Virology 275, 444–451 (2000) doi:10.1006/viro.2000.0512, available online at http://www.idealibrary.com on View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Soluble, Template-Dependent Extracts from Nicotiana benthamiana Plants Infected with Potato Virus X Transcribe both Plus- and Minus-Strand RNA Templates Carol A. Plante,* Kook-Hyung Kim,*,1 Neeta Pillai-Nair,* Toba A. M. Osman,† Kenneth W. Buck,† and Cynthia L. Hemenway*,2 *Department of Biochemistry, North Carolina State University, Raleigh, North Carolina 27695; and †Department of Biology, Imperial College of Science, Technology, and Medicine, London, SW7 2AZ, United Kingdom Received May 4, 2000; returned to author for revision June 22, 2000; accepted July 7, 2000 We have developed a method to convert membrane-bound replication complexes isolated from Nicotiana benthamiana plants infected with potato virus X (PVX) to a soluble, template-dependent system for analysis of RNA synthesis. Analysis of RNA-dependent RNA polymerase activity in the membrane-bound, endogenous template extracts indicated three major products, which corresponded to double-stranded versions of PVX genomic RNA and the two predominant subgenomic RNAs. The endogenous templates were removed from the membrane-bound complex by treatment with BAL 31 nuclease in the presence of Nonidet P-40 (NP-40). Upon the addition of full-length plus- or minus- strand PVX transcripts, the corresponding-size products were detected. Synthesis was not observed when red clover necrotic mosaic dianthovirus (RCNMV) RNA 2 templates were added, indicating template specificity for PVX transcripts. Plus-strand PVX templates lacking the 3Ј terminal region were not copied, suggesting that elements in the 3Ј region were required for initiation of RNA synthesis. Extracts that supported RNA synthesis from endogenous templates could also be solublized using sodium taurodeoxycholate and then rendered template-dependent by BAL 31 nuclease/NP-40 treatment. The solubilized preparations copied both plus- and minus-strand PVX transcripts, but did not support synthesis from RCNMV RNA 2. These membrane-bound and soluble template-dependent systems will facilitate analyses of viral and host components required for PVX RNA synthesis. © 2000 Academic Press INTRODUCTION (Bates et al., 1995), and turnip crinkle virus (Song and Simon, 1994). Some of these template-dependent sys- The development of template-dependent, RNA-depen- tems have been utilized for analyses of host and viral dent RNA polymerase (RdRp) preparations from tissues or cells infected with plus-strand RNA viruses has facil- protein components contained in the RdRp complexes itated analyses of the components and reactions neces- (for reviews, see Buck, 1996; and Lai, 1998). Several of sary for RNA synthesis. Such systems have been devel- the plant viral RdRp preparations have been useful for oped for several viruses that infect a variety of hosts, the determination of template requirements for initiation including bacteriophage Q␤ (Blumenthal and Car- of minus-strand RNA synthesis (for reviews, see Buck, michael, 1979), black beetle virus (Saunders and Kaes- 1996; and Dreher, 1999). The BMV and TCV RdRp prep- berg, 1985), flockhouse virus (Wu et al., 1992), polio virus arations have additionally been utilized for analysis of (van Dyke and Flanegan, 1980; Baron and Baltimore, plus-strand RNA synthesis in vitro (Miller et al., 1985; 1982), Sindbis virus (Lemm et al., 1998), and the plant Adkins et al., 1997; Guan et al., 1997, 2000a, b; Siegel et viruses alfalfa mosaic virus (Houwing and Jaspars, 1986; al., 1997; Wang and Simon, 1997; Sivakumaran and Kao, Quadt et al., 1991), brome mosaic virus (Miller and Hall, 1999; Sivakumaran et al., 1999) and for studying recom- 1983; Quadt and Jaspars, 1990), cucumber mosaic virus bination mechanisms (Nagy et al., 1998; Nagy and Si- (Hayes and Buck, 1990; Quadt and Jaspars, 1991), turnip mon, 1998a, b). For the most part, such requirements yellow mosaic virus (Mouches et al., 1974; Garbouri- have been investigated for plant viruses containing ei- Bouzid et al., 1991; Deiman et al., 1997; Singh and Dreher, ther a tRNA-like 3Ј terminus or other predicted struc- 1997), tobacco mosaic virus (Osman and Buck, 1996; tures, but not for viruses containing polyadenylated ter- Watanabe et al., 1999), red clover necrotic mosaic virus mini such as those in the Potexvirus genus. Potato virus X (PVX), the type member of the Potexvirus genus, is a flexuous rod-shape particle containing a 1 Current address: School of Applied Biology and Chemistry, College capped and polyadenylated genomic RNA of 6.4 kb of Agriculture and Life Science, Seoul National University, Suwon, (Skryabin et al., 1988a, b). This RNA contains an 84- Korea 441-744. 2 Ј To whom correspondence and reprint requests should be ad- nucleotide (nt) 5 nontranslated region (NTR), five open dressed. reading frames (ORFs), and a 72-nt 3Ј NTR (Fig. 1A). The 0042-6822/00 $35.00 Copyright © 2000 by Academic Press 444 All rights of reproduction in any form reserved. TEMPLATE-DEPENDENT EXTRACTS FROM N. benthamiana 445 tabacum (NT-1) protoplasts and plants (Kim and Hemen- way, 1996; Miller et al., 1998, 1999). Conserved oc- tanucleotide sequence elements located upstream of the two major PVX sgRNAs are important for sgRNA accu- mulation (Kim and Hemenway, 1997, 1999), and interac- tions between these elements and complementary ter- minal sequences are important for optimal plus-strand RNA accumulation. Elements in the 3Ј NTR also effect PVX RNA accumulation in NT-1 cells (Pillai-Nair, Kim, and Hemenway, unpublished data). The development of a soluble, template-dependent system would allow for mechanistic studies of PVX RNA synthesis in vitro and identification of protein compo- nents and activities necessary for PVX replication. An RdRp preparation from barley plants infected with an- other potexvirus, foxtail mosaic virus (FMV), was devel- oped, but treatment of these preparations with micrococ- cal nuclease for removal of endogenous template ren- dered them inactive (Rouleau et al., 1993). Previously, we isolated extracts containing membrane-bound PVX RdRp from Nicotiana tabacum, but also were not able to elim- inate the endogenous viral RNA (Doronin and Hemen- way, 1996). Li et al. (1998) recently demonstrated tem- plate-dependent RdRp activity in Escherichia coli trans- FIG. 1. PVX genome organization and transcripts derived from PVX formed with the PVX replicase protein using short PVX cDNA clones. A illustrates the PVX genome, depicting five open read- templates; however, this system cannot be utilized to ing frames as boxes and the two major subgenomic RNAs as arrows study the in vivo composition of the replication complex. below the genome. Transcripts utilized for template-dependent RdRp assays were derived from various PVX cDNA clones shown in B. Here we report the isolation and solubilization of a tem- Plus-strand RNA templates were derived from wild-type pMON8453, plate-dependent RdRp activity isolated from Nicotiana p32 (containing an internal deletion of most of ORF 1, as noted by benthamiana plants infected with PVX that supports RNA dotted line), and ⌬84 (lacking the first 84 nucleotides of the 5Ј end) by synthesis from both plus- and minus-strand PVX tem- transcription with bacteriophage T7 RNA polymerase (shown as arrow plates. to left) of SpeI-digested plasmids. These transcripts contain 17 A residues and a G residue (from the SpeI site) at their 3Ј termini. Truncated plus-strand transcripts lacking the 3Ј region of the PVX RESULTS AND DISSUSSION genome were generated by transcription of ⌬84 digested with BstEII. Full-length minus-strand transcripts were obtained by transcription of Products produced from endogenous templates are BstBI-digested p75 with with bacteriophage SP6 RNA polymerase. primarily double-stranded With the ultimate goal of developing an extract con- first ORF encodes a 165-kDa protein (P1) that has ho- taining a template-dependent RdRp, we first developed a mology to other known RNA-dependent RNA polymerase membrane-containing RdRp preparation from N. proteins (Rozanov et al., 1992) and is the only viral protein benthamiana plants inoculated with PVX. Similar to the required for RNA synthesis in tobacco (Longstaff et al., procedure published for TMV (Osman and Buck, 1996), a 1993). Three internal reading frames (ORFs 2–4), referred P30 pellet was resuspended and purified through a su- to as the triple gene block, encode proteins involved in crose density gradient. Similar to the TMV system, anal- cell-to-cell movement (Beck et al., 1991; Angell et al., ysis of RdRp activity on endogenous PVX templates in- 1996). ORF 5 encodes the coat protein, which functions dicated that the near-colorless, sucrose gradient fraction in cell-to-cell movement (Chapman et al., 1992; Baul- 3 (F3) and the dark green fraction 4 (F4) contained the combe et al., 1995) and encapsidation. Replication of peak of activity (data not shown). As indicated in Fig. 2 PVX in plant tissue results in the production of genomic- (lane 3), analysis of the RNA products using F3 from length plus- and minus-sense RNAs, two major plus- infected plants (Inf) on a denaturing gel indicated RNAs sense subgenomic RNAs, and corresponding double- migrating at approximately 6.4, 2.5, and 0.9 kb. Products stranded RNAs (Dolja et al., 1987). We have demon- treated with S1 nuclease prior to electrophoresis exhib- strated that sequence elements, as well as a stem–loop ited a similar profile (Fig. 2, lane 4), indicating that these structure located in the 5Ј nontranslated region of the products are primarily double-stranded. In contrast, ex- PVX genome, are required for both genomic and sub- tracts from mock inoculated plants (M), did not support genomic plus-strand RNA accumulation in Nicotiana synthesis of these reaction products (lane 2). This profile 446 PLANTE ET AL. nuclease or treatment with KCl in the presence of deter- gents did not significantly remove the endogenous tem- plates (data not shown).