Cowpea Mosaic Virus 32- and 60-Kilodalton Replication Proteins

JOURNAL OF VIROLOGY, June 2002, p. 6293–6301 Vol. 76, No. 120022-538X/02/$04.00�0 DOI: 10.1128/JVI.76.12.6293–6301.2002Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Cowpea Mosaic Virus 32- and 60-Kilodalton Replication ProteinsTarget and Change the Morphology of Endoplasmic

Reticulum MembranesJan E. Carette,1† Jan van Lent,2 Stuart A. MacFarlane,3 Joan Wellink,1* and Ab van Kammen1

Laboratory of Molecular Biology1 and Laboratory of Virology,2 Wageningen University, Wageningen, The Netherlands, andDepartment of Virology, Scottish Crop Research Institute, Invergowrie, Dundee, United Kingdom3

Received 11 December 2001/Accepted 22 March 2002

Cowpea mosaic virus (CPMV) replicates in close association with small membranous vesicles that areformed by rearrangements of intracellular membranes. To determine which of the viral proteins are respon-sible for the rearrangements of membranes and the attachment of the replication complex, we have expressedindividual CPMV proteins encoded by RNA1 in cowpea protoplasts by transient expression and in Nicotianabenthamiana plants by using the tobacco rattle virus (TRV) expression vector. The 32-kDa protein (32K) and60K, when expressed individually, accumulate in only low amounts but are found associated with membranesmainly derived from the endoplasmic reticulum (ER). 24K and 110K are freely soluble and accumulate to highlevels. With the TRV vector, expression of 32K and 60K results in rearrangement of ER membranes. Besides,expression of 32K and 60K results in necrosis of the inoculated N. benthamiana leaves, suggesting that 32K and60K are cytotoxic proteins. On the other hand, during CPMV infection 32K and 60K accumulate to high levelswithout causing necrosis.

It has become increasingly clear that replication of positive-strand RNA viruses takes place in association with intracellularmembranes (for a review see reference 6). Often these mem-branes are induced upon infection by vesiculation or rear-rangement of membranes from different organelles includingthe early and late endomembrane system (24, 27, 36). Theimportance of membranes for viral replication is evident fromvarious studies. In vitro synthesis of positive-strand RNAs ofpicornaviruses and nodaviruses depends on membranes (25,49). Poliovirus, Semliki Forest virus, and cowpea mosaic virus(CPMV) RNA replication is inhibited in cells treated withcerulenin, an inhibitor of lipid biosynthesis (7, 17, 28). Bromemosaic virus RNA replication is severely inhibited in yeast cellsthat carry a mutation in the host �9 fatty acid desaturase,which affects the lipid composition of the membranes (21).Despite the central role of membranes in viral replication, itremains poorly understood how viral replication complexes aretargeted to and assembled on specific membrane sites.

Upon infection of cowpea (Vigna unguiculata L.) or Nicoti-ana benthamiana cells with CPMV, typical cytopathologicalstructures are formed which consist of an amorphous matrix ofelectron-dense material traversed by rays of small membra-nous vesicles (7, 10, 20). The membranous vesicles are closelyassociated with CPMV RNA replication, as was revealed byautoradiography in conjunction with electron microscopy onsections of CPMV-infected leaves treated with [3H]uridine(10). Fractionation of homogenates from infected leaves fur-

ther demonstrated that the viral replicase activity is present inthe crude membrane fraction, which corroborated the notionthat viral replication complexes are physically associated withmembranes (13). Based on the observation that the endoplas-mic reticulum (ER) undergoes a drastic proliferation uponCPMV infection and that the rearranged ER membranes co-localize with the cytopathological structure, we have proposedthat the small membranous vesicles originate from the ER (7).

CPMV is the type member of the comoviruses and bearsstrong resemblance to animal picornaviruses both in gene or-ganization and in the amino acid sequence of replication pro-teins. Both RNA1 and RNA2 express large polyproteins, whichare proteolytically cleaved into different proteins by the 24-kDa proteinase (24K) (Fig. 1A). The proteins encoded byRNA1 are necessary and sufficient for virus replication,whereas RNA2 codes for the capsid proteins and the move-ment protein. Based on amino acid sequence comparison withother viruses and different experimental data, functions havebeen attributed to the different RNA1-encoded proteins. The32-kDa protein (32K) is a hydrophobic protein which containsno motifs common to other positive-strand RNA viruses out-side the Comoviridae. It is involved in regulation of the RNA1polyprotein processing and is required as a cofactor in thecleavage of the RNA2 polyprotein (32). 60K (58K � VPg) isable to bind ATP via a conserved Walker nucleotide-bindingmotif and has been proposed elsewhere to be a viral helicase(31). 87K has a domain specific to RNA-dependent RNA poly-merases (RdRp); however, 110K (87K plus 24K) is the onlyviral protein present in highly purified RdRp capable of elon-gating nascent viral RNA chains (13), suggesting that fusion to24K is required for replicase activity.

During infection, the bulk of the nonstructural proteins en-coded by RNA1 is present in electron-dense structures adja-cent to the small membranous vesicles (48). It was proposed

* Corresponding author. Mailing address: Laboratory of MolecularBiology, Wageningen University, Dreijenlaan 3, 6703 HA Wagenin-gen, The Netherlands. Phone: 31-317483266. Fax: 31-317483584. E-mail: [email protected].

† Present address: Division of Gene Therapy, Department of Med-ical Oncology, VU University Medical Center, Amsterdam, The Neth-erlands.

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elsewhere that the replication complexes are deposited ontothese electron-dense structures after taking part in RNA rep-lication located on the membranes (43). Aggregation of thevirus nonstructural proteins in this structure complicates theanalysis of the membrane binding properties of the individualproteins during normal CPMV infection. Expression of RNA1-encoded proteins in insect cells, by using the baculovirus ex-pression system, showed that 60K but not 110K is able toinduce and associate with small membranous vesicles in thecytoplasm in these cells (41).

In this study, the nature of the interaction of differentRNA1-encoded proteins and membranes was further exploredin plant cells. By using a virus vector-based expression system,selected RNA1 proteins were expressed alone or in a fusionwith a reporter protein, which allowed us to identify and visu-alize specific membranes interacting with the viral proteins inlive plant cells. In addition, expression of certain viral proteinsled to drastic rearrangements of intracellular membranes andultimately cell death.

MATERIALS AND METHODS

Construction of the transient expression vectors. Plasmid pMON999 is a plantexpression vector that contains the enhanced cauliflower mosaic virus (CaMV)35S promoter, followed by a multiple cloning site and the terminator of thenopaline synthase gene (43). pMON-HA is derived from pMON999 and containsthe coding region of the hemagglutinin (HA) epitope tag (YPYDVPDYA)between the promoter and terminator (44). The individual CPMV RNA1 pro-teins were PCR amplified with gene-specific primers (Table 1) and cloned inthese vectors. Because the individual proteins are expressed in a polyprotein, theprimers were designed to add a start codon at the 5� end and a stop codon at the3� end of each gene when appropriate. Furthermore, the primers containedrestriction sites to facilitate cloning. pM-HA-32K was constructed by amplifica-tion of the 32K coding region from pTB1G (14) with primers EcoRI_32K_F andBamHI_32K_R and insertion in pMON-HA. pM-HA-60K was constructed byPCR amplification of the 60K coding region from pTB1G with EcoRI_60K_Fand BamHI_60K_R, and pM-HA-GFP was constructed by amplification of thegreen fluorescent protein (GFP) coding region from pM19GFP10 (16) withprimers EcoRI_GFP_F and BamHI_GFP_R. pM-60K was constructed by am-plification of the 60K coding region from pTB1G (14) with primers XbaI_60K_Fand BamHI_60K_R and insertion in pMON999. pM-32K was constructed byinsertion of an XbaI-SstI fragment of pTB32*, which contains the 32K codingsequence followed by a stop codon and the SstI restriction site (47), intopMB200K. pMB200K contains the entire RNA1-encoded 200K polyprotein fol-lowed by the SstI restriction site in pMON999 (43). The XbaI restriction site ispresent internally in the coding sequence of 32K.

Construction of TRV expression vectors. Tobacco rattle virus (TRV) vectorscarrying CPMV RNA1-encoded proteins were constructed with TRV-GFPc.This plasmid contains cDNA from TRV RNA2 and an engineered subgenomicpromoter followed by the GFP gene (23). First this plasmid was modified tocontain a start codon followed by the coding sequence of the HA epitope tag anda multiple cloning site. This was achieved by replacement of the NcoI fragmentreleased from TRV-GFPc with a PCR fragment amplified from pM-HA-32Kwith primers NcoI_HA_F and SstI_TAA_32K_R digested with the same enzyme.This intermediate plasmid was designated TRV-HA-�32K and contains the SalI,PstI, and EcoRI sites directly following the coding sequence for the HA epitope.These restriction sites and the EcoRI, SstI, or KpnI present at the 3� end can beused to insert a gene of choice as an N-terminal fusion with the HA tag.

TRV-HA-24K was constructed by PCR amplification of the 24K coding

FIG. 1. Schematic diagram of CPMV RNA1 and the TRV RNA2-based expression vector used to express the CPMV nonstructural pro-teins. (A) Genetic organization and translational expression of CPMVRNA1. Open reading frames in the RNA molecules (open bars), VPg(black square), cleavage sites (Q/G, Q/M, and Q/S), and nucleotidepositions of start and stop codons are indicated. Abbreviations: pro,proteinase; co-pro, cofactor for proteinase; ntb, nucleotide bindingprotein; pol, core RdRp. (B) Organization of the constructs in theplant expression vector pMON-HA used to transiently express CPMVRNA1-encoded proteins in cowpea protoplasts. Expression is drivenby an enhanced CaMV 35S promoter (P-e35S), and transcription isterminated at a nopaline synthase terminator sequence (Tnos). An HAepitope tag was added to facilitate immunodetection of the expressedproteins (hatched box). 60K is composed of the 58K nucleotide-bind-ing protein and VPg. (C) Organization of the expression vector TRV-GFPc and the TRV constructs used to express CPMV RNA1-encodedproteins in N. benthamiana leaves. The duplicated promoter region

with downstream cloning sites (arrow) permits transcription of a sub-genomic RNA for expression of foreign gene inserts. The 110K poly-merase is composed of the 24K proteinase and the 87K core polymer-ase as indicated. Additional constructs were made that contain a fusionwith GFP. VAP27 is an A. thaliana protein that is inserted in the ERmembrane. The position of the HA epitope tag is indicated with ahatched box.

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sequence from pTB1G with primers NcoI_24K_F and SstI_TAA_24K_R. ThisPCR product was digested with NcoI (filled in with Klenow polymerase) and SstIand cloned in TRV-HA-�32K digested with EcoRI (filled in with Klenow poly-merase) and SstI. TRV-HA-32K was constructed by PCR amplification of the32K coding sequence from pTB1G with primers NcoI_32K_F andSstI_TAA_32K_R. This PCR product was digested with HindIII and SstI and wascloned in TRV-HA-�32K digested with HindIII and SstI. TRV-HA-60K wasconstructed by PCR amplification of the 60K coding sequence from pMON-HA-60K with primers NcoI_60K_F and EcoRI_TAA_60K_R. This PCR product wasdigested with EcoRI and cloned in TRV-HA-�32K digested with EcoRI. Toconstruct TRV-HA-110K, the 110K coding sequence was PCR amplified frompTB1G with primers NcoI_110K_F and SstI_TAA_110K_R, digested with NcoIand SstI, and cloned in TRV-GFPc digested with the same enzymes, resulting inclone TRV-110K. Subsequently TRV-HA-24K was digested with KpnI, and thereleased fragment was replaced with the KpnI fragment of TRV-110K to con-struct TRV-HA-110K. The fusion constructs of the different viral proteins withGFP were made by using the SalI restriction site directly downstream of the HAepitope sequence. The GFP coding sequence was PCR amplified frompM19GFP10 (16) with primers SalI_GFP5_F and SalI_GFP5_R. For the con-struction of TRV-HA-VAP27, the complete coding sequence of the 27-kDaVAMP-associated protein (VAP27) was released as an EcoRI fragment frompGAD10-VAP27, a clone isolated from an Arabidopsis thaliana cDNA library.This fragment was ligated in TRV-HA-�32K digested with the same enzyme.

Transfection of cowpea protoplasts and infection of plants. Cowpea mesophyllprotoplasts were prepared and transfected by polyethylene glycol-mediatedtransformation of plasmid DNA as described previously (43). Five-week-oldwild-type N. benthamiana plants or N. benthamiana plants carrying themGFP5-ER transgene (34) were dusted with carborundum and infected withTRV RNA1 together with transcripts of the TRV RNA2 recombinants as de-scribed previously (23).

Subcellular fractionation and immunoblotting. Protoplasts transfected withthe transient expression vectors were collected 16 h posttransfection and centri-fuged at 30,000 � g, and proteins from the supernatant and pellet fraction wereelectrophoresed in 10% polyacrylamide gels and immunoblotted as describedpreviously (5) with the mouse monoclonal antibody anti-HA (clone 12CA5;Boehringer) recognizing the HA epitope tag.

For immunoblot analysis of the proteins produced by the TRV expressionvector, infected leaves were collected 1 day postinfection and ground with amortar and pestle with 2 ml of extraction buffer (50 mM Tris-acetate [pH 7.4], 10mM potassium acetate, 1 mM EDTA, 5 mM dithiothreitol, 0.5 mM phenylmeth-ylsulfonyl fluoride per g of fresh weight). After extraction, the homogenates werecentrifuged, electrophoresed, and immunoblotted as described earlier (5) withanti-HA or anti-GFP (Clontech). In some cases, homogenates were not centri-fuged, but total protein was extracted with sample buffer (10% glycerol, 5%�-mercaptoethanol, 2% sodium dodecyl sulfate, 0.01% bromophenol blue, 75mM Tris-HCl, pH 6.8), boiled for 3 min, and loaded on the gel.

Fluorescence microscopy. A Zeiss LSM 510 confocal microscope was used toobtain images. GFP fluorescence was observed with standard settings (excitationwavelength, 488 nm; emission band-pass filter, 505 to 550 nm). Red chlorophyllfluorescence was detected with an excitation wavelength of 543 nm and an

emission long-pass filter of 560 nm. To observe GFP fluorescence in livingepidermal cells, large samples (2 by 2 cm) were cut from N. benthamiana leavesinfected with the TRV recombinants 1 day postinfection. The samples wereplaced on a glass slide, and after a drop of water was applied on top of thesamples, they were covered with a coverslip. The samples were directly analyzedby confocal laser scanning microscopy.

RESULTS

Expression of individual CPMV RNA1-encoded proteins inplant cells. In previous studies several individual CPMV pro-teins were transiently expressed in cowpea protoplasts by usingthe plant expression vector pMON999, which carries the en-hanced CaMV 35S promoter (30, 43). Those studies concernedthe viral RdRp and therefore were focused on the C-terminal110K region of the 200K polyprotein. In the present study theproteins contained in the N-terminal region were includedalso. For this purpose, the coding sequences for the 32K pro-teinase cofactor and the 60K nucleoside triphosphate (NTP)binding protein were inserted into the multiple cloning site ofpMON999 to produce pM-32K and pM-60K. The constructswere used to transfect protoplasts, which were then harvested16 h posttransfection. Homogenates of the protoplasts weresubjected to centrifugation at 30,000 � g, crudely separatingsoluble proteins from membrane-bound proteins. It was shownpreviously by immunoblotting procedures that 110K and 87Kaccumulate to high levels in protoplasts and are found mainlyin the soluble fraction (S30) (43). 32K and 60K were notdetectable in either S30 or the pellet fraction (P30) with anti-32K and anti-VPg sera (data not shown). The failure to detect32K and 60K might be due to low expression levels of theseproteins and/or to low titers of the antisera. To avoid the latterpossibility, constructs were made with the HA epitope tagfused to the N terminus of 32K and 60K (Fig. 1B). As a positivecontrol, the HA epitope was also fused to GFP. Western blotanalysis of extracts from transfected protoplasts with mousemonoclonal antibodies raised against the HA epitope revealedthat HA-GFP accumulated to readily detectable levels and waspresent mainly in the S30 fraction (Fig. 2). HA-60K accumu-lated to much lower levels and was present mainly in the P30fraction (Fig. 2), whereas 32K was not detectable in either S30or P30.

TABLE 1. Oligonucleotides used in this study to insert coding sequences of CPMV replication proteins and GFP in differentexpression vectors

Primer Sequence

EcoRI_32K_F ..................................................................................................................GAGAATTCGGTCTCCCAGAATATGAGGBamHI_32K_R ................................................................................................................GGGGATCCTCACTGTGCATTGTTCTTTTCACEcoRI_60K_F ..................................................................................................................CGGAATTCAGTAGTCCTGTTATCCTCTTAGBamHI_60K_R ................................................................................................................CCGGATCCTTATTGTGCGTCTGCCCAAAEcoRI_GFP_F .................................................................................................................CGGAATTCATGAGTAAAGGAGAAGAACTTTTCACTBamHI_GFP_R ...............................................................................................................CCGGATCCTTATTTGTATAGTTCATCCATGCNcoI_HA_F......................................................................................................................GGGACCATGGCTTATCCATACGATGTTCCANcoI_24K_F .....................................................................................................................GGGGCCATGGCTTCTTTGGATCAGAGTAGTGTTSstI_24K_R.......................................................................................................................CCCGAGCTCTTATTGCGCTTGTGCTATTGGNcoI_32K_F .....................................................................................................................GGGGCCATGGGTCTCCCAGAATATEcoRI_TAA_32K_R .......................................................................................................CCCGAATTCTTACTGTGCATTGTCCTTTTCACCNcoI_60K_F .....................................................................................................................GGGGCCATGGCTAGTAGTCCTGTTATCCTCTTAEcoRI_TAA_60K_R .......................................................................................................CCCGAATTCTTATTGTGCGTCTGCCCAAACTCSstI_TAA_110K_R..........................................................................................................CCCGAGCTCCTAAACATCAGAAAAAGCGAAATTGASalI_GFP5_F....................................................................................................................GGGGGGTCGACGATGAGTAAAGGAGAAGAACTTTTCSalI_GFP5_R ...................................................................................................................CCCCCGTCGACTCTTTGTATAGTTCATCCATGC

VOL. 76, 2002 CPMV 32K AND 60K TARGET ER 6295

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To confirm the results obtained in the protoplast system, theindividual CPMV RNA1-encoded proteins were expressed inplants by using the TRV expression system (23). TRV does notreplicate and spread in cowpea plants, which are the naturalhost of CPMV. However, in N. benthamiana, which is also asystemic host for CPMV, the TRV-based expression systemwas shown previously to give high levels of foreign proteinexpression (23). Since TRV does not cause visible morpholog-ical changes to the host endomembrane system as observed byelectron microscopy (9) and is not related to CPMV, a TRVinfection probably does not interfere with proper targeting of

the CPMV proteins. Vector TRV-GFPc contains the codingsequence of GFP in a cDNA clone of TRV RNA2 in whichGFP can be replaced with other genes of choice (Fig. 1C) (23).To facilitate immunodetection of the CPMV proteins, TRV-GFPc was modified to encode the HA epitope followed by amultiple cloning site. Subsequently, the coding sequences forthe 24K proteinase, the 32K proteinase cofactor, the 60K NTPbinding protein, and the 110K polymerase were each insertedinto the TRV vector immediately downstream of the HA tag,thereby replacing the GFP gene (Fig. 1C). In vitro transcriptsof these constructs were combined with TRV RNA1 and usedas inoculum on N. benthamiana. Homogenates of infectedleaves were separated into P30 and S30 fractions. Sodiumdodecyl sulfate-polyacrylamide gel electrophoresis was per-formed on samples of these fractions followed by Western blotanalysis with mouse monoclonal antibodies recognizing theHA epitope present at the N terminus of the proteins. 24K and110K were present predominantly in the soluble fraction,whereas 60K was present in the crude membrane fraction (Fig.3A). We were unable to detect 32K in either fraction (Fig. 3A).To confirm that membrane-associated proteins are localized tothe pellet during fractionation, cDNA of an A. thaliana integralmembrane protein, VAP27, was cloned into TRV as a fusionwith the HA epitope tag. VAP27 is a SNARE-like protein thatis inserted posttranslationally into the ER membrane via aC-terminal transmembrane domain (37). Homogenates ofTRV-VAP27-infected leaves were fractionated, and like 60K,VAP27 was detected predominantly in the P30 fraction (Fig.3A).

These data suggest that 60K associates with membraneswhen expressed in the absence of other CPMV-encoded pro-teins whereas 24K and 110K behave as cytoplasmic, solubleproteins. We were unable to detect the 32K protein by immu-noblotting either expressed in N. benthamiana leaves with theTRV expression system or transiently expressed in cowpeaprotoplasts.

FIG. 2. Immunodetection of CPMV RNA1-encoded proteins indifferent subcellular fractions of cowpea protoplasts transfected withtransient expression vectors. Protoplasts were harvested 16 h post-transfection, and homogenates were separated in the supernatant (s)and pellet fraction (p) by centrifugation at 30,000 � g. Detection of theproteins was done with a mouse monoclonal anti-HA antibody. 60Kand GFP are indicated by arrows. Cowpea proteins that cross-reactwith this antibody are indicated by asterisks. “Water” indicates laneswith no DNA added.

FIG. 3. Immunodetection of CPMV RNA1-encoded proteins, either tagged with HA (A) or fused to GFP (B), in different subcellular fractionsof N. benthamiana leaves infected with TRV expression vectors. Infected leaves were collected 1 dpi, and total homogenates (t) were prepared thatwere either loaded directly for TRV-GFP-60K and TRV-GFP-110K or centrifuged at 30,000 � g to separate proteins from the supernatant (s) andpellet fraction (p) for the other constructs. Detection of the proteins was done with mouse monoclonal anti-HA antibody (A) or with a rabbitpolyclonal anti-GFP serum (B). The bands corresponding to the CPMV proteins are indicated by arrows, while N. benthamiana proteins thatcross-react with anti-HA are indicated by asterisks.


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Intracellular localization of the replication proteins. To vi-sualize and characterize the protein-membrane interaction inlive cells, TRV expression vectors encoding fusions of theCPMV proteins with GFP were constructed. First it was es-tablished by immunoblotting that the constructs were properlyexpressed. Homogenates of infected leaves were separated bycentrifugation in the P30 and S30 fractions. By using anti-GFPserum, GFP-24K was found present mainly in the S30 fraction(Fig. 3B). In contrast to native 32K, GFP-32K was detectableby immunoblotting and was found to be present mainly in theP30 fraction (Fig. 3B). Expression of GFP-60K and GFP-110Kwas detected in the leaf homogenates (Fig. 3B); however, afterfractionation in the P30 and S30 fractions no specific bandscould be visualized for unknown reasons (data not shown).

The intracellular localization of the fusion proteins was ex-amined in infected leaf epidermal cells 1 day postinoculation(dpi) by confocal laser scanning microscopy. Cells containingGFP-24K (data not shown) or GFP-110K (Fig. 4A) displayeda pattern of fluorescence similar to that of nonfused GFP (Fig.4B), and fluorescence was present diffusely in the cytoplasmand in the nucleus. Since GFP is excluded from the vacuolethat takes up more than 90% of the cell volume in epidermalcells, cytoplasmic GFP is confined to a small layer between thevacuole and the cell wall (the cortex) and to a small region nearthe nucleus (Fig. 4B). The occurrence of GFP in the nucleus isin line with earlier reports that indicate that GFP alone orfused to a soluble protein accumulates partly in the nucleus,probably by means of passive diffusion through the nuclearpore (46). The fluorescence pattern of GFP-60K differed fromthat of nonfused GFP. GFP-60K fluorescence was most obvi-ous in a ring surrounding the nucleus, in small rings surround-ing spherical organelles in the cytoplasm, and in one or severalspherical aggregates often near the nucleus, while no fluores-cence was present in the nucleus (Fig. 4C). The fluorescentring around the nucleus was reminiscent of what we haveobserved in transgenic N. benthamiana plants expressing ER-GFP (7), a GFP fusion protein that contains an N-terminalcleavable signal peptide and a C-terminal HDEL retentionsignal and therefore is targeted to the lumen of the ER (18)(compare Fig. 4C with Fig. 5B). It thus appears that GFP-60Klocalizes to the ER membranes that are contiguous with thenuclear envelope. Alternatively, 60K is present in the nuclearenvelope itself. Red chlorophyll autofluorescence was ob-served in the small spherical organelles (Fig. 4C, inset), sug-gesting that GFP-60K is also present in the outer membrane ofplastids. The nature of the spherical aggregates near the nu-cleus (Fig. 4C, arrow) is unknown, but it might be a structurederived from ER membranes, as pointed out below. Also thefluorescence observed with GFP-32K differed markedly fromthat for nonfused GFP. The fluorescence was most obvious atthe cortex of the infected cells and was present in a clearreticulate pattern (Fig. 4D) closely resembling the pattern ob-served with ER-GFP in transgenic plants (compare Fig. 4Dwith Fig. 5A). It thus appears that GFP-32K localizes to thecortical ER network. In some cells the reticulate pattern wasless bright and GFP-32K was additionally present in largehighly fluorescent aggregates at the cortex, which may be de-rived from ER membranes (Fig. 4E). No fluorescence of GFP-32K was observed in the nuclear envelope (data not shown),which is in contrast to the fluorescence observed in transgenic

plants that express ER-GFP or in TRV-GFP-60K-infectedplants.

These data corroborate the cell fractionation data and indi-cate that individually expressed 24K and 110K are cytosolic,soluble proteins and that 32K and 60K associate with mem-branes mainly derived from the ER.

Expression of 32K and 60K modifies ER morphology.CPMV infection induces an extensive rearrangement of ERmembranes, a process independent of RNA2-encoded pro-teins (7). To investigate whether a specific RNA1-encodedprotein is responsible for these membrane modifications, theTRV constructs each carrying an individual RNA1-encodedprotein (not fused to GFP) were used to infect N. benthamianaplants transgenic for GFP targeted to the lumen of the ER (18,34). At 1 dpi the morphology of the ER membranes in infectedepidermal cells was observed and compared to that of mock-infected cells. TRV infection itself had no influence on ERmembrane morphology, since, in plants infected with TRV,ER-GFP fluorescence in epidermal cells did not differ fromthat in mock-infected cells and fluorescence was detected inthe typical cortical ER network (Fig. 5A) and the nuclearenvelope (Fig. 5B). In TRV-24K- and TRV-110K-infectedcells the fluorescence pattern of ER-GFP was also indistin-guishable from that of mock-infected cells (data not shown). Incontrast, the ER in cells infected with TRV-32K or TRV-60Kdiffered radically from the ER in mock-infected cells. In TRV-32K-infected cells, ER-GFP fluorescence was present in largeamorphous aggregates that were connected with the corticalER network (Fig. 5C) and in spherical aggregates (Fig. 5D)which were often near the nucleus. While in TRV-60K-in-fected cells these spherical aggregates were also found near thenucleus (Fig. 5E), cortical ER aggregates were not observed(data not shown). Both types of aggregates resembled theregions of proliferative ER membranes induced in CPMV-infected cells, although the extent of ER proliferation inCPMV-infected cells was generally larger (Fig. 5F). The ERmodifications seen with TRV-32K and with TRV-60K may beartifacts that occur upon expression of any ER membrane-associated protein with the TRV-based expression system. Thiswas tested by expressing the integral ER membrane proteinVAP27 with the TRV vector. When TRV-VAP27 was used toinfect ER-GFP transgenic plants, no changes were found in theER-GFP fluorescence pattern from that of mock-infected cells(data not shown).

From these data we conclude that expression of 32K and60K induced drastic morphological changes in the ER, resem-bling CPMV-induced ER membrane proliferation, whereas24K and 110K expression did not influence ER morphology.

Expression of 32K and 60K induces necrosis. During thecourse of the experiments a severe effect of prolonged expres-sion of 32K and 60K on cell viability was observed. TRVinfection caused no visible symptoms on inoculated leaves ofN. benthamiana, but a slight curling of the systemically infectedleaves was apparent 4 dpi. Infection of leaves with TRV-GFPallowed us to monitor the spread of TRV from the initialinfection sites to the systemic leaves by using a handheld UVlamp. One day after infection GFP fluorescence was visible asgreen spots on inoculated leaves. With time these spots grewlarger, and 3 dpi fluorescence was observed in large patchesand in the major veins of the inoculated leaves as well as in the


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veins of the systemically infected leaves (Fig. 6A�). In contrastto TRV and TRV-GFP (Fig. 6A), infection with TRV-32K orTRV-60K triggered formation of necrotic spots on the inocu-lated leaves. The first sign of lesion formation occurred fromapproximately 2 dpi onward when infected tissue developed aglassy appearance. At 3 dpi the developing lesions were moredistinct from the surrounding tissue, appearing more clearlynecrotic and collapsed (Fig. 6B and C). The necrotic tissue wastypically confined to the inoculated leaves, but occasionallysome necrosis was observed in the upper, noninoculated leavesat 4 to 6 dpi (data not shown). This suggests that movement ofthe recombinant TRV RNA2 is severely inhibited. Also theTRV construct carrying the GFP-32K fusion produced forma-tion of necrotic spots, but TRV-GFP-60K did not trigger ne-crosis (data not shown). Infection with TRV-24K, TRV-110K,TRV-GFP-24K, TRV-GFP-110K, and TRV-VAP27 did notresult in the formation of necrotic lesions, although a slightyellowing of infection sites was observed for TRV-110K at 6dpi (data not shown). These results suggest that prolongedexpression of 32K or 60K or the fusion of 32K with GFP fromthe TRV expression vector leads to cell death. This was rathersurprising, since CPMV does not cause necrosis on N.benthamiana plants and moves systemically to the upper leavesand high amounts of 32K and 60K accumulate during theinfection. To determine whether 32K and 60K expression alsoproduced necrosis in other host plants, Nicotiana clevelandii,Nicotiana tabacum, and Nicotiana glutinosa plants were in-fected with TRV-32K, TRV-60K, and TRV-GFP as control. Inall these Nicotiana species, necrosis was observed at 2 to 3 dpion inoculated leaves infected with TRV-32K and TRV-60K,whereas leaves infected with TRV-GFP were symptomless(Fig. 6D to G and data not shown).

DISCUSSION

In this study, the CPMV 32K and 60K replication proteinswere shown to associate with membranes mainly derived fromthe ER, when expressed in isolation in plant cells. Moreover,expression of the 32K protein and to a lesser extent 60K ef-

fected proliferation of the ER resembling what occurs uponCPMV infection. Other RNA1-encoded proteins, the 110Kpolymerase and the N-terminal cleavage product 24K protein-ase, behaved as freely soluble proteins when expressed in iso-lation. We propose that localization signals in 32K and 60Ktarget the replication complexes to ER membranes and thatthis physical association causes an ER membrane rearrange-ment that may result in formation of the small membranousvesicles that are the site of CPMV RNA synthesis.

During CPMV infection the replication proteins accumulatein both electron-dense structures and the adjacent small mem-branous vesicles where CPMV replication occurs (10, 48). Toanalyze the properties of the individual RNA1-encoded non-structural proteins, they were expressed separately with a tran-sient expression system in cowpea protoplasts and with theTRV expression vector in N. benthamiana. In contrast to the110K (87K plus 24K) polymerase or the 24K proteinase, whichaccumulated to high levels in both systems, the 60K nucleotide-binding protein accumulated poorly, while the 32K cofactor forthe protease was not detectable by immunoblot analysis, whichmay suggest that 32K and 60K have a high turnover rate. Thisis in line with previous findings in which expression of 32K and60K in insect cells or in Escherichia coli cells yielded consid-erably lower protein levels than did expression of 110K (29, 33,41, 42). The high levels of 32K and 60K accumulating duringCPMV infection may be the result of aggregation in the elec-tron-dense material. The role of the electron-dense structuresin the viral life cycle and how the replication proteins end upin this structure remain unclear. Alternative explanations forthe low expression levels of 32K and 60K may be the cytotox-icity of the proteins or the possibility that sequences in theRNA either inhibit mRNA translation or stimulate mRNAdegradation. Fusion of GFP to 32K improved the expressionlevels, and GFP-32K, like 60K, was found to be present mainlyin the crude membrane fraction. Comparison of the GFP-32Kfluorescence with that of GFP-60K revealed that 32K waspresent mainly in the cortical ER whereas GFP-60K was foundmainly in the nuclear envelope, the plastidial membrane, and

FIG. 6. Necrosis of leaves from different host plants infected with TRV-32K and TRV-60K. Leaves were infected with the TRV constructs andphotographed 3 dpi. (A and A�) N. benthamiana leaf infected with TRV-GFP photographed either under white light (A) or under UV light (A�),where GFP fluorescence appears green. The red fluorescence in A� is due to chlorophyll. (B to G) Leaves were photographed under white light.(B) N. benthamiana leaf infected with TRV-32K. (C) N. benthamiana leaf infected with TRV-60K. (D) N. tabacum leaf infected with TRV-GFP.(E) N. tabacum leaf infected with TRV-32K. (E�) Close-up of panel E. (F) N. clevelandii infected with TRV-GFP. (G) N. clevelandii infected withTRV-60K.

FIG. 5. Effect of expression of different CPMV proteins on the morphology of the ER. N. benthamiana plants transgenic with GFP targetedto the lumen of the ER (ER-GFP) were infected with TRV expression vectors. Fluorescent signals were visualized by confocal microscopy 1 dpi.In epidermal cells infected with TRV the pattern of ER-GFP fluorescence is not distinguishable from that in mock-infected cells and is presentin a cortical reticulate pattern (A) and in a ring around the nucleus (arrowhead; B). In cells infected with TRV-32K ER-GFP fluorescence isadditionally present in large aggregates connected to the cortical network (C) and in one or several spherical aggregates (arrow; D) often near thenucleus (arrowhead). In TRV-60K-infected cells the latter structure is also present (arrow; E). Extensive ER proliferation near the nucleus(arrowhead) of a CPMV-infected cell is shown (F). The images shown in panels B, D, E, and F correspond to single optical sections of 1 �m, whilepanels A and C correspond to projections of serial optical sections. Bars, 10 �m.

FIG. 4. Subcellular distribution of CPMV proteins fused to GFP in N. benthamiana epidermal cells infected with TRV expression vectors.Fluorescent signals were visualized by confocal microscopy 1 dpi. GFP-110K (A) is present in the nucleus (arrowheads) and in a diffuse patternin the cytoplasm, as is nonfused GFP (B). GFP-60K (C) localizes in a ring surrounding the nucleus (arrowhead), in one or several bodies (arrow)often near the nucleus, and in a ring surrounding plastids that autofluoresce in red (inset). GFP-32K localizes in a cortical reticulated network(D) and in cortical fluorescent aggregates (E). The images shown in panels A to C correspond to single optical sections of 1 �m, while panels Dand E correspond to projections of serial optical sections. Bars, 10 �m.


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aggregates presumably derived from the ER. This suggests that32K is more specifically targeted to and/or retained in thecortical ER, in contrast to 60K, which displays affinity forvarious intracellular membranes. It should be noted, however,that fusion of 60K to GFP abolished the ability of 60K toinduce necrosis, which may suggest that 60K could not engagein the proper interactions when fused to GFP. Therefore, thelocalization pattern observed with GFP-60K might not repre-sent the actual localization of the nonfused 60K.

Expression of either 32K or 60K was shown to influence ERmorphology, with 32K inducing proliferation of the cortical ERand formation of one or several small bodies of proliferatedER near the nucleus, whereas 60K induced only the latterstructure. The alterations of the ER morphology resembledthe proliferations that occur in CPMV-infected cells, althoughthe regions of proliferated ER are generally larger in the lattercase. Maybe the combined action of 32K and 60K is necessaryto induce proliferations of the ER that mimic those observedfor CPMV-infected cells. The observation that CPMV non-structural proteins, when expressed in isolation, target ERmembranes and cause ER membrane rearrangements is inagreement with the previously proposed notion that the smallmembranous vesicles that are involved in virus replication orig-inate from the ER (7).

Previous studies showed that 60K, when expressed in insectcells by the baculovirus expression system, induced formationof small membranous vesicles (41). Here we make the firstreport that 32K is also involved in the process of membraneattachment and rearrangement. Unlike the other replicationproteins of CPMV, the 326-amino-acid hydrophobic 32K con-tains no conserved domains such as an NTP-binding motif(present in 60K), a proteinase domain (24K), or a polymerasedomain (110K). In vitro translation studies pointed to a rolefor 32K as a cofactor required for processing of the RNA2-encoded polyprotein (32). Deletion of 32K from the RNA1-encoded 200K polyprotein resulted in more rapid cleavage ofthe 170K processing intermediate both in vivo and in vitro,suggesting that 32K inhibits processing of 200K (32). It is likelythat targeting of and insertion in the membrane of replicaseproteins, polyprotein cleavage, and the initiation of viral RNAsynthesis occur in a coordinated fashion during CPMV repli-cation. Since it was shown previously that 32K remains asso-ciated with 170K after cleavage from the 200K polyprotein(15), 32K might first target the 32K/170K complex to the mem-branous site of replication where, after further processing,initiation of replication occurs.

The properties of 32K and 60K in ER membrane attach-ment and modification resemble those of replication proteinsencoded by different viruses of which poliovirus is the best-studied example. Membrane association of the poliovirus pro-teins encoded by the 2BC3AB region was shown previously tobe mediated by a stretch of hydrophobic amino acids (3AB) orby an amphipathic helix (2BC and 2C) (4, 26, 40). 32K containsthree stretches of hydrophobic amino acids at the C-terminalend (unpublished observations), and 60K contains an amphi-pathic helix at the N terminus (amino acids 45 to 61 [32]) anda 22-amino-acid stretch of hydrophobic amino acids (aminoacids 544 to 565 [unpublished observations]) at the C terminusimmediately upstream of VPg. These domains are conservedamong the comoviruses. Further experiments will be necessary

to verify that these regions are involved in membrane binding,and the TRV expression system might provide a suitable ex-perimental system for that purpose. Apart from binding tointracellular membranes, expression of poliovirus proteins indifferent cell types has revealed that proteins from the2BC3AB region could extensively modify the endomembranesystem. 2BC and 2C induced formation of small membranousvesicles probably derived from the ER that were morphologi-cally similar to poliovirus-induced vesicles (2, 3, 8). The 3Aprotein, when expressed in isolation, associated with ER mem-branes, causing swelling of these membranes, but no smallmembranous vesicles were formed (11, 12). Recently, it wasreported that coexpression of 3A and 2BC resulted in forma-tion of vesicles that were similar, both in ultrastructure and inbiochemical properties, to the vesicles induced in a poliovirusinfection (39). Also, for the equine arteritis virus it was shownelsewhere that coexpression of the membrane proteins nsp2and nsp3 induces the formation of double membrane vesiclesprobably derived from the ER (38). The tobacco etch virus6-kDa protein has been shown to localize specifically to ERmembranes when expressed as a fusion with GFP or glucuron-idase and was proposed to cause the ER modifications that areobserved during tobacco etch virus infection (35). Such mem-brane-associated proteins may play a dual role. Firstly, theinduction and accumulation of vesicles could compartmental-ize viral RNA synthesis. Secondly, these proteins could pro-mote the interaction of viral RdRp with the proliferating mem-branes.

Infection with TRV-32K and TRV-60K led to formation ofnecrotic tissue in inoculated leaves from N. benthamiana andother Nicotiana species, suggesting that 32K and 60K are cy-totoxic proteins that cause cell death. Fusion of 60K with GFPabolished its ability to induce necrosis for unknown reasons.The cytotoxic properties of 60K have been reported before forinsect cells expressing 60K with the baculovirus system (42).These cells showed abnormal cytopathic effects and rapid celllysis usually within 48 h postinfection. In the same systemexpression of 32K did not lead to clear effects on cell viability(41). It is remarkable that 32K and 60K induce necrosis onlywhen expressed separately from other RNA1-encoded pro-teins, since CPMV infection of N. benthamiana leads to highaccumulation of 32K and 60K but no visible necrosis. Theaggregation of 32K and 60K in electron-dense structures dur-ing CPMV infection may somehow prevent induction of celldeath by these proteins. The significance of the cytotoxic prop-erties of 32K and 60K for the viral life cycle is unclear. Unlikeanimal viruses, plant viruses do not rely on lysis of host cells fortheir spread through the plant. It has been reported that ex-pression in mammalian cells of poliovirus 2B and 2BC or 2Bfrom the related coxsackievirus resulted in an increase in mem-brane permeability ultimately leading to cell lysis, suggestingthat these proteins play an important role in the release of viralprogeny (1, 45).

Recently it was reported that tobacco mosaic virus modifiedto express the 2B protein of tomato aspermy virus triggered theinduction of a hypersensitive response (HR) resulting in tissuenecrosis, despite the fact that infection of this plant specieswith either wild-type tobacco mosaic virus or wild-type tomatoaspermy virus did not lead to necrosis (22). The HR is a plantdefense mechanism that occurs upon attack by certain patho-


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gens and involves rapid death of plant cells in association withthe restriction of pathogen spread (for a review see reference19). At the moment we cannot exclude the possibility that 32Kand 60K are recognized by the plant and elicit an HR ratherthan causing cell death directly by increasing membrane per-meability.

ACKNOWLEDGMENTS

We gratefully acknowledge Jan Verver and Jeroen Pouwels fortechnical assistance and comments and Janneke Saaijer and Aart vanOmmeren for growing the N. benthamiana plants.

J.E.C. was supported by The Netherlands Foundation for ChemicalResearch (CW) with financial aid from The Netherlands Foundationfor Scientific Research (NWO).

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Cowpea Mosaic Virus 32- and 60-Kilodalton Replication Proteins

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