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J. Biol. Chem., Vol. 280, Issue 8, 7398-7406, February 25, 2005
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From the Instituto de Microbiología Bioquímica, Consejo Superior de Investigaciones Científicas/Universidad de Salamanca, Avda. del Campo Charro s/n Salamanca 37007, Spain
Received for publication, October 25, 2004 , and in revised form, December 10, 2004.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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replicase, consisting of a viral-encoded RdRp and host proteins, which can synthesize the positive and negative viral RNAs in vitro (1). Unlike the holoenzyme of coliphage, most of the replicases of eukaryotic viruses are associated with intracellular membranous structures (2, 3). This makes their identification and characterization difficult. Although biochemical and genetic approaches have been attempted, replication mechanisms of eukaryotic viruses remain to be elucidated. If a membrane-free replication system is available, it may provide direct information on the replication mechanism and also on the components involved in this process.
Most laboratory strains of Saccharomyces cerevisiae harbor 20 S RNA virus. The virus belongs to the genus Narnavirus of the family Narnaviridae (4). It is a persistent virus and has no extracellular transmission pathway. Typical of fungal viruses, 20 S RNA virus does not kill the host nor render phenotypic changes to the host. This virus was originally described as an RNA species induced under nitrogen starvation conditions (5, 6). The copy number of 20 S RNA genome in some strains becomes almost equivalent to those of rRNAs under the induction conditions. The complimentary (negative) strands count less than 1–2% compared with the amount of positive strands, a ratio similar to those found among positive strand RNA viruses (7). The viral genome is small (2514 nt) and has no 3'-poly(A) tail and perhaps no 5'-cap structure (8). It encodes only a single protein of 91 kDa (p91) (9, 10). p91 contains four amino acid motifs well conserved among RdRps. Fewer strains of yeast harbor another narnavirus called 23 S RNA virus. Its genome is slightly larger than 20 S RNA genome (2891 nt) and encodes a single protein of 104 kDa (p104) (8, 11). The RdRp motifs of p91 and p104 are most closely related to those of the RdRps of RNA coliphages (12). These viruses are compatible in the same host and do not exclude each other. When yeast cells harboring these viruses are grown at high temperature (37 °C) the cells accumulate double-stranded RNAs (dsRNAs) called T and W (13). T and W are the dsRNA forms of 23 S RNA and 20 S RNA, respectively (8). Because the viral genomes do not encode coat proteins, the RNA genomes are not encapsidated into viral particles (14–16). Instead, they form ribonucleoprotein complexes with their cognate RNA polymerases in a 1:1 stoichiometry and reside in the host cytoplasm (17).
Previously we detected a 20 S RNA positive strand-synthesizing activity in lysates prepared from 20 S RNA-induced cells (18). The activity was not associated with host cell membranes and migrated in sucrose gradients almost the same as 20 S RNA/p91 complexes. In this study, we analyzed replication intermediates of 20 S RNA synthesis in vitro and found that the native replication intermediates have a single-stranded RNA (ssRNA) backbone, consisting of a positive strand less than unit length and a template negative strand loosely associated, perhaps held by p91. Deproteination with phenol converted the RNA in the intermediate to a double-stranded form. This indicates that W, the dsRNA form of 20 S RNA, is not a replication intermediate but a by-product of 20 S RNA replication. The completion of RNA synthesis in vitro resulted in the release of the synthesized positive strand as well as the negative strand template from the replication complex. Our results suggest that the positive and negative strand ssRNAs released from replication complexes are associated with protein.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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RNA Polymerase Assay—RNA-synthesizing activity was assayed by incubating 
1–2 µl of the lysate in the standard reaction mixture (12.5 µl) that contained 50 mM Tris·HCl, pH 8.75, 5 mM MgCl2, 150 mM NaCl, 1% polyethylene glycol 4000, 0.2% bentonite, 4 mM dithiothreitol, 0.1 mM EDTA, 0.5 mM each of ATP, GTP, and CTP, and 20 µM UTP. 2–10 µCi of [32P]UTP (3000 Ci/mmol, Amersham Biosciences) were also included in the reactions. After incubation at 30 °C, the reaction was stopped by the addition of 0.3% SDS and 5 mM EDTA. In pulse-chase experiments, an excess amount of non-labeled UTP (final concentration, 2 mM) was added at 5 min, and the incubation was continued until the time indicated in each experiment. RNA was extracted with phenol, phenol:chloroform, and chloroform, precipitated with ethanol, and washed once with 80% ethanol. The precipitate was suspended in 1x loading buffer (0.04% bromphenol blue, 0.04% xylene cyanol, 1 mM EDTA), or 1x loading buffer containing 7 M urea and heated at 95 °C for 1 min. Then RNA was separated in a 1% agarose gel in TAE buffer (40 mM Tris acetate, pH 8.0, 1 mM EDTA) containing 0.5 µg/ml ethidium bromide. After electrophoresis, the gel was dried and the synthesis of RNA was detected by autoradiography.
Gel-filtration Columns—Elimination of unincorporated NTPs from an RNA polymerase reaction mixture was done by passing the mixture through a Sephadex G-50 mini-spin column (Worthington). Briefly, an RNA polymerase reaction was carried out at 30 °C in the standard reaction mixture supplemented with [32P]UTP and 0.15 unit/µl RNasin (Promega). The reaction was stopped at 5 min by the addition of 15 mM EDTA, and the mixture was passed through the column pre-equilibrated with 50 mM Tris·HCl, pH 8.75, 150 mM NaCl, 10 mM EDTA, and 4 mM dithiothreitol. After centrifugation, the filtrate was divided into aliquots. The aliquots were incubated again at 30 °C for 60 min with or without 15 mM MgCl2. Some aliquots were further supplemented with one of non-labeled NTPs (0.5 mM) or all of them together. After incubation, RNA was extracted and analyzed in an agarose gel without heat denaturation.
Northern Hybridization—Northern hybridization with 32P-labeled RNA polymerase reaction products as probe has been described (18). Briefly, non-labeled RNAs containing 20 S RNA or 23 S RNA sequences made in vitro by T7 or T3 RNA polymerase were separated in an agarose gel. 20 S RNA positive and negative strands (2514 nt) were made by T7 RNA polymerase using SmaI-digested pALI-17 (8) and by T3 RNA polymerase using SmaI-digested pALI-18, respectively. Similarly, 23 S RNA positive and negative strands (2891 nt) were made by T7 transcription from SmaI-digested pALI-37 and by T3 transcription from SmaI-digested pALI-38 (17), respectively. pALI-1 and pPAZ-11 contain DNA fragments of 20 S RNA from nt 10 to 178 and from nt 2288 to 2504, respectively, inserted between the T7 and T3 promoters of SK+ vector (Stratagene). Therefore, T7 or T3 run-off transcription from these plasmids pre-digested with appropriate restriction enzymes will produce transcripts containing 169 or 217 nt of 20 S RNA positive or negative strand terminal sequences as shown in the diagram of Fig. 2B. After electrophoresis the RNA was denatured in the gel and blotted onto a nylon membrane as described previously (19). The membrane was hybridized with heat-denatured 32P-labeled RNA products, made in vitro by cell lysates in the standard RNA polymerase reaction mixture at 30 °C for 1 h. After hybridization, the membrane was washed, and the hybrids were detected by autoradiography (19). In the experiments shown in Fig. 2B, the membrane was treated, before the final stringent wash, with 5 µg/ml RNase A at 37 °C for 15 min in a buffer containing 10 mM Tris·HCl, pH 8.0, 0.5 M NaCl, and 1 mM EDTA, and washed twice 15 min each at room temperature with the same buffer containing 0.5% SDS but without RNase A. Then, the final wash was done with a buffer containing 10 mM NaPO4, pH 7.7, 0.18 M NaCl, 1 mM EDTA, and 0.1% SDS at 60 °C for 30 min.
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RESULTS |
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2% of that of positive strands, roughly the same as the ratio of negative to positive strands present in the lysate. When parts of the sample shown in Fig. 1A were analyzed without heat denaturation, we found another, slower moving band whose mobility corresponded to W, a dsRNA form of 20 S RNA (Fig. 1B). During incubation, radioactive nucleotides were first incorporated into the dsRNA form with a peak at 20 min, and then the label in the dsRNA form decreased gradually (Fig. 1B). On the other hand, the appearance of the labeled ssRNA form was slightly delayed compared with that of the dsRNA form, but its amount increased steadily during the incubation (Fig. 1B). These results suggest a precursor-product relationship between them. This was confirmed by the pulse-chase experiments shown in Fig. 1D. During the first 5-min labeling period, most of labeled nucleotides were incorporated into the dsRNA form. After the addition of an excess amount of non-radioactive nucleotide, the label in the dsRNA form was converted into the final ssRNA products. When the same samples were heat-denatured in the presence of 7 M urea, the label in the dsRNA intermediates disappeared and was converted to ssRNA forms (Fig. 1C). This indicates that the labeled positive strands were non-covalently associated with negative strands. In the early labeling period we observed heterogeneous molecules moving faster than full-length 20 S RNA in the gel (Fig. 1C). During the chasing period these molecules became full-length. Therefore, they are 20 S RNA positive strand molecules shorter than full-length. In the absence of heat denaturation, however, these molecules were not detected at the same position in the gel (Fig. 1D). Instead, we observed a broad smear band between the dsRNA and ssRNA forms. During the following chasing period, this broad band moved upwards until reaching the position of the dsRNA. Furthermore, there was no incorporation into materials larger than the dsRNA form (Fig. 1D). These observations thus suggest that (i) the dsRNA form consists of a full-length template negative strand and a single molecule of full-length or almost full-length positive strand and (ii) that the smear broad band between the dsRNA and ssRNA forms has the same RNA composition as the dsRNA form but with a positive strand much shorter than full length and heterogeneous in size.
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The RNA in Native Replication Complexes Is Mostly Single-stranded—As shown in Fig. 4, lane 1, phenol-extracted in vitro products resolved into two bands corresponding to the dsRNA intermediates and the final ssRNA products in an agarose gel. RNase A treatment under high salt conditions completely digested the ssRNA products, whereas the dsRNA intermediates remained undigested (lanes 4 and 5). When the same sample but without phenol extraction was treated with the enzyme under high salt conditions, both RNA bands disappeared (lanes 2 and 3). This indicates that RNA in native replication complexes is mostly single-stranded and that deproteination with phenol converted the negative strand template and the positive strand product in the complex into the dsRNA intermediates. Hereafter we call these native intermediates "replication complexes" to distinguish them from phenol-extracted dsRNA intermediates. This implies that they also contain proteins, at least its RdRp p91.
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Although native replication complexes moved as a broad band, the lower edge of the band never ran faster than the ssRNA products, thus suggesting that the RNA content in the replication complex is more than a single molecule of 20 S RNA. After the addition of non-labeled UTP, the lower edge of the broad band shifted upwards, converging to the upper edge of the band in the first 10-min incubation (Fig. 5A). During the following incubation, all of the label in the broad band was converted to the ssRNA products. There is a good correlation between the mobility of four phenol-extracted ssRNAs (18 S rRNA, 20 S RNA, 23 S RNA, and 25 S rRNA) in the gel (Fig. 5D) and their nucleotide sizes (Fig. 5E). By extrapolation, the upper edge of the broad band in panel A was estimated to correspond to the mobility of an ssRNA 5 kb in size (Fig. 5E). Because 20 S RNA is 2514-nt long, this is equivalent to two molecules of 20 S RNA. This suggests that these intermediates at the upper edge contain a full-length negative strand and an almost full-length positive strand per complex. When the same native samples shown in Fig. 5A were electroblotted to a nitrocellulose membrane, we again observed a broad band corresponding to the replication intermediates (Fig. 5C). The upward movement of the lower edge of the band during incubation became more evident. Because nitrocellulose membranes are slightly negatively charged, phenol-extracted ssRNA products did not bind to the membrane under the blotting conditions used (17; also compare the far right lanes in Fig. 5, B and C). On the contrary, the same products but without phenol extraction bound to the membrane. This suggests that the native ssRNA products released from replication complexes were associated with protein. Because p91 associates with 20 S RNA positive strands in non-replicating complexes in a 1:1 stoichiometry and the replication intermediate should contain at least one molecule of p91, p91 is a good candidate for the protein bound to the ssRNA products in vitro.
Release of the Negative Strand Template from a Replication Complex in Vitro—We were interested in the fate of the negative strand templates in replication complexes during in vitro positive strand synthesis. Because the templates do not incorporate radioactive nucleotides in the reaction, we visualized them by hybridization using a radioactive probe specific to the negative strand (Fig. 6). A cell lysate was incubated in the standard RNA polymerase reaction mixture from which the radioactive nucleotide was omitted. After 5-min incubation, an excess amount (2 mM) of non-labeled UTP was added, and the reaction was kept another 55 min. During the incubation samples were taken at the times indicated and loaded directly onto a non-denaturing agarose gel. Therefore, the experimental conditions were almost identical to the ones used in Fig. 5. After electrophoresis, RNA in the gel was electroblotted onto either a positively charged nylon membrane (Fig. 6A) or a nitrocellulose membrane (Fig. 6B) and 20 S RNA negative strands on the membranes were detected by hybridization. As shown in Fig. 6A, most of negative strands in the lysates prior to incubation (time 0) migrated as a broad band, and this band had the same mobility as the broad band of replication complexes shown in Fig. 5. Therefore, they were the template strands present in replication complexes. During incubation these negative strands decreased and we observed a concomitant increase of negative strands in the single-stranded form. This increase appears to coincide with the release of newly synthesized positive strands from replication complexes (compare Fig. 6A with Fig. 5A). Because the lysate predominantly synthesized 20 S RNA positive strands over negative strands in vitro (the latter synthesis counted <2% compared with that of the former ones), and replication complexes engaging positive strand synthesis are expected to contain negative strand templates equimolecular to the products, most of the increase of negative strands in the single-stranded form was not caused by its synthesis in vitro but derived from negative strand templates released from replication complexes. During incubation we observed a 3- to 5-fold increase of negative strands in the single-stranded form. This suggests that most of negative strands in 20 S RNA-induced cells were present in replication complexes, thus engaging in positive strand synthesis. When negative strands were electroblotted onto a nitrocellulose membrane (Fig. 6B), we observed again an increase in the amount of negative strands in the single-stranded form and a concomitant decrease of negative strands present in the replication complexes. Interestingly, when a sample taken at 10 min was phenol-extracted and loaded onto the same gel, the single-stranded form of negative strands did not bind to the nitrocellulose membrane (compare the far right lane with the third lane from the left in Fig. 6B) but to the positively charged nylon membrane (Fig. 6A, far right lane). Therefore, these results suggest that the negative strand templates released from replication complexes are also associated with protein.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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RNA Constituents of Replication Complexes—The broad band of the native replication complexes seen in the early labeling period reflects the heterogeneity in length of the growing positive strands in the complexes (Fig. 5, A and C). In the following chasing period the lower edge of the band shifted quickly to the upper edge of the band, and all the label was eventually converted to the ssRNA form. This indicates that the upward shift of the label during this period was due to positive strand RNA synthesis. Consistently, the front edge of the broad band ran as fast as 20 S RNA (or 20 S RNA/p91 resting complexes, see below), and the upper edge of the band migrated as a ssRNA of 5 kb in size, twice the size of 20 S RNA (2514 nt). These results, thus suggest that the replication complexes contain, as RNA components, one full-length negative strand template and one growing, but less than full-length, 20 S RNA positive strand. There are several lines of evidence that support this. First, as shown in Fig. 2B, most of the in vitro synthesis was done by chain elongation of positive strands pre-initiated in vivo. Thus there are no multiple rounds of positive strand synthesis in vitro from the same template. Consistently, vigorous RNA synthesis ceased after 10-min incubation (Fig. 1E), accompanied with the accumulation of the dsRNA form. Second, in pulse-chase experiments, we observed molecules smaller than the dsRNA form during the early incubation period (Fig. 1D). Heat denaturation with urea converted them into molecules smaller than full-length positive strands (Fig. 1C). There was no incorporation into materials larger than the dsRNA form. Therefore, these observations indicate that the total positive strand content in replication complexes never exceeds a single molecule of 20 S RNA per complex. This implies that a strand-replacement mechanism is not responsible for the conversion of dsRNA intermediates to the final ssRNA products. If a full-length positive strand is displaced by a newly synthesized positive strand, then the total amount of positive strand RNA in the complex should be more than a single molecule of 20 S RNA. Finally, we detected negative strand templates in the replication complexes. When RNA in lysates was separated in non-denaturing agarose gels, a negative strand-specific probe detected two bands (Fig. 6). The faster moving band is the single-stranded form of negative strands. The second broad band corresponds to the negative strand templates present in replication complexes. During incubation, the second band disappeared and a concomitant increase in the single-stranded form was observed. Because negative strand synthesis in vitro was almost negligible, the increase of the single-stranded form was mostly derived from the negative strand templates released from replication complexes. In good agreement, the increase of negative strands in the single-stranded form coincided with the appearance of positive strand products released from replication complexes during incubation.
Although the replication complexes contain p91, the contribution of protein components to their mobility in the gel appears to be minimal. The mobility of protein-stripped 20 S RNA (808 kDa) is hardly distinguishable from that of 20 S RNA/p91 resting complexes (Ref. 17; see also Fig. 5, A and B). Interestingly, both the positive and negative strands released from replication complexes bound to a nitrocellulose membrane by electroblotting but did not bind to the same membrane when extracted with phenol. This suggests that both strands were associated with protein.
Comparison with Replication Complexes from Other Positive Strand RNA Viruses—The single-stranded nature of the RNA components of 20 S RNA replication intermediates is quite similar to that of native replication intermediates of some positive strand RNA viruses. It has been well documented that the RNA in native replication intermediates of the RNA coliphage Q is fully single-stranded (20). Deproteination with phenol converts it into a form that is largely double-stranded. Once extracted with phenol, the annealed RNA is unable to infect protoplasts of the host cells nor to serve as template for Q replicase. However, both activities can be restored by heating the RNA to melt it (21, 22). Although multistranded intermediates have been observed, a single Q replicase is capable of synthesizing RNA multiple rounds. After completion of product-strand elongation, both the product and template strands are released from the replication complex as single-stranded RNA. Then the replicase is free to bind to a new template to proceed the next cycle of RNA synthesis (23).
In polio virus-infected cells, a short exposure to [3H]uridine predominantly labels a minor virus-related RNA species called replicative intermediate (RI). The RI consists of a genome-length negative strand template RNA and complementary RNA with an average of five to seven nascent single-stranded tails (24, 25). Although deproteinated RI has a backbone mostly double-stranded, a careful electron microscope analysis using a membrane-permeable cross-linking reagent demonstrated that the native RI in vivo has a predominantly single-stranded backbone attached to several nascent RNA chains with few or no regions of extensive base-pairing (26). Despite similarity in their single-stranded nature, replication intermediates of 20 S RNA virus do not have multiple nascent RNA chains. Because 20 S RNA virus is not infectious, this may reflect its slower replication rate compared with the infectious counterparts. Alternatively, 20 S RNA virus may have some mechanism to prevent a second round of initiation from templates still engaging in the synthesis of complementary strands.
W dsRNA Is a By-product of 20 S RNA Replication—20 S RNA virus-harboring cells, when grown at 37 °C, accumulate W dsRNA. As shown in this work, however, native replication complexes have a single-stranded RNA backbone and deproteination with phenol converted them to dsRNA intermediates. Therefore, W dsRNA is not an intermediate of 20 S RNA replication but a by-product. Interestingly, W dsRNA isolated from cells grown at 37 °C had the intact 3'-end sequences in both the positive and negative strands (8). Growth at the higher temperature may simply accelerate duplex formation between single-stranded positive and negative strands. Alternatively, the higher temperature might affect replication complexes in such a way that the nascent strand is allowed more easily to re-hybridize with the template strand behind the site of RNA synthesis without affecting the catalytic activity of the polymerase machinery. In contrast to W dsRNA, the dsRNA intermediates detected in vitro after phenol treatment were formed only when the labeled positive strands were still associated with replication complexes. Furthermore, their release from replication complexes required net RNA synthesis. Therefore, these observations suggest that the positive strands in dsRNA intermediates were not full-length. It appears that RNA synthesis slows down considerably near the 3'-end of the positive strand. It is interesting to know if there is a specific pausing site(s) for positive strand synthesis. Such a pause(s) could be caused by the structure of the template RNA or by binding of protein to the template RNA. Considering the importance of the formation of ribonucleoprotein complexes in narnavirus replication (see below), the latter case may be of particular interest. 20 S and 23 S RNA genomes do not encode capsid proteins. Thus their RNA genomes are not encapsidated into intracellular viral particles. Instead these positive strand RNAs form resting ribonucleoprotein complexes with their cognate RdRps in a 1:1 stoichiometry and reside in the cytoplasm. These RNAs have no poly(A) tails at the 3'-ends and perhaps no 5'-cap structures, thus resembling intermediates of mRNA degradation. Therefore, we expect these RNAs, if alone without forming complexes with their RdRps, to be susceptible to the exonucleases involved in mRNA catabolism. 20 S and 23 S RNA genomes share the same 5' nt inverted repeats at both ends (5'-GGGGC... GCCCC-OH) (8). Recently we have succeeded in generating 23 S RNA virus in vivo from a vector containing the entire viral cDNA (27). Using this system we found that the cluster of 4 Cs at the 3'-end of the positive strands is a part of a bipartite 3' cis signal for replication (28). We also found that this bipartite signal is essential for the formation of a ribonucleoprotein complex in vivo between the 23 S RNA positive strand and its RdRp, p104 (29). It points out the importance of the ribonucleoprotein complex formation in the life of 23 S RNA virus. In the case of 20 S RNA virus we have developed a similar virus launching system and found that the cluster of four Cs at the 3'-end of the 20 S RNA positive strand is also a part of a 3' cis signal essential for replication.2 On the analogy of 23 S RNA virus, we expect that p91 interacts with this 3' signal to form a ribonucleoprotein complex. In fact, when p91/20 S RNA resting complexes were extensively digested with RNase A, anti-p91 antiserum immunoprecipitated 3'-end fragments as well as 5'-end fragments of the 20 S RNA positive strands. We located the 5' p91 binding site on the second stem structure from the 5'-end (nt 72–104, numbered from the 5'-end).3 These data strongly suggest that a single p91 molecule circularizes a 20 S RNA positive strand in a resting complex by binding at both ends of the RNA. This may give a good explanation of why 20 S RNA virus can survive in the yeast cytoplasm without its RNA genome being digested by exonucleases, provided that the negative strand genome is also protected by a similar mechanism. Our available data indicate that negative strands can be immunoprecipitated with anti-p91 antiserum from a sucrose gradient fraction containing non-replicating 20 S RNA genomes (30). Therefore, 20 S RNA negative strands also form ribonucleoprotein complexes with p91. Recently we found that 20 S RNA negative strands transcribed from a vector can also generate the virus in vivo if an active p91 is provided in trans from a second vector.2 Using this two-vector system we observed that the cluster of 4 Cs at the 3'-end of the 20 S RNA negative strand again is a part of a 3' cis signal for replication. It is likely that this signal is also involved in the formation of complexes with p91. So far our knowledge on the negative strand is limited, because the low amount of 20 S RNA negative strands in the cells makes it difficult to study them in detail. In this context, this two-vector system can be exploited, especially to analyze cis signals on the negative strands to form complexes with p91 by expressing a higher amount of these RNAs from a vector in vivo. It will be more difficult to understand the structural features of replication complexes, given their heterogeneity and complexity compared with resting complexes. Some of the features, however, may not be fundamentally far from those seen in resting complexes. For example, if negative strands in resting complexes are circularized by p91 for protection, as suggested for positive strands, then such protection might also occur in negative strand templates in replication complexes. We have observed that positive strand synthesis in vitro apparently decelerated when the replication machinery approached the 5'-end of the template. This slow-down might be caused by p91 bound to the 5'-end region of the template strand, because the polymerase machinery has to displace this p91 from the template to complete the positive strand synthesis. This possibility could be verified experimentally by comparing p91 binding sites on the negative strand in resting complexes, with pausing sites of positive strand synthesis located on the template strand. If it is the case, then this scenario leads to another interesting question of whether the "replicating" p91 in the polymerase machinery is the same molecule "bound" to the negative strand template. If the same single molecule has these two functions, then either the positive strand product or the negative strand template released from a replication complex will need an additional p91 molecule to form resting complexes. Alternatively, if there are two p91 molecules with distinct functions in a replication complex, then, we may expect their non-random distribution to the respective strands released from the complex, thus resulting in (or leading to) the formation of two resting complexes. As we have observed in this work, both the positive and negative strands released from replication complexes in vitro are associated with protein. Because each replication complex contains at least a single molecule of p91, either the positive or negative strand released should be associated with p91 (or p91 might be distributed between them randomly). To answer this question, therefore, we need to know which strand released from the replication complex is associated with p91 and what is the stoichiometry of their association. Although we expect that these experiments are technically challenging, the membrane-free nature of 20 S RNA virus replication complexes may allow us to answer these questions. These results will provide fundamental insights to understand the replication mechanism of positive strand RNA viruses in yeast.
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FOOTNOTES |
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Recipient of a fellowship from the Spanish Ministry of Education and Science. Present address: Dept. of Microbiology, Box 1124, One Gustave L. Levy Place, Mount Sinai School of Medicine, New York, NY 10029.
To whom correspondence should be addressed. Tel.: 34-923-120673; Fax: 34-923-224876; E-mail: tfujimura{at}usal.es.
1 The abbreviations used are: RdRp, RNA-dependent RNA polymerase; ssRNA; single-stranded RNA; dsRNA, double-stranded RNA; nt, nucleotide(s); Cs, cytidines; RI, replicative intermediate.
2 R. Esteban, L. Vega, and T. Fujimura, manuscript in preparation.
3 T. Fujimura and R. Esteban, manuscript in preparation.
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HindIII markers is also shown. From the autoradiogram it is estimated that the amounts of 23 S RNA-positive strands and 20 S RNA-negative strands synthesized in vitro are 8 and 2%, respectively, compared with that of 20 S RNA positive strands. A diagram of the non-labeled 20 S RNA and 23 S RNA positive and negative strands used is shown under the panels. B, small RNAs containing the 5'- or 3'-end terminal sequence of the positive and negative strands of 20 S RNA were separated in an agarose gel and processed as in A. After hybridization the membrane was treated with RNase A to digest the single-stranded portions of the 32P-labeled products bound to the small RNAs before the final stringent wash. The upper panels show the ethidium bromide-staining of the gel (left) and an autoradiogram of the membrane (right). A diagram of the positive (+) and negative (-) strands of 20 S RNA and the small terminal fragments used (indicated by the arrows) with their nucleotide sizes is shown under the panels.
