI/Hsp70-F (5′-AACCGGTTGGCGCGCCATGGCAGGAAAAGGTGAAGG-3′) plus AscI/Hsp70-R (5′-AACCGGTTGGCGCGCCAACACCAACAGCTTAGTC-3′) and AscI/Hsp90-F (5′-AAGGCGCGCCATGGCGGACACAGAGACGTTTGC-3′) plus AscI/Hsp90-R (5′-AAGGCGCGCCTTAGTCAACCTCCTCCATCTTGC3′), respectively. The amplified DNAs were digested with AscI and inserted into pBYL2 that had been cut with the same restriction enzyme.

(ii) pTVHsp70.The partial fragment of NbHsp70c-1 was amplified from pPVX.NbHsp70c-1 (38) using SmaI/70-F (5′-GGGGGGCCCGGGTAACGAGAAGGTGCAGG-3′) and BamHI/70-R (5′-CGCGGATCCATTGGCGTCGATGTCAAAG-3′). The amplified DNA was digested with SmaI and BamHI and inserted into pTV00 that had been cut with the same restriction enzymes.

(iii) pBICAscII.An AscI linker (5′-GATCTGGCGCGCC-3′) was treated with T4 polynucleotide kinase (New England Biolabs, Ipswich, MA), followed by annealing. The annealed linker was then inserted into pBICp35, which had been digested with BamHI.

(iv) pBICHsp70.The Hsp70 sequence was amplified from pBYLHsp70 using BamHI/Hsp70-F (5′-ACGGGGATCCAAGGAGATATAACAATGGCAGGAAAAGGTGAAGGA-3′) and BamHI-Hsp70-R (5′-ACGGGGATCCTTAGTCGACCCTCAATC-3′), digested with BamHI, and inserted into pBICp35 that had been cut with the same restriction enzyme.

(v) pBICHsp90.pBYLHsp90 was cut with AscI, and then the resulting 2.1-kb fragment was ligated with AscI-digested pBICAscII.

(vi) pBICHA:cYFP and pBICmyc:nYFP.The sequence of hemagglutinin (HA)-tagged yellow fluorescent protein C-terminal fragment (cYFP) or myc-tagged yellow fluorescent protein N-terminal fragment (nYFP) was amplified from pBICp27-HA:cYFP using StuI-HA/cYFP-F (5′-GAGAGGCCTACGGGGATCCAAGGAGATATAACAATGTACCCATACGATGTTCC-3′) and KpnI-HA/cYFP-R or from pBICp27-myc:nYFP using StuI-myc/nYFP-F (5′-GGAGAGGCCTACGGGGATCCAAGGAGATATAACAATGGAGCAGAAGCTGATCAGC-3′) and KpnI-myc/nYFP-R, respectively. The amplified DNA fragments were digested with StuI and KpnI and inserted into StuI/KpnI-digested pBICp35 to construct pBICHA:cYFP and pBICmyc:nYFP.

(vii) pBICHA:cYFP-Hsp70 and pBICHA:cYFP-Hsp90.The sequence of HA-tagged cYFP was amplified from pBICHA:cYFP using StuI-HA/cYFP-F and StuI-HA/cYFP-R (5′-GTAGGCCTCTTGTACAGCTCGTCCATGCCGAG-3′). The amplified DNA was digested with StuI and cloned into StuI-digested pBICHsp70 and pBICHsp90 to construct pBICHA-cYFP:Hsp70 and pBICHA-cYFP:Hsp90.

(viii) pBICmyc:nYFP-Hsp70 and pBICmyc:nYFP-Hsp90.The sequence of myc-tagged nYFP was amplified from pBICmyc-nYFP using StuI-myc/nYFP-F and StuI-myc/nYFP-R (5′-GTAGGCCTGGCCATGATATAGACGTTGTGG-3′), respectively. The amplified DNA was digested with StuI and cloned into StuI-digested pBICHsp70 to construct pBICmyc:nYFP-Hsp70 and pBICmyc:nYFP-Hsp90.

(ix) pBICp27-HA:cYFP.The sequence of the C-terminal half of YFP was amplified from pBI_C-CY using HA/cYFP-F (5′-TACCCATACGATGTTCCTTACTTGTACAGCTCGTCCATG-3′) and KpnI-HA/cYFP-R (5′-AGCGGGGTACCTTACTTGTACAGCTCGTCCATG-3′). A PCR fragment was then amplified from pBICp27 using p27-22R (5′-AGCAGATGGAACGTGTAG-3′) and HA/p27-R (5′-AGCGTAATCTGGAACATCGTATGGGTAAAAATCCTCAAGGGATTTGA-3′). A recombinant PCR product was then amplified from the mixture of these fragments using p27-22R and KpnI-HA/cYFP-R, digested with EcoRI and KpnI, and inserted into the corresponding region of pBICp27.

(x) pBICp27-myc:nYFP.The sequence of the N-terminal half of YFP was amplified from pBI_C-NY using myc/nYFP-F (5′-GAGCAGAAGCTGATCAGCGAGGAGGACCTGGCCGGTGGTGGAGGAGCCGGC-3′)and KpnI-myc/nYFP-R (5′-AGCGGGGTACCTTAGGCCATGATATAGACGTTG-3′). A PCR fragment was then amplified from pBICp27 using p27-22R and myc/p27-R (5′-CAGGTCCTCCTCGCTGATCAGCTTCTGCTCAAAATCCTCAAGGGATTTGA-3′). A recombinant PCR product was then amplified from the mixture of these fragments using p27-22R and KpnI-myc/nYFP-R, digested with EcoRI and KpnI, and inserted into the corresponding region of pBICp27.

(xi) pBICp88-HA:cYFP.The sequence of the C-terminal half of YFP was amplified from pBI_C-CY using HA/cYFP-F and KpnI/HA/cYFP-R, and a PCR fragment was amplified from pBICp88 using p88-167R (5′-AGTGCGAGCTTCGTTGG-3′) and HA/p88-R (5′-AGCGTAATCTGGAACATCGTATGGGTATCGGGCTTTGATTAGATCTTTG-3′). A recombinant PCR product was then amplified from the mixture of these fragments using p88-167R and KpnI-HA/cYFP-R, digested with XhoI and KpnI, and inserted into the corresponding region of pBICp88.

(xii) pBICp88-myc:nYFP.The sequence of the N-terminal half of YFP was amplified from pBI_C-NY using myc:nYFP-F and KpnI-myc/nYFP-R, and a PCR fragment was amplified from pBICp88 using p88-167R and myc/p88-R (5′-CAGGTCCTCCTCGCTGATCAGCTTCTGCTCTCGGGCTTTGATTAGATCTTTG-3′). A recombinant PCR product was then amplified from the mixture of these fragments using p88-167R and KpnI-myc/nYFP-R, digested with XhoI and KpnI, and inserted into the corresponding region of pBICp88.

(xiii) pBICsGFP-Hsp70 and pBICsGFP-Hsp90.The green fluorescent protein (GFP) sequence was amplified from pBICRMsG using StuI-sGFP-F (5′-GGAGAGGCCTACGGGGATCCAAGGAGATATAACAATGGTGAGCAAGGGCGAGGAGCTG-3′) and StuI-sGFP-R (5′-CCGTAGGCCTCTTGTACAGCTCGTCCATG-3′), digested with StuI and inserted into StuI-digested pBICHsp70 and pBICHsp90 to construct pBICsGFP-Hsp70 and pBICsGFP-Hsp90, respectively.

(xiv) pBICDRm-p27.A PCR fragment from pDsRed-monomer-actin was amplified using Bam/DRm-F (5′-GGGGATCCGGATGGACAACACCGAGGACGTCATC-3′) and p27/DRm-R (5′-ATTTATAAAACCCATGCCCCCCTGGGAGCCGGAGTGGCGG-3′), and a PCR fragment from pUCR1 was amplified using DRm/p27-F (5′-CACTCCGGCTCCCAGGGGGGCATGGGTTTTATAAATCTTT-3′) and Kpn/p27-R (5′-GGGGTACCCTAAAAATCCTCAAGGGATTT-3′). A recombinant PCR product was then amplified from the mixture of these two fragments by the use of Bam/DRm-F and Kpn/p27-R, digested with BamHI and KpnI, and inserted into the corresponding region of pBICp35.

(xv) pBICp27-DRm.A PCR fragment from pBICp27 was amplified using p27-47R (5′-AGATGACATGGGAAAGG-3′) and DRm-p27-R (5′-CCATGCCCCCAAAATCCTCAAGGGATTTGA-3′), and a PCR fragment from pBICER-DRm (35) was amplified using p27-DRm-F (5′-TTTTGGGGGCATGGACAACACCGAGGACGT-3′) and KpnI-DRm-R (5′-CGGGGTACCCTACTGGGAGCCGGAGTGGCGGG-3′). A recombinant PCR product was then amplified from the mixture of these two fragments by the use of p27-47R and KpnI-DRm-R, digested with EcoRI and KpnI, and inserted into the corresponding region of pBICp27.

(xvi) pUEGFP2.pBE2113-GUS (54) was cut with SacI, treated with T4 DNA polymerase, cut with SmaI, and self-ligated to eliminate the β-glucuronidase (GUS) gene. The small HindIII/EcoRI fragment of the resultant plasmid was inserted into the HindIII/EcoRI site of pUC118 (TaKaRa Bio), creating pUC2113. A GFP gene was PCR amplified from pBICB3aGFP (35) using the Xba/5LGFP5′ primer (5′-GGTGGCTCTAGAAAGGAGATATAACAATGAGTAAAGGAGAAGAACT-3′) and the Bam/GFP3′ primer (5′-GGGGGGATCCTTATTTGTATAGTTCATCC-3′). The amplified fragment was cut with XbaI and BamHI and inserted into the XbaI/BamHI site of pUC2113, creating pUEGFP2.

(xvii) pUBp27-HA.A PCR fragment was amplified from pBICp27-HA using BamHI-p27-F (5′-GGAGAGGCCTACTCTAGAGGATCCGGATGGGTTTTATAAATCTT-3′) and KpnI-p27-HA-R (5′-TTCAGCGGGGTACCCTAAGCGTAATCTGGAACATCGTATGGGTA-3′), digested with BamHI and KpnI, and inserted into corresponding region of pUBp35.

(xviii) pUBp88-HA.The sequence of p88 was amplified from pUBp88 using p88-XhoI-F (5′-CCTGTCGATGTACTCGAGAAGGTGGCGTTT-3′) and p88-HA-R (5′-TTAAGCGTAATCTGGAACATCGTATGGGTATCGGGCTTTGATTA-3′), and a PCR fragment from pUBp88 was amplified using p88-HA-F (5′-TACCCATACGATGTTCCAGATTACGCTTAAGGTACCCCGCTGAA-3′) and M13-rev (5′-CAGGAAACAGCTATGACCATG-3′). A recombinant PCR product was then amplified from the mixture of these fragments using p88-XhoI-F and M13-rev, digested with XhoI and KpnI, and inserted into the corresponding region of pUBp88.

In vitro transcription.The plasmids with the prefix “pUC” or “pRC” were digested with SmaI. The linearized plasmids were used as templates for in vitro transcription by T7 RNA polymerase as described previously (58). If required, capped transcripts were prepared using the ScriptCap m7G capping system according to the manufacturer's instruction (Epicentre Biotechnologies, Madison, WI).

Inhibitor treatments.2-Phenylethynesulfonamide (PES), methanesulfonamide (MS), geldanamycin (GDA), and MG132 were purchased from EMD Biochemicals, Inc. (Gibbstown, NJ), Wako (Osaka, Japan), Sigma-Aldrich (St. Louis, MO), and Merck (Darmstadt, Germany), respectively. The inhibitors were diluted from stock solutions in dimethyl sulfoxide (DMSO) (Sigma-Aldrich). For the control experiments, an equivalent concentration of DMSO was applied.

Protoplast experiments.Protoplasts were prepared from tobacco BY-2 suspension cultured cells as described previously (84). BY-2 protoplasts (∼3 × 105) were inoculated with in vitro-transcribed RNA1 and RNA2 (1 μg each), with RCNMV virion (2 μg), with pUBRC1 and pUBRC2 (5 μg each), or with pUBp27-HA, pUBp88-HA, and in vitro-transcribed RNA2 (10 μg, 5 μg, and 500 ng, respectively) as described previously (21). The inoculated protoplasts were incubated at 17°C for 16 h in the presence of PES, MS, or GDA. Viral RNAs were detected by Northern blotting.

Agrobacterium infiltration.The plasmids with the prefix “pBIC” or “pTV” were introduced by electroporation into Agrobacterium tumefaciens GV3101 (pMP90) or A. tumefaciens GV3101 (pSoup), respectively. Agrobacterium suspensions were mixed at a final optical density at 600 nm (OD600) of 0.2 each for bimolecular fluorescence complementation (BiFC) experiments, at an OD600 of 0.4 each for subcellular localization assays, and at an OD600 of 0.5 for gene silencing. Agrobacterium suspensions harboring an empty vector, pBICp35, were used as the filler. The mixtures were infiltrated into Nicotiana benthamiana leaves essentially as described by Takeda et al. (84).

Silencing of Hsp70 and Hsp90 genes in N. benthamiana plants.Appropriate combinations of silencing vectors were expressed by Agrobacterium infiltration in 3- to 4-week-old N. benthamiana plants as described previously (73). At 10 days after agroinfiltration (dai), the leaves above the agroinfiltrated leaves were inoculated with in vitro-transcribed RNA1 and RNA2. The inoculated plants were incubated at 22°C for 2 days. Total RNAs were extracted using TRIzol reagent (Invitrogen), treated with RQ1 RNase-free DNase (Promega, Madison, WI), purified by phenol-chloroform and chloroform extractions, and precipitated with ethanol. Viral RNAs were detected by Northern blotting. The mRNA levels of NbHsp70c-1 (6, 38) were examined by semiquantitative RT-PCR using Nb70c-F (5′-CTAGAATCCCAAAGGTGCAACAGC-3′) and Nb70c-R (5′-CTTCTCATCTTTCACAGTGTTCCTC-3′). The mRNA levels of NbHsp90 were examined as described previously (85). As a control to show equal amounts of cDNA templates in each reaction mixture, the ribulose 1,5-biphosphate carboxylase small-subunit gene (rbcS), a constitutively expressed gene, was amplified by RT-PCR using NbRbcS-F (5′-CCTCTGCAGTTGCCACC-3′) and NbRbcS-R (5′-CCTGTGGGTATGCCTTCTTC-3′).

BiFC and subcellular localization assays.Appropriate combinations of yellow fluorescent protein (YFP) fragment-fused proteins or fluorescent protein-fused proteins were expressed in N. benthamiana leaves by Agrobacterium infiltration. Fluorescence of YFP, green fluorescent protein (GFP), DsRed monomer (DRm), and mCherry was visualized with confocal microscopy at 3 dai for subcellular localization assay and at 4 dai for BiFC. Protein expression was confirmed by Western blotting. Accumulations of viral RNAs were analyzed by Northern blotting.

Confocal microscopy.The fluorescence signals of GFP, YFP, DRm, and mCherry were observed using an Olympus FluoView FV500 confocal microscope (Olympus Optical Co., Tokyo, Japan) equipped with an argon laser, an He-Ne laser, and a 40× Plan Apo oil immersion objective lens. The samples were excited with the argon laser for GFP/YFP and with the He-Ne laser for DsRed-monomer/mCherry. We used a dichroic mirror, DM488/543, a beam splitter, SDM560, and two emission filters, BA505-525 for GFP/YFP and BA560IF for DsRed-monomer/mCherry. Scanning was performed in sequential mode to minimize signal bleedthrough. Images were processed using Adobe Photoshop CS3 software.

GST pulldown assays.E. coli Rosetta 2(DE3) (Invitrogen) transformed by pET42a, pET42a-Hsp70, or pET42a-Hsp90 was grown overnight at 37°C in LB medium containing kanamycin (100 μg/ml). Overnight cultures of the transformed E. coli were diluted to1:50 in LB medium containing kanamycin (100 μg/ml). After incubation for 2 h at 37°C, protein expression was induced by the addition of 0.3 mM isopropyl-β-d-thiogalactopyranoside. The cells expressing glutathione S-transferase (GST), GST-Hsp70, or GSR-Hsp90 were cultured at 28°C for 1 h, 4 h, or 4 h, respectively. The induced cells were harvested by centrifugation at 5,000 × g for 5 min.

Cells collected from 5 ml (pET-42a) or 10 ml (pET42a-Hsp70 and pET42a-Hsp90) of medium were resuspended in 500 μl of phosphate buffer saline (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4) and sonicated on ice to disrupt the cells. After sonication, Triton X-100 was added at the final concentration of 0.5% and centrifuged at 15,000 × g for 10 min at 4°C. The supernatant was added to a 12.5-μl bed volume of equilibrated glutathione-Sepharose 4B (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom) and incubated at 4°C for 1 h with gentle rotation. The resin was washed three times with 1 ml of binding buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 10 mM 2-mercaptoethanol, and 0.5% Triton X-100). After washing, the resin was incubated for 2 h at 4°C in 200 μl of binding buffer containing 500 ng of the purified His-p27-FLAG (52). After incubation, the resin was washed three times with 500 μl of binding buffer. The bound proteins were eluted by addition of Laemmli sample buffer (42), followed by incubation for 3 min at 95°C. Protein samples were subjected to SDS-PAGE and then blotted onto a polyvinylidene difluoride (PVDF) membrane. The separated proteins were analyzed by Western blotting and stained with Ponceau S.

Cell-free in vitro translation and replication experiments.Cell extracts of evacuolated BY-2 protoplasts (BYL) were prepared, and in vitro translation/replication reactions were performed essentially as described previously (28, 40, 52, 53). BN-PAGE analysis was performed as described previously (53) except that the total protein samples solubilized with 0.5% Triton X-100 were subjected to BN-PAGE. StreptoTag affinity purification was performed essentially as described previously (27). Immunopurification of p27-FLAG is described below.

Immunopurification of p27-FLAG.p27-FLAG was expressed in BYL by adding an in vitro transcript to a concentration of 20 nM. After 2 h of incubation, BYL expressing p27-FLAG was incubated on ice for 30 min with 500 μM PES, MS, or GDA. At this time, ADPβS or ATPγS, which stabilizes the binding of Hsp70 or Hsp90 to substrate proteins, respectively, was also added to a concentration of 2 mM. Then, a 10-μl bed volume of anti-FLAG M2-agarose affinity gel (Sigma-Aldrich) was added to the BYL and further incubated for 90 min with occasional mixing. The resin was washed three times with 200 μl of TR buffer (40) supplemented with 500 mM NaCl and 0.1% Triton X-100 for the analysis of p27-Hsp70 interaction or with TR buffer supplemented with 0.1% Triton X-100 for the analysis of p27-Hsp90 interaction. The bound proteins were eluted by addition of Laemmli sample buffer, followed by incubation for 3 min at 95°C. Protein samples were subjected to SDS-PAGE, followed by Western blotting with appropriate antibodies.

Northern blot analysis.Northern blot analysis was performed essentially as previously described (28). Digoxigenin-labeled RNA probes specific for the 3′ untranslated regions (UTRs) of RCNMV RNA1 and RNA2 and the full-length negative-strand RNA1 and RNA2 were described previously (56, 57). The RNA signals were detected with a luminescent-image analyzer (LAS-1000 plus; Fuji Photo Film, Japan), and the signal intensities were quantified using the NIH Image program.

Western blot analysis.Western blot analysis was performed essentially as described previously (53). Protein samples were subjected to SDS-PAGE or BN-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Immobilon-P; Millipore, Bedford, MA). Anti-p27 rabbit polyclonal antibody (84), anti-NtHsp90 rabbit polyclonal antibody (82), anti-Hsp70/Hsc70 mouse monoclonal antibody (Stressgen, Victoria, British Columbia, Canada), anti-c-myc mouse polyclonal antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA), anti-HA rat monoclonal antibody (Roche Diagnostics, Penzberg, Germany), and anti-FLAG M2 mouse monoclonal antibody (Sigma-Aldrich) were used as the primary antibodies. Alkaline phosphatase (AP)-conjugated anti-rabbit IgG antibody (Cell Signaling Technology, Beverley, MA), AP-conjugated anti-mouse IgG antibody (KPL, Gaithersburg, MD), AP-conjugated anti-rat IgG antibody (Santa Cruz Biotechnology Inc.), horseradish peroxidase (HRP)-conjugated anti-rabbit IgG antibody (Cell Signaling Technology), and HRP-conjugated anti-mouse IgG antibody (KPL) were used as the secondary antibodies. To detect p88-T7, AP-conjugated anti-T7 monoclonal antibody (Merck) was used. Signals were detected using a luminescent-image analyzer (LAS-1000 Plus), and the signal intensities were quantified using the NIH Image program.

Nucleotide sequence accession numbers.The GenBank accession numbers for the Nicotiana tabacum Hsp70 and Hsp90 cDNA sequences reported in this paper are AB689673 and AB689674, respectively.

RESULTS

Hsp70 and Hsp90 are host factors required for RCNMV RNA replication.In a previous study, we identified many host proteins that were copurified with RCNMV replicase complexes from virus-infected Nicotiana benthamiana tissues. These included Hsp70- and Hsp90-related proteins (53). This led us to investigate whether Hsp70 and Hsp90 are involved in RCNMV infection. We applied Tobacco rattle virus (TRV)-based gene silencing to downregulate cytosolic Hsp70 and Hsp90 genes in N. benthamiana plants. The TRV vectors harboring the partial fragment of the Hsp70 (TRV:Hsp70) (38) or Hsp90 (TRV:Hsp90) gene (85) were expressed by Agrobacterium-mediated expression. As a control, the empty TRV vector (TRV:00) (73) was expressed. The newly developed leaves were inoculated with in vitro-transcribed RCNMV RNA1 and RNA2 at 10 dai. Note that any morphological defects such as chlorotic and stunted phenotypes were not observed at this stage (Fig. 1A), although such phenotypes became visible after 16 dai (data not shown). Total RNA was extracted from the inoculated leaves 2 days after inoculation (at 12 dai). Semiquantitative RT-PCR analysis confirmed the specific reduction of Hsp70 and Hsp90 mRNAs in plants infiltrated with TRV:Hsp70 and TRV:Hsp90, respectively (Fig. 1B and C, lower panels). Importantly, the accumulation of RCNMV RNAs was impaired in Hsp70- and Hsp90-silenced plants as assessed by Northern blotting (Fig. 1B and C, upper panels). These results suggest that Hsp70 and Hsp90 play positive roles during RCNMV infection.

Fig 1
Fig 1

Knockdown of Hsp70 or Hsp90 mRNA levels via gene silencing inhibits accumulation of RCNMV RNAs in N. benthamiana plants. (A) The Tobacco rattle virus (TRV) vector harboring the partial fragment of N. benthamiana Hsp70c-1 (TRV:Hsp70) or Hsp90 (TRV:Hsp90) was expressed in N. benthamiana by Agrobacterium infiltration. The empty TRV vector (TRV:00) was used as a control. Pictures were taken at 10 days after agroinfiltration (dai). Note that the infiltrated plants show no morphological defects at this stage. (B and C) The newly developed leaves were inoculated with RCNMV RNA1 and RNA2 10 dai. Total RNAs were extracted from the inoculated leaves 2 days after inoculation. Accumulation of RCNMV RNAs was analyzed by Northern blotting. Ethidium bromide (EtBr)-stained rRNAs are shown below the Northern blots as loading controls. Hsp70 and Hsp90 mRNA levels were assessed by RT-PCR with primers that allow the amplification of the regions of Hsp70 and Hsp90 not present in the TRV:Hsp70 and TRV:Hsp90, respectively. RT-PCR results for the ribulose 1,5-biphosphate carboxylase small-subunit gene (rbcS) gene demonstrate equal amounts of total RNAs used for the RT and the equal efficiencies of the RT reaction in the samples.

To test whether Hsp70 and Hsp90 function in RCNMV RNA replication in a single cell, we treated tobacco BY-2 protoplasts with specific inhibitors of Hsp70 or Hsp90, i.e., PES or GDA, respectively. These inhibitors have been used successfully to analyze the functions of Hsp70 and Hsp90 in plant and animal systems (22, 23, 31, 43, 44, 82). MS, an analogue of PES, was used as a negative control for PES. Protoplasts inoculated with in vitro-transcribed RNA1 and RNA2 were incubated in the presence of various amounts of the inhibitors. Northern blot analysis showed that PES had an inhibitory effect on the accumulation of RCNMV RNAs; 40 μM PES inhibited viral RNA accumulation by about 95%, but MS had no effect (Fig. 2A and B). GDA had little effect on the accumulation of RCNMV RNAs (data not shown). However, GDA inhibited viral RNA accumulation when protoplasts were inoculated with the plasmids that transcribe RCNMV RNAs under the control of the Cauliflower mosaic virus (CaMV) 35S promoter: 50 μM GDA inhibited the accumulation of RCNMV RNAs by about 70% (Fig. 2C and D). Importantly, GDA did not reduce the accumulation of GFP mRNAs (Fig. 2C), indicating that the reduction of viral RNA accumulation was not caused by the inhibitory effect of GDA on CaMV 35S promoter-driven transcription under the assay conditions used. The inhibitory effect of GDA on viral RNA accumulation was also observed in protoplasts inoculated with RCNMV virions (Fig. 2E and F). Overall, these data suggest that both Hsp70 and Hsp90 are required for RCNMV RNA replication in a single cell.

Fig 2
Fig 2

Inhibitors of Hsp70 or Hsp90 impair RCNMV RNA replication in a single cell. (A and B) Tobacco BY-2 protoplasts were inoculated with in vitro-transcribed RNA1 and RNA2. The inoculated protoplasts were incubated for 16 h at 17°C in the presence of various concentrations of 2-phenylethynesulfonamide (PES), a specific inhibitor of Hsp70. Methanesulfonamide (MS) is a nonfunctional analogue of PES. Total RNAs were analyzed by Northern blotting. EtBr-stained rRNAs as loading controls are shown below the Northern blots (A). The accumulation levels of RNA1 and RNA2 from three separate experiments using PES were quantified using NIH Image and are plotted in the graphs (B). (C and D) BY-2 protoplasts were inoculated with pUBRC1 and pUBRC2, which transcribe RNA1 and RNA2, respectively, under the control of the Cauliflower mosaic virus (CaMV) 35S promoter. pUEGFP, which expresses GFP mRNA driven by the CaMV 35S promoter, was also inoculated as an internal control. The inoculated protoplasts were incubated for 16 h at 17°C in the presence of various concentrations of geldanamycin (GDA), a specific inhibitor of Hsp90. Total RNAs were analyzed by Northern blotting. EtBr-stained rRNAs are shown as loading controls (C). The accumulation levels of RNA1 and RNA2 from three separate experiments were quantified using NIH Image and are plotted in the graphs (D). (E and F) BY-2 protoplasts were inoculated with RCNMV virions and incubated with GDA as described above. Total RNAs were analyzed by Northern blotting. EtBr-stained rRNAs are shown as loading controls (E). The accumulation levels of RNA1 and RNA2 from three separate experiments were quantified using NIH Image and are plotted in the graphs (F).

Hsp70 and Hsp90 interact with p27 within the virus-induced aggregated structures of the ER in the context of viral RNA replication.Because Hsp70 and Hsp90 are well-known protein chaperones, these chaperones might bind directly or indirectly to p27, p88, or both. To characterize these interactions in living cells, we used a BiFC assay. We isolated cDNAs encoding Hsp70 and Hsp90 from tobacco BY-2 cultured cells as described in Materials and Methods. We first tested whether p27 interacts with Hsp70 and Hsp90. p27 fused to the N- or C-terminal half of YFP at the C terminus was expressed together with Hsp70 or Hsp90 fused to the other halves of YFP at the N terminus (i.e., p27-nYFP plus cYFP-Hsp70 or cYFP-Hsp90 and p27-cYFP plus nYFP-Hsp70 or nYFP-Hsp90) in the presence of p88, RNA2, mCherry containing an ER targeting signal (ER-mCherry) (34), and the silencing suppressor p19 of Tomato bushy stunt virus (TBSV) in N. benthamiana by Agrobacterium infiltration. At 4 dai, large aggregated fluorescent structures of YFP were detected (Fig. 3A and B, left panels). This YFP fluorescence was merged with the large aggregated fluorescent structures of ER-mCherry (Fig. 3A and B, middle and right panels), a characteristic feature of morphological changes of ER induced by RCNMV infection (41, 87). Little or no YFP fluorescence was detected in control experiments in which YFP fragments, p27, Hsp70, or Hsp90 were expressed separately instead of their fusion protein counterparts (Fig. 3C and D). Western blot analysis confirmed the accumulation of YFP fragment-fused proteins in Agrobacterium-infiltrated leaves (Fig. 3E and F). Northern blot analysis showed that p27-cYFP but not p27-nYFP supported the accumulation of positive- and negative-strand RNA2 (Fig. 3E and F), indicating that p27-cYFP participated in the replication of RNA2 under the assay conditions used. Together, these results suggest that p27 interacts with Hsp70 and Hsp90 in planta in the absence and presence of viral RNA replication.

Fig 3
Fig 3

p27 interacts with Hsp70 and Hsp90 in planta. (A and B) Bimolecular fluorescence complementation (BiFC) analysis of the interactions of p27 with Hsp70 and Hsp90. p27 fused to the N- or C-terminal half of YFP at the C terminus (p27-nYFP and p27-cYFP) was expressed together with Hsp70 or Hsp90 fused to the other half of YFP at the N terminus (cYFP-Hsp70, nYFP-Hsp70, cYFP-Hsp90, and nYFP-Hsp90) in the presence of p88, RNA2, ER-mCherry, and the suppressor p19 of Tomato bushy stunt virus in N. benthamiana leaves by Agrobacterium infiltration. The right panels show the merging of mCherry and YFP as yellow color. Arrowheads indicate the large fluorescent aggregates. Bars, 20 μm. (C and D) Control experiments for the BiFC analysis. YFP fragments and p27, Hsp70, or Hsp90 were separately expressed instead of their fusion protein counterparts in the presence of p88, RNA2, ER-mCherry, and p19 in N. benthamiana leaves by Agrobacterium infiltration. Merged images of the YFP and ER-mCherry visualized by confocal microscopy at 4 days after agroinfiltration are shown. Arrowheads indicate the large fluorescent aggregates. Bars, 20 μm. (E and F) Functional analysis of YFP fragment-fused p27 in viral RNA replication. Total RNAs and proteins extracted from agroinfiltrated leaves were analyzed by Northern and Western blotting, respectively. EtBr-stained rRNAs as loading controls are shown below Northern blots.

We also used BiFC to test whether p88 interacts with Hsp70 and Hsp90, but we failed to observe these interactions. Appropriate expression of C-terminally YFP fragment-fused p88 in combination with the other YFP fragment-fused Hsp70 or Hsp90 showed no or little YFP fluorescence (data not shown). We note that both p88 derivatives were functional in supporting the replication of RNA2, although C-terminally cYFP-fused p88 did not accumulate to detectable levels (data not shown). Thus, it appears that the interactions of p88 with Hsp70 and Hsp90 do not occur, or if they occur, the interactions are too weak to be detected by BiFC.

Because the reconstitution of YFP is irreversible (49), the above-described BiFC experiments did not rule out the possibility that the reconstitution of YFP occurred only in the cytoplasm and that the YFP signals detected in the aggregated ER structures reflected artificial tethering of Hsp70 and Hsp90 to p27 localized to the ER. To check whether Hsp70 and Hsp90 colocalize with p27 without the artificial tethering, we first examined the subcellular localization of Hsp70 and Hsp90 in the absence of RCNMV infection by expressing GFP-fused Hsp70 (GFP-Hsp70) or Hsp90 (GFP-Hsp90) together with ER-mCherry in N. benthamiana. At 3 dai, fluorescence of GFP-Hsp70 and GFP-Hsp90 was observed in the nucleus and cytoplasm (Fig. 4A and C, left panels). The fluorescence of ER-mCherry showed an ER distribution that included the nuclear envelope (Fig. 4A and C, middle panels). The fluorescence of GFP-Hsp70 and GFP-Hsp90 was merged only partially with the fluorescence of ER-mCherry (Fig. 4A and C, right panels), indicating that Hsp70 and Hsp90 hardly localized to the ER in the absence of RCNMV infection. However, when coexpressed with C-terminally DRm-fused p27 (p27-DRm), p88, and RNA2, the large aggregated fluorescence of GFP-Hsp70 and GFP-Hsp90 was detected in addition to the cytoplasmic and nuclear fluorescence (Fig. 4B and D, left panels), and it merged with the fluorescent aggregates of p27-DRm (Fig. 4B and D, middle and right panels). These results show that GFP-Hsp70 and GFP-Hsp90 colocalized with p27-DRm in the large aggregated structures of the ER. We note that the p27-DRm was functional in supporting the replication of RNA2 (data not shown). Together, these data suggest that Hsp70 and Hsp90 are recruited by p27 to the large aggregate structures of the ER in the context of viral RNA replication.

Fig 4
Fig 4

RCNMV RNA replication affects the subcellular localization of Hsp70 and Hsp90. Green fluorescent protein (GFP)-fused Hsp70 (GFP-Hsp70) plus ER-mCherry (A), GFP-Hsp70 plus C-terminally DsRed monomer-fused p27 (p27-DRm) plus p88 plus RNA2 (B), GFP-fused-Hsp90 (GFP-Hsp90) plus ER-mCherry (C), and GFP-Hsp90 plus p27-DRm plus p88 plus RNA2 (D) were expressed in N. benthamiana leaves by Agrobacterium infiltration. Fluorescence was visualized by confocal microscopy. The merging of the green and red fluorescence is shown as yellow color. Bars, 20 μm.

We used an in vitro GST pulldown assay to characterize further the interactions of p27 with Hsp70 and Hsp90. We purified p27 with an N-terminal His tag and a C-terminal FLAG tag (His-p27-FLAG), as described previously (52). N-terminally GST-fused Hsp70 (GST-Hsp70) or Hsp90 (GST-Hsp90) captured on glutathione-bound beads was incubated with purified His-p27-FLAG. After extensive washing, the bound proteins were analyzed by Western blotting with an anti-FLAG antibody. His-p27-FLAG was pulled down by both GST-Hsp70 and GST-Hsp90 but not by GST alone, which was used as a negative control (Fig. 5A). Thus, like other substrate proteins, Hsp70 and Hsp90 bind to p27 via protein-protein contacts.

Fig 5
Fig 5

p27 interacts with Hsp70 and Hsp90 in vitro. (A) Glutathione resin-bound GST-fused Hsp70 (GST-Hsp70) or GST-Hsp90 was incubated with the purified recombinant N-terminally His and C-terminally FLAG-tagged p27 (His-p27-FLAG). After washing, pulled-down complexes were subjected to SDS-PAGE and blotted onto a membrane. Pulled-down His-p27-FLAG was detected by Western blotting with anti-FLAG antibody. After detection, the separated proteins on the membrane were visualized by Ponceau S staining (Ponceau S). (B and C) A capped transcript that expresses p27-FLAG (20 nM) was added to BYL. After 2 h of incubation at 17°C, BYL expressing p27-FLAG was incubated for 30 min at 17°C with DMSO, PES (500 μM), or MS (500 μM) in the presence of a 2 mM concentration of a nonhydrolyzable analogue of ADP (ADPβS) for the analysis of p27-Hsp70 interaction (B). Alternatively, BYL expressing p27-FLAG was incubated for 30 min at 17°C with DMSO or GDA (500 μM) in the presence of a 2 mM concentration of a nonhydrolyzable analogue of ATP (ATPγS) and 3 mM MgCl2 for the analysis of p27-Hsp90 interaction (C). The BYL was then mixed with a 10-μl bed volume of anti-FLAG M2 antibody agarose and incubated further on ice for 90 min. After washing, Hsp70 and Hsp90 copurified with p27-FLAG were analyzed by Western blotting with appropriate antibodies.

Hsp70 and Hsp90 promote the assembly of the 480-kDa replicase complex of RCNMV by binding to p27.Because PES and GDA block interactions of Hsp70 and Hsp90, respectively, with their client proteins (23, 31, 43, 44), we hypothesized that these chemicals inhibit RCNMV RNA replication by blocking the interactions of Hsp70 and Hsp90 with p27. To test this, we used BYL, an in vitro translation/replication system (40). BYL has been used successfully to recapitulate the RNA replication processes of RCNMV (1, 21, 27–29, 52, 53, 57, 74, 81). We first tested the effects of PES and GDA on the interactions of p27 with Hsp70 and Hsp90, respectively. BYL expressing C-terminally FLAG-tagged p27 (p27-FLAG), which is functional in RCNMV RNA replication (53), was subjected to immunopurification with anti-FLAG antibodies in the presence of inhibitors. Western blot analysis showed that PES and GDA blocked the copurification of endogenous Hsp70 and Hsp90, respectively, with p27-FLAG (Fig. 5B and C). These results confirmed the inhibitory effects of PES and GDA on the interactions of p27 with Hsp70 and Hsp90, respectively.

Next, we tested the effects of PES and GDA on the negative-strand RNA synthesis of RCNMV RNAs. Both chemicals inhibited the accumulation of p27 and negative-strand RNAs in BYL (Fig. 6), suggesting that Hsp70 and Hsp90 are required for the translation/accumulation of p27 protein as well as the negative-strand RNA synthesis. We then designed an assay using BYL to investigate the roles of Hsp70 and Hsp90 in RCNMV RNA replication other than in protein translation and folding (Fig. 7A). This assay is based on the replication mechanism of RNA2, which exploits p27 and p88 supplied in trans, in contrast to the translation-coupled replication of RNA1 (27, 66). This assay enabled us to monitor the assembly of the 480-kDa replicase complex and the subsequent negative-strand synthesis of RNA2 after the completion of translation and folding of p27 and p88 proteins. In this assay, p27-FLAG and C-terminally T7-tagged p88 (p88-T7) were translated individually in BYL. These BYL were mixed together following the addition of the protein synthesis inhibitor cycloheximide. RNA2 was then added with PES or GDA to the mixed BYL, followed by further incubation. Northern blot analysis showed dose-dependent inhibition of the negative-strand RNA2 synthesis by PES and GDA (Fig. 7B, E and F). Western blot analysis of the protein complexes separated by BN-PAGE revealed that PES and GDA inhibited the formation of the 480-kDa replicase complex in a dose-dependent manner (Fig. 7B to D). Interestingly, PES but not GDA increased the accumulation of large complexes (∼1,024 kDa) (Fig. 7B to D). MS had no effects on the accumulation of the 480-kDa complexes and negative-strand RNA2 (Fig. 7B). PES affected the stability of p88-T7 (Fig. 7B and C). However, this reduction in p88-T7 was not the major reason for the inhibitory effect of PES on the negative-strand synthesis of RNA2 because more negative-strand RNA2 accumulated in the presence of MS than in the presence of PES even when the amount of p88-T7 in MS-treated BYL was adjusted to be similar to or even less than that in PES-treated BYL (Fig. 7G). Taken together, these results suggest that Hsp70 and Hsp90 facilitate the assembly of RCNMV replicase complex likely through interactions with p27 and thereby promote subsequent negative-strand RNA synthesis.

Fig 6
Fig 6

PES and GDA inhibit accumulations of p27 and negative-strand RNAs in BYL. BYL was incubated for 4 h at 17°C with in vitro-synthesized RNA1 (7.5 nM) and RNA2 (20 nM) in the presence of various concentrations of inhibitors. Accumulations of negative-strand RNA1 and RNA2 were detected by Northern blotting. Accumulations of p27 were analyzed by Western blotting with anti-p27 antisera. EtBr-stained rRNAs and Coomassie brilliant blue (CBB)-stained cellular proteins are shown as loading controls.

Fig 7
Fig 7

Hsp70 and Hsp90 promote the assembly of the RCNMV replicase complex. (A) Depiction of the assay system using BYL to monitor the assembly of the 480-kDa replicase complex and the synthesis of negative-strand RNA2 independently of the translation and folding of p27 and p88 replicase proteins (1). Capped transcripts expressing p27-FLAG (20 nM) or p88-T7 (20 nM) were incubated separately in BYL for 2 h at 17°C (2). After the addition of cycloheximide at a concentration of 100 μg/ml, BYL expressing p27-FLAG and BYL expressing p88-T7 were mixed at a 2:1 ratio (3). RNA2 (20 nM) was added together with DMSO, PES, MS, or GDA to the mixed BYL and incubated for an additional 2 h at 17°C. (B) Effects of PES (500 μM) and GDA (100 μM) on the accumulation of 480-kDa complexes and the negative-strand synthesis of RNA2. Accumulation of negative-strand RNA2 was analyzed by Northern blotting. EtBr-stained rRNAs are shown below the Northern blots as loading controls. Accumulation of the 480-kDa replicase complexes was analyzed by Western blotting with anti-p27 antisera after the separation of protein complexes by blue native polyacrylamide gel electrophoresis (BN-PAGE). Accumulation of p27-FLAG and p88-T7 was analyzed by Western blotting with anti-FLAG and anti-T7 antibodies, respectively, after separation of the denatured proteins by SDS-PAGE. CBB-stained cellular proteins are shown below the Western blots as loading controls. (C to F) Dose-dependent effects of PES and GDA on the accumulation of the 480-kDa replicase complex and negative-strand RNA synthesis. BYL expressing p27-FLAG and p88-T7 were prepared and mixed as described for panel A, and then the mixed BYL was incubated with RNA2 (20 nM) for 2 h at 17°C in the presence of increasing amounts of PES (C and E) or GDA (D and F). Western blotting with anti-FLAG antibodies in combination with BN-PAGE detected the 480-kDa replicase complexes and nonfunctional large complexes. Western blotting in combination with SDS-PAGE detected p27-FLAG and p88-T7 components. CBB-stained cellular proteins are shown as loading controls. Northern blots showed accumulations of negative-strand RNA2. EtBr-stained rRNAs are shown below the Northern blots as loading controls. (G) Effects of the reduced accumulation of p88 on negative-strand RNA2 synthesis. BYL expressing p27-FLAG, BYL expressing p88-T7, and mock-treated BYL were mixed at 2:1:0, 2:0.67:0.33, 2:0.5:0.5, and 2:0.33:0.67 in the presence of MS, and BYL expressing p27-FLAG and BYL expressing p88-T7 were mixed at 2:1 in the presence of PES (from left to right). Accumulations of viral RNAs and proteins were analyzed by Northern and Western blotting, respectively. EtBr-stained rRNAs and CBB-stained cellular proteins are shown below the Northern and Western blots, respectively, as loading controls.

Hsp90 regulates the assembly of the 480-kDa replicase complex by promoting the specific binding of p27 to YRE.We have shown previously that the addition of RNA2 increases the accumulation of the 480-kDa complexes in BYL (53) and that mutations in p27 that compromise the binding activity of p27 to YRE of RNA2 affect the assembly of the 480-kDa complexes in BYL (21). An aptamer-based pulldown assay using p27 produced in BYL showed the interaction between p27 and YRE, whereas no such interaction was observed using purified recombinant p27 produced in Escherichia coli in an electrophoretic mobility shift assay (27), suggesting that an unknown plant factor(s) is required for the binding of p27 to YRE. These findings led us to hypothesize that the interaction between p27 and YRE is a key step in the assembly of the 480-kDa replicase complex on RNA2 and that this step is regulated by Hsp70 and/or Hsp90.

To test this hypothesis, we first used an RNA2 mutant (RNA2 LM8) that is unable to interact with p27 (27) to evaluate its ability to support the assembly of the 480-kDa complex. After addition of cycloheximide, BYL expressing p27-FLAG was mixed with BYL expressing p88-T7, as illustrated in Fig. 7A. Wild-type RNA2 or RNA2 LM8 was then added to the mixed BYL, followed by further incubation. Northern blot analysis confirmed the requirement of p27-YRE interaction for the negative-strand synthesis of RNA2 (Fig. 8A) (1, 21, 27). Western blot analysis in combination with BN-PAGE showed that the accumulation of the 480-kDa complex was increased by the addition of wild-type RNA2, whereas little increase in the accumulation of the 480-kDa complex was observed when RNA2 LM8 was added (Fig. 8A). These results indicate that the binding of p27 to YRE is one of the key steps for the assembly of the 480-kDa replicase complex.

Fig 8
Fig 8

Hsp90 regulates the specific binding of p27 to the Y-shaped RNA element (YRE) of RNA2, which is critical for the assembly of the RCNMV replicase complex. (A) Effects of a mutation in YRE that inhibits the interaction between p27 and YRE on the assembly of the 480-kDa complex. BYL expressing p27-FLAG and BYL expressing p88-T7 were mixed as shown in Fig. 7A. RNA2 or its mutant, LM8, which cannot interact with p27 (27), was added to the mixed BYL, and the mixture was incubated for 2 h. Accumulation of viral RNAs was analyzed by Northern blotting. Accumulation of the 480-kDa complexes and its viral components was analyzed by Western blotting in combination with BN-PAGE and SDS-PAGE, respectively. (B) Effects of inhibitors on the binding of p27 to YRE. Membrane-depleted BYL (BYLS20) expressing p27-FLAG was incubated for 20 min on ice with STagT-fused YRE in the presence of PES, MS, or GDA (500 μM each). Heparin was added to the BYLS20 at a final concentration of 20 μg/ml, and the mixture was incubated for 40 min. The BYLS20 was subjected to StreptoTag affinity purification, followed by Western blotting with anti-FLAG antibody. The affinity-purified YRE was detected with EtBr staining.

We next used a pulldown assay based on the high affinity of an RNA aptamer termed “STagT” with streptomycin to test the effects of PES and GDA on the interaction between p27 and YRE (2, 9). This RNA-based pulldown assay in combination with the membrane-depleted BYL (BYLS20), which can support negative-strand synthesis of RCNMV RNAs, was used to analyze the interaction between p27 and YRE (21, 27). STagT-fused YRE was incubated with BYLS20 expressing p27-FLAG in the presence of the inhibitors. The p27-FLAG proteins interacting with STagT-fused YRE were pulled down by streptomycin-conjugated beads. Similar amounts of bait RNAs were purified and indicated by ethidium bromide staining. Interestingly, Western blotting with anti-FLAG antibodies showed that GDA but not PES inhibited the copurification of p27-FLAG with STagT-fused YRE (Fig. 8B). These results suggest that the interaction of p27 with Hsp90, but not with Hsp70, is critical for the interaction between p27 and YRE of RNA2, which then promotes the formation of the 480-kDa replicase complex. These results also illustrate the differential contributions of Hsp70 and Hsp90 in the assembly of the 480-kDa replicase complex.

DISCUSSION

In this study, we showed that Hsp70 and Hsp90 play critical roles in different steps of the assembly of the RCNMV replicase complex, likely through interactions with p27. Inhibition of the p27-Hsp70 interaction resulted in the assembly of p27 into large complexes that were nonfunctional in viral RNA synthesis. In contrast, inhibition of p27-Hsp90 interaction caused the loss of the ability of p27 to bind to viral genomic RNA2, which is critical for the assembly of the 480-kDa replicase complex. These findings reveal the regulatory mechanism for the assembly of viral replicase complexes and suggest the potential roles of Hsp70 and Hsp90 in controlling the assembly of ribonucleoprotein complexes.

Hsp70 regulates the correct assembly of the RCNMV replicase complex.Hsp70 interacts with viral replicase proteins of various eukaryotic positive-strand RNA viruses (5, 11, 64, 90). For example, Pogany et al. (71) successfully reconstituted the RdRP activities of TBSV replicase complexes using viral replicase proteins, p33 and p92, produced in E. coli with the membrane fractions of yeast extracts and purified yeast Hsp70. They suggested that Hsp70 plays an integral role in the early replication process of TBSV, including the assembly of TBSV replicase complexes.

In the present study, we analyzed the assembled RCNMV replicase complex itself and found that inhibition of p27-Hsp70 interaction by PES causes the formation of large aggregates containing p27 and reduces accumulation of the 480-kDa replicase complex, which in turn impairs viral RNA synthesis (Fig. 7). These data lead us to propose that one of the major roles of Hsp70 in viral RNA replication is to control the proper assembly of viral replicase complexes.

How does Hsp70 regulate the assembly of the RCNMV replicase complex? Based on the accumulation of nonfunctional large complexes upon the inhibition of p27-Hsp70 interaction, it is possible that the Hsp70 prevents the aggregation of p27 protein and thereby facilitates the assembly of the RCNMV replicase complex. In fact, it has been reported that Hsp70 family proteins promote the assembly of cellular multiprotein complexes such as immunoglobulin G antibody and synaptic SNARE complex by preventing aggregations of their component proteins (13, 75). Alternatively, the large complexes could be intermediates for the assembly of the RCNMV replicase complex, and Hsp70 might assist the assembly process from these complexes to functional replicase complexes. Such functions are reported for the chaperones dedicated to the assembly of proteasome (60). Further studies are needed to gain mechanistic insights into how Hsp70 regulates the assembly of RCNMV replicase complex.

Hsp90 regulates the key interaction between p27 and YRE, which is required for the assembly of the RCNMV replicase complex.Gene silencing in plants and pharmacological inhibition in protoplasts showed that Hsp70 and Hsp90 are host factors required for RCNMV replication (Fig. 1 and 2). Interestingly, however, an inhibitory effect of GDA in protoplast experiments was observed when RCNMV RNAs were supplied from plasmids or virions but not when in vitro-synthesized RCNMV RNAs were directly introduced (Fig. 2C to F and data not shown). These results suggest that Hsp90 is required for plasmid- or virion-dependent processes such as efficient transcription and virion uncoating. An alternative, but not mutually exclusive, interpretation is that Hsp90 plays a role in a very early step in the RCNMV replication process, because the time required for transcription from plasmids or virion uncoating must delay the initiation of viral RNA replication. The latter interpretation was supported by the finding that GDA inhibits two distinct steps in the early replication process of RCNMV in vitro (Fig. 6 to 8). The first step is the accumulation of p27 protein (Fig. 6). An inhibitory effect of GDA on p27 accumulation as well as viral RNA accumulation was also observed when BY-2 protoplasts were inoculated with plasmids that express hemagglutinin (HA)-tagged p27 and p88 together with RNA2 (data not shown). The roles of Hsp90 in the accumulation of RCNMV replicase proteins are discussed below. The second step is the binding of p27 to YRE of RNA2, which is a critical step in the assembly of the 480-kDa replicase complex (Fig. 8). A similar mode of assembly was proposed for TBSV replicase complexes: p33 auxiliary protein interacts with the internal replication element (IRE) located in the coding region of p92 RdRP (72), and this interaction is required for the RdRP activities of the TBSV replicase complexes in yeast and its cell extracts (70–72). However, unlike p27, p33 can bind to the IRE without other viral and host proteins (72). Thus, it appears that these two related viruses have evolved different strategies for the interactions of viral replicase proteins with replication templates and the subsequent assembly of the viral replicase complexes.

Recently, Huang et al., (20) showed that Hsp90 of N. benthamiana binds to the 3′ UTR of BaMV genomic RNA and is required for the efficient accumulation of the genomic RNA during the early stages of infection. This finding suggests a potential function of Hsp90 in regulating viral RNA replication through interaction with viral RNAs. However, it is unlikely that Hsp90 promotes the assembly of the 480-kDa replicase complex by binding to RCNMV RNA2, because we failed to detect any interactions between Hsp90 and RNA2 in BYL based on STagT-aptamer pulldown assays (data not shown). It is still possible that Hsp90 binds to RNA1 as well as p27 and/or p88, which may facilitate translation and subsequent assembly of the replicase complex. Interestingly, the Lsm1-7 complex, which is involved in mRNA degradation in processing bodies, is required for translation and replication of Brome mosaic virus (BMV) genomic RNAs in yeast (3, 50, 65). The Lsm1-7 complex binds directly to tRNA-like structures and the intergenic region of BMV RNA3 (14).

Roles of Hsp70 and Hsp90 in the translation/stability of RCNMV replicase proteins.PES and GDA decreased the accumulation of p27 in BYL when the inhibitors were present throughout the experiments (Fig. 6). However, the deleterious effects were not observed when these inhibitors were added after the addition of cycloheximide (Fig. 7B to D), suggesting that Hsp70 and Hsp90 are dispensable for the stability of p27 after translation. It is likely that Hsp70 and Hsp90 promote the synthesis of p27, as seen for the synthesis of flock house virus protein A (4, 91). Alternatively, it is possible that the translating p27 is protected from degradation by a ribosome-associated chaperone system, in which Hsp70 is thought to interact directly with nascent polypeptides emerging from ribosomes (37, 92). Cotranslational binding of Hsp70 and/or Hsp90 might facilitate the subsequent function of these chaperones during the assembly of the RCNMV replicase complex.

In contrast, PES decreased the stability of p88 in BYL (Fig. 7B, C and E). The proteasome inhibitor MG132 did not restore the stability of p88 (data not shown), suggesting that Hsp70 might protect p88 from proteasome-independent degradation. However, it is also possible that the effect of PES on the stability of p88 is an artifact of the in vitro condition, in which p88 tends to form aggregates because of its overexpression (53). This situation never occurs during the actual infection process of RCNMV because p88 is translated by a programmed −1 ribosomal frameshifting at a quite low frequency (39, 81) and because the translation and accumulation of p88 are tightly coupled to viral RNA replication (27, 53, 66).

In conclusion, this study has demonstrated the multiple roles of Hsp70 and Hsp90 in the early replication process of RCNMV. These chaperones promote the translation of p27 and interact directly with p27 (Fig. 5 to 7). These interactions lead to the recruitment of Hsp70, Hsp90, and RNA2 to the ER membrane (Fig. 3, 4 and 8) (21, 27), where Hsp70 and Hsp90 regulate the assembly of the 480-kDa replicase complex (Fig. 7 and 8). Finally, the properly assembled 480-kDa replicase complex initiates viral RNA replication via cRNA synthesis (53).

ACKNOWLEDGMENTS

We thank Ryohei Terauchi for pPVX.NbHsp70c-1, Takashi Araki for BiFC constructs, Ichiro Mitsuhara and Yuko Ohashi for the anti-NtHsp90 antibody and pBE2113-GUS, and David C. Baulcombe for TRV vectors. We are also grateful to Hiro-oki Iwakawa for helpful discussion.

This work was supported in part by Grants-in-Aid for Scientific Research (A) (22248002) from the Japan Society for the Promotion of Science.

FOOTNOTES

    • Received 28 June 2012.
    • Accepted 21 August 2012.
    • Accepted manuscript posted online 29 August 2012.

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