JOURNAL OF VIROLOGY, July 1996, p. 4795–4799 Vol. 70, No. 7 0022-538X/96/$04.0010 Copyright q 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 b-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 3 g. The supernatant was centrifuged for 20 4), referred to as the triple block (TB) genes, encode proteins min at 12,000 3 g and, subsequently, for 40 min at 80,000 3 g. involved in cell-to-cell movement (2). ORF 5 encodes the coat The 80,000 3 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 3 g. The final pellet was resuspended in buffer II and tion of genomic-length plus- and minus-sense RNAs, several stored at 2808C. 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 378C for 60 min in 60 mlof approximately 2.1 and 0.9 kb in size, presumably correspond to reaction mixture containing 6 ml 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 mCi of [a- 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 428C. Although maxi- RNA replication, very few studies have focused on the bio- mum levels of these products were observed at 328C, we chose chemistry of membrane-associated replicative complexes (4, to use 378C, 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 378C 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 synthe- sized 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 65% 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 (preferential- ly 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.