Synthesis of Potato Virus X Rnas by Membrane- Containing Extracts

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JOURNAL OF VIROLOGY, July 1996, p. 4795–4799 Vol. 70, No. 7 0022-538X/96/$04.00ϩ0 Copyright ᭧ 1996, American Society for Microbiology

Synthesis of Potato Virus X RNAs by Membrane- Containing Extracts

SERGEY V. DORONIN AND CYNTHIA HEMENWAY* Department of Biochemistry, North Carolina State University, Raleigh, North Carolina 27695-7622

Received 16 October 1995/Accepted 30 March 1996

Membrane-containing extracts isolated from tobacco plants infected with the plus-strand RNA virus, potato virus X (PVX), supported synthesis of four major, high-molecular-weight PVX RNA products (R1 to R4). Nuclease digestion and hybridization studies indicated that R1 and R2 are a mixture of partially single- stranded replicative intermediates and double-stranded replicative forms. R3 and R4 are double-stranded products containing sequences typical of the two major PVX subgenomic RNAs. The newly synthesized RNAs were demonstrated to have predominantly plus-strand polarity. Synthesis of these products was remarkably stable in the presence of ionic detergents.

Potato virus X (PVX), the type member of the Potexvirus tracts were derived from Nicotiana tabacum leaves at 7 days genus, is a flexuous rod-shaped particle containing a single, postinoculation by a procedure similar to that described by genomic RNA of 6.4 kb that is capped and polyadenylated (21, Lurie and Hendrix (15). Inoculated and upper leaves (50 g) 27). Of the five open reading frames (ORFs), the first encodes were homogenized in 150 ml of buffer I (50 mM Tris-HCl [pH a 165-kDa protein (P1) that has homology to other known 7.5], 250 mM sucrose, 5 mM MgCl2, 1 mM EDTA, 10 mM RNA-dependent RNA polymerase (RdRp) proteins (23). P1 is ␤-mercaptoethanol, 10 mM phenylmethanesulfonyl fluoride), the only viral protein required for RNA synthesis in tobacco filtered through two layers of cheesecloth, and centrifuged for protoplasts (14). The three internal reading frames (ORFs 2 to 10 min at 1,000 ϫ g. The supernatant was centrifuged for 20 4), referred to as the triple block (TB) genes, encode proteins min at 12,000 ϫ g and, subsequently, for 40 min at 80,000 ϫ g. involved in cell-to-cell movement (2). ORF 5 encodes the coat The 80,000 ϫ g pellet was washed twice with buffer II (50 mM protein (CP), which is necessary for assembly and cell-to-cell Tris-HCl [pH 7.5], 15% glycerol, 5 mM MgCl2, 1 mM EDTA, movement and may play a role in modulation of replication 1 mM 2-mercaptoethanol) and centrifuged for 40 min at (3). Replication of PVX in plant tissue results in the produc- 100,000 ϫ g. The final pellet was resuspended in buffer II and tion of genomic-length plus- and minus-sense RNAs, several stored at Ϫ80ЊC. This solution is referred to as the membrane- species of plus-sense subgenomic RNAs, and corresponding containing extract. double-stranded RNAs (6). The two major subgenomic RNAs, RNA synthesis was assayed at 37ЊC for 60 min in 60 ␮lof approximately 2.1 and 0.9 kb in size, presumably correspond to reaction mixture containing 6 ␮l of membrane-containing plus-strand RNAs for the TB and CP genes, respectively. Al- extract, 50 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 1 mM ATP, though P1 is required for synthesis of these RNAs, little is 32 1 mM GTP, 1 mM UTP, 20 ␮Ci of [␣- P]CTP (800 Ci/mmol), known about other key components or the biochemical reac- 0.05 mg of actinomycin D per ml, 40 U of placental ribonucle- tions involved in PVX replication. ase inhibitor, and 5 mM vanadyl ribonucleoside complex. RNA On the basis of studies with other plus-strand viruses that products isolated from these reaction mixtures by the addition infect plants, replication typically requires host and viral com- of sodium dodecyl sulfate (SDS) to 0.1%, extraction with phe- ponents that copurify in complexes with viral RNA and cellular nol-chloroform, and elution through Sephadex G-50 spin col- membranes (5). In several cases, treatment of membrane-con- umns were analyzed by agarose gel electrophoresis. As indi- taining extracts from infected plants with nonionic detergents cated in Fig. 1, extracts from infected plants (lane 2) supported and micrococcal nuclease resulted in isolation of soluble RdRp synthesis of high-molecular-weight RNAs (R1 to R4) and a preparations that are template dependent (1, 8–10, 19, 20, 24, lower-molecular-weight product (R5). The largest products, 25, 28). However, complete replication of exogenously added R1 and R2, generally migrated as diffuse bands that overlap. template RNA was observed only for cucumber mosaic virus Both of these products migrated more slowly than the single- (9). For some plant viral systems, treatment of membrane- stranded PVX RNA, whereas R3 and R4 migrated faster than containing extracts with detergents and nucleases was not suf- this marker. In extracts derived from mock-inoculated plants ficient to generate template-dependent preparations (7, 22, 30, (Fig. 1, lane 1), only the R5 product was detected. Product 32). Although these studies indicate that soluble systems gen- profiles were similar and levels of synthesis differed by only erally do not contain all components necessary to support 15% over a temperature range of 25 to 42ЊC. Although maxi- RNA replication, very few studies have focused on the bio- mum levels of these products were observed at 32ЊC, we chose chemistry of membrane-associated replicative complexes (4, to use 37ЊC, because fewer low-molecular-weight products 11, 16, 31). were observed at this temperature (data not shown). At the We have initiated studies to understand the components and 37ЊC incubation temperature, maximum synthesis was detected reactions necessary for replication of PVX RNA by isolating at 60 min (data not shown). These data indicated that mem- membrane-containing extracts from PVX-infected tissue. Ex- brane-containing extracts from PVX-infected plants synthesized specific RNAs that were not observed in extracts from mock-inoculated plants. * Corresponding author. Phone: (919) 515-5719. Fax: (919) 515- Several detergents were added to extracts from infected 2047. plants to determine if conditions that potentially disrupt pro-

4795 4796 NOTES J. VIROL.

of R3 and R4, whereas Sarkosine (Fig. 1, lane 6) increased the levels of R1 and R2, with no detectable change in the levels of R3 or R4. However, when the concentration of Sarkosine was increased to 1% (Fig. 2A, lane 9), the product profile was similar to that observed with 0.1% SDS (Fig. 2A, lane 13). This enhancement of R1 and R2 levels and reduction in R3 and R4 levels may indicate that these detergents inhibit synthesis of R3 and R4, suggesting that these products are produced in a manner different from that of R1 and R2. In addition, the synthesis machinery in these extracts must be very stable under conditions that disrupt membranes, because product levels in 3% Sarkosine and 1% SDS (as measured by counts per minute incorporated) were Ϯ5% of levels observed in the absence of detergent (data not shown). The stability of the products in the extracts was further analyzed by the addition of micrococcal nuclease (preferentially cleaves single strands [33]), V1 nuclease (preferentially FIG. 1. Effects of detergents on RNA synthesis in extracts derived from cleaves double-stranded regions [13]), and benzonase (cleaves PVX-infected N. tabacum. RNA products (R1 to R5) synthesized in extracts both single- and double-stranded molecules [18]) in the ab- from mock-inoculated (lane 1) and PVX-infected (lanes 2 to 6) plants were sence of detergent or at detergent concentrations likely to purified from reaction mixtures and analyzed by agarose gel electrophoresis. disrupt membranes. After nuclease digestion, the products Lanes 1, 2, and 4 depict RNA products synthesized in the absence of detergents. Products isolated from reaction mixtures containing 0.1% SDS, 0.1% Nonidet were isolated from reaction mixtures and analyzed by electro- P-40 (NP 40), or 0.1% Sarkosine (Sark) are shown in lanes 3, 5, and 6, respec- phoresis and autoradiography, as shown in Fig. 2. Because the tively. The arrowhead at left denotes the position of purified PVX RNA. nuclease inhibitor vanadyl ribonucleoside complex was ex- cluded from reaction mixtures for these experiments, addi- tional products or intermediates were observed (compare Fig. tein complexes and/or membranes alter product levels or types 1, lane 2, with Fig. 2A, lane 1). Although one of these products of products synthesized. All RNA product levels increased exhibited a mobility similar to that of PVX RNA, this result after the addition of 0.1% Nonidet P-40 (Fig. 1, lane 5). Treat- was not consistently observed among different extracts and the ment with either 0.1% SDS (Fig. 1, lane 3) or 0.1% Sarkosine identity of this product has not been verified. In the absence of (Fig. 1, lane 6) resulted in decreased production of R5. The detergent (Fig. 2A and B, lanes 1 to 4), all three nucleases addition of SDS (Fig. 1, lane 3) caused an increase in the level digested products (Fig. 2A) and cellular RNAs (Fig. 2B) to of R1 relative to that of R2 and a marked decrease in the levels varying extents, but none resulted in complete digestion. Prod-

FIG. 2. Susceptibility of RNA products to nuclease digestion. (A and B) RNAs were synthesized in the absence of detergent (lanes 1 to 4) or in the presence of 1% Nonidet P-40 (NP 40) (lanes 5 to 8), 1% Sarkosine (lanes 9 to 12), or 0.1% SDS (lanes 13 to 16). The products were either purified directly (lanes 1, 5, 9, and 13) or digested with 60 U of micrococcal nuclease (lanes 2, 6, 10, and 14),1UofV1nuclease (lanes 3, 7, and 11),1Uofbenzonase (lanes 4, 8, 12, and 15), or 60 U of benzonase (lane 16) prior to purification. V1 nuclease was not included in the 0.1% SDS panel, because it is inhibited by this detergent (data not shown). (C and D) RNAs synthesized in the absence (Ϫ) or presence of 0.1% SDS were purified and analyzed directly (lanes 1 and 2) or digested with 25 U of S1 nuclease (lanes 3 and 4). Autoradiograms are shown in panels A and C, and the corresponding stained gels are depicted in panels B and D. Arrowheads at left denote the positions of purified PVX RNAs. VOL. 70, 1996 NOTES 4797 ucts synthesized in extracts containing 1% Nonidet P-40 (Fig. 2A and B, lanes 5 to 8), 1% Sarkosine (Fig. 2A and B, lanes 9 to 12), and 0.1% SDS (Fig. 2A and B, lanes 13 to 16) were highly susceptible to V1 nuclease (Fig. 2A and B, lanes 7 and 11) and benzonase (Fig. 2A and B, lanes 8, 12, 15, and 16). Although V1 is reported to preferentially cleave double- stranded RNAs (13), it digested all products under these conditions. As shown in Fig. 2B, digestion of cellular RNA in the extracts was more effective in 1% Sarkosine and 0.1% SDS. These data are not surprising, since disruption of complexes and membranes with ionic detergents would be expected to make RNAs more accessible. It is likely that the stable RNA species migrating at the same position of PVX RNA in all samples in Fig. 2B is packaged PVX RNA, because virions were observed in electron micrographs of extracts (data not shown). Micrococcal nuclease digested most of the cellular RNAs (Fig. 2B, lanes 10 and 14) in the presence of these ionic detergents, but R2, R3, and R4 products remained resistant to micrococcal nuclease digestion (Fig. 2A, lanes 10 and 14). These products were also stable in the presence of 200 U of micrococcal nuclease and 0.1% SDS (data not shown). These FIG. 3. Analysis of RNA product sizes. RNA products were synthesized in data suggest that micrococcal nuclease preferentially digested the absence of detergent (lanes 4 and 10) or in the presence of 0.1% SDS (lanes 5 and 11) or were treated with micrococcal nuclease after synthesis in the single-stranded RNAs, thereby indicating that R2, R3, and R4 presence of 0.1% SDS (lanes 6 and 12). The products were purified and analyzed are double-stranded products. It can also be concluded that the directly (lanes 1 to 6) or after denaturation by incubation in glyoxal (lanes 7 to R1-R2 products synthesized in the presence of ionic detergents 12). RNA markers of 6.4 (lanes 1 and 7), 2.7 (lanes 2 and 8), and 0.96 (lanes 3 and 9) kb were synthesized by runoff-transcription of different PVX cDNA include partially single-stranded RNAs that can be hydrolyzed 32 by micrococcal nuclease to yield products similar to R2, R3, templates in the presence of [␣- P]CTP. and R4. To further demonstrate which products are double or single stranded, RNAs synthesized in the presence or in the absence results indicate that products generated by nucleolytic cleavage of SDS were purified and subsequently treated with nuclease of R1-R2 are similar in size to R2, R3, and R4 synthesized in S1 (Fig. 2C and D). The R2, R3, and R4 products isolated from the absence of detergent. reaction mixtures with no detergent were similar before (Fig. The electrophoretic patterns of products R2, R3, and R4 2C and D, lane 1) and after (Fig. 2C and D, lane 3) treatment suggested that they may correspond to double-stranded ver- with S1. However, S1 digestion resulted in sharper bands and sions of the full-length genome, subgenomic RNA for the TB reduced smearing between bands, indicating that single- proteins, and subgenomic RNA for CP, respectively. To inves- stranded products or partially single-stranded intermediates tigate this possibility, individual R2, R3, and R4 products were were degraded by the nuclease. Treatment of RNA synthesized isolated and used as hybridization probes for Southern blots in the presence of SDS (Fig. 2C and D, lane 2) with S1 nucle- containing different PVX cDNA fragments obtained by diges- ase (Fig. 2C and D, lane 4) resulted in conversion of the R1-R2 tion of the clone pMON8448 (Fig. 4A). The extent of hybrid- products to species migrating at positions similar to those of ization of probes to a SacI digest of pMON8448 is shown in R2, R3, and R4. Hence, R2, R3, and R4 are primarily double- Fig. 4B. This digest generated three fragments: one of 4.4 kb, stranded molecules, and R1-R2 is a mixture of conformational containing the PVX 5Ј leader and replicase gene (nucleotides isomers that contains partially single-stranded and double- 1 to 4454), one of 3.0 kb, containing pGEM3Zf(Ϫ) vector stranded products. sequence, and one of 2.0 kb, containing the TB genes, CP gene, The sizes of the PVX products isolated from reaction mix- and 3Ј untranslated region (nucleotides 4455 to 6400). None of tures were analyzed by denaturation with glyoxal (17). As in- the R2, R3, or R4 probes hybridized to the 3.0-kb vector dicated in Fig. 3, nondenatured markers (lanes 1 to 3) and fragment. Product R2 hybridized to both the 4.4- and 2.0-kb products (lanes 4 to 6) were compared with denatured markers fragments, suggesting that it contains PVX sequences from (lanes 7 to 9) and products (lanes 10 to 12) on a nondenaturing different regions of the PVX genome and may represent full- agarose gel. As expected, the native R1-R2 products synthe- length, double-stranded PVX RNA. In contrast, products R3 sized in the absence (Fig. 3, lane 4) or presence (Fig. 3, lane 5) and R4 hybridized predominantly to the 2.0-kb fragment. The of detergent migrated above the single-stranded, 6.4-kb PVX low level of hybridization to the 4.4-kb fragment was probably transcript (Fig. 3, lane 3). After denaturation with glyoxal, a due to contamination of the R3 and R4 probes with R2 and/or substantial portion of the products (Fig. 3, lanes 10 to 12) R2 degradation products during isolation. Thus, R3 and R4 migrated at a position similar to those of the denatured PVX contain PVX sequences downstream of the replicase gene. To RNA transcripts (Fig. 3, lane 9). The diffuse bands above this further distinguish the identity of R3 and R4 sequences, a position may correspond to unmodified or incompletely mod- second blot containing a purified 0.8-kb NheI-SpeI (CP gene) ified products or may be higher-molecular-weight species. The fragment and a 0.3-kb Bsu36I-BglII (first of the TB genes) two smaller products that migrated below the 2.7-kb marker fragment was probed (Fig. 4C). The R2 and R3 probes hybrid- and above the 0.96-kb marker were similar in size to the 2.1- ized to both fragments, whereas R4 hybridized only to the and 0.9-kb sizes reported for the two major PVX subgenomic 0.8-kb CP fragment. These data indicated that R3 contains RNAs in infected plants (6). The products generated by mi- sequences found in subgenomic RNAs encoding TB and CP crococcal nuclease treatment of SDS-containing reaction mix- genes and that R4 contains sequences typical of the CP sub- tures (Fig. 3, lane 12) were similar in size to those observed in genomic RNA. reaction mixtures without detergent (Fig. 3, lane 10). These The polarity of the newly synthesized RNAs was determined 4798 NOTES J. VIROL.

The R1-R2 products include partially single-stranded and double-stranded RNAs that are likely to be replicative intermediates and replicative forms. R3 and R4 are double-stranded products containing sequences found in the two major subgenomic RNAs. Although the P1 protein was detected in our membrane-containing extracts from infected plants and not in mock-inoculated plants (data not shown), the extent of P1 involvement in the synthesis of these RNAs remains to be determined. However, it is unlikely that R1 to R4 were synthesized by a host RdRp, because they were not synthesized in extracts from mock-inoculated plants. Typically, host RdRp preparations synthesize RNAs that are small in size (26, 29). Because most products were double stranded, it is unlikely that they were synthesized by terminal transferases. R5 was the only product found in extracts from both infected and mock-inoculated plants and may represent a product synthesized by a host RdRp and/or terminal transferase. We observed remarkable stability of the PVX RNA synthesis machinery to ionic detergents. In the presence of SDS or Sarkosine, a decrease in R3 and R4 levels was accompanied by an increase in R1-R2 accumulation. Hydrolysis of R1-R2 by nucleases to yield products similar in size to R2, R3, and R4 may indicate that these detergents interfere with metabolism of the products or stabilize intermediate forms. Thus, ionic detergents may be useful for studying intermediates in the replication process. A clear understanding of RNA replication will require detailed structural and biochemical analyses of replicative complexes, as well as purification and characterization of activities contributed by individual components. The membrane-containing PVX extracts will be a valuable starting material for such studies. FIG. 4. Hybridization of individual RNA products to PVX cDNA fragments and transcripts. Infected extracts were utilized for synthesis in the presence of This work was supported by Public Health Service grant GM 49841 0.1% SDS and subsequently treated with micrococcal nuclease (as described in to C.H. the legend to Fig. 3). RNA was purified and subjected to native, agarose gel electrophoresis. Products R2, R3, and R4 were excised, purified, and used to REFERENCES probe Southern blots (B and C) containing digests of a PVX cDNA clone and a 1. Bates, H. J., M. Farjah, T. A. M. Osman, and K. W. Buck. 1995. Isolation and Northern blot (D) containing plus- and minus-strand PVX transcripts. (A) Par- characterization of an RNA-dependent RNA polymerase from Nicotiana tial restriction map of the PVX cDNA region in pMON8448. The five ORFs clevelandii plants infected with red clover necrotic mosaic dianthovirus. J. corresponding to replicase, the TB genes, and the CP gene are drawn as open Gen. Virol. 76:1483–1491. rectangles. (B) Southern blot of pMON8448 samples digested with SacI. Indi- 2. Baulcombe, D. C., S. Chapman, and S. Santa Cruz. 1995. Jellyfish green vidual strips were probed with RNA product R2, R3, or R4. The sizes of the fluorescent protein as a reporter for virus infections. Plant J. 7:1045–1053. expected products are noted at left. The 3.0-kb pGEM3Zf(Ϫ) vector (Promega) 3. Chapman, S., T. Kavanagh, and D. Baulcombe. 1992. Potato virus X as a band generated in these digests does not hybridize with the probes. (C) Purified vector for gene expression in plants. Plant J. 2:549–557. fragments containing the PVX CP gene (NheI-SpeI) and a region from the first 4. De Graaff, M., L. Coscoy, and E. M. J. Jaspars. 1993. Localization and of the TB genes (Bsu36I-BglII) were electrophoresed, blotted, and probed with biochemical characterization of alfalfa mosaic virus replication complexes. RNA products R2, R3, and R4. (D) A Northern blot containing plus- and Virology 194:878–881. minus-strand PVX RNAs (ϩ and Ϫ, respectively), transcribed in vitro was cut 5. 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