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Journal of Virology, April 2007, p. 4371-4373, Vol. 81, No. 8
0022-538X/07/$08.00+0     doi:10.1128/JVI.02672-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Distinct Sites on the Sindbis Virus RNA-Dependent RNA Polymerase for Binding to the Promoters for the Synthesis of Genomic and Subgenomic RNA

Mei-Ling Li¹ and Victor Stollar^1,2*

Department of Molecular Genetics, Microbiology, and Immunology, University of Medicine and Dentistry of New Jersey—Robert Wood Johnson Medical School, Piscataway, New Jersey 08854,¹ Cancer Institute of New Jersey, New Brunswick, New Jersey 08901²

Received 4 December 2006/ Accepted 29 January 2007

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Sindbis virus-infected cells make two positive-strand RNAs, a genomic (G) RNA and a subgenomic (SG) RNA. Here we report the amino acid sequence in nonstructural protein 4 (nsP4), the viral RNA-dependent RNA polymerase, that binds to the promoter for the synthesis of G RNA. In addition, using a cell-free system that makes both G and SG RNA, we show that specific amino acid changes in nsP4 that abolish the synthesis of SG RNA have no effect on the synthesis of G RNA. Our findings indicate that nsP4 has distinct sites for the recognition of the G and SG promoters.

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Viruses in the Togavirus family, such as Sindbis virus (SV), make two species of plus-strand RNA in infected cells, a genomic (G) RNA and a subgenomic (SG) RNA (10, 11). However, the mechanisms whereby these two species are made differ. Whereas synthesis of the G RNA must begin by copying the 3' end of the minus-strand RNA template, synthesis of the SG RNA involves internal initiation on the minus-strand RNA. A critical question is how the appropriate balance between the syntheses of G and SG RNA is achieved. One possibility is that there are different sites on the viral RNA-dependent RNA polymerase (RDRP) for the recognition of the G and SG promoters and that the interactions between the RDRP and these two promoters are critical. The promoter sequence for the synthesis of SV SG RNA is well defined (13), and we reported recently that it is recognized by the amino acid sequence 329-LVRRLT-334 in nonstructural protein 4 (nsP4), the viral RDRP (6). Here we present new information concerning the G promoter and its recognition by a specific amino acid sequence in nsP4. We also show that specific amino acid changes in nsP4 prevent the synthesis of SG RNA without affecting the synthesis of G RNA.

A computer-predicted stem-loop (SL) structure has been described at the 5' end of the SV G RNA which involves the 44 5' nucleotides (nt) (SL1); and a complementary SL structure (cSL1) has been described at the 3' end of the minus-strand RNA (1, 7, 8). A report by Frolov et al. (1) provided strong evidence that the nucleotides at positions 2 to 5 from the 3' end of the minus-strand RNA are a critical part of the RNA sequence that functions as the G promoter. These results imply that the promoter for G RNA synthesis lies within the cSL1 sequence.

To determine what length of minus-strand RNA sequence is necessary to bind the SV replicase assembled in cells infected with recombinant vaccinia virions which express the four SV nsPs (4), we carried out electrophoretic mobility shift assays. To obtain the minus-strand probes, we made PCR products which contained the appropriate regions of pToto (9) and an SP6 promoter at one end; these enabled us to transcribe the desired negative-strand RNA sequence. Each RNA probe was 5' labeled with ³²P and incubated with a P15 fraction prepared from cells infected with recombinant vaccinia virions expressing the four SV nsPs which make up the SV replicase/transcriptase. Slowing of the electrophoretic mobility of the labeled probe indicated that the SV replicase complex bound to the probe and likely contained the promoter for the synthesis of G RNA.

Initial experiments demonstrated a shift in mobility when the 3'-terminal 210-, 155-, and 60-nt sequences of the minus-strand RNA were used but not the corresponding sequences of the positive-strand RNA (not shown). Figure 1 shows that a mobility shift was also observed with the 3'-terminal 45 nt of the minus-strand RNA; however, no mobility shift was noted with the 5'-terminal 45 nt of the plus-strand RNA. We conclude that the promoter for the synthesis of G RNA is contained within the sequence that corresponds to the cSL1 structure. This result is consistent with the findings of Frolov et al. (1), Gorchakov et al. (2), and Thal et al. (12). The report of Thal et al. included a direct demonstration that deletion of the 3'-terminal 42 nt of the minus-strand template eliminated synthesis of plus-strand RNA in an in vitro sysytem.

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FIG. 1. Electrophoretic mobility shift assays using different SV RNA probes. Electrophoretic mobility shift assay experiments were carried out as described earlier (6). 5'-labeled RNA probes were incubated with P15 fractions prepared from cells infected with recombinant vaccinia virions expressing the four SV nonstructural proteins. Lane 1, an unrelated 31-mer oligoribonucleotide alone; lane 2, an unrelated 31-mer oligoribonucleotide plus nsP1234 (nsP1, -2, -3, and -4); lane 3, the 5'-terminal 45 nt of SV plus-strand RNA alone; lane 4, the 5'-terminal 45 nt of SV plus-strand RNA plus nsP1234; lane 5, the 3'-terminal 45 nt of SV minus-strand RNA alone; lane 6, the 3'-terminal 45 nt of SV minus-strand RNA plus nsP1234.

To identify the sequence in nsP4 that recognized the G promoter, we used a procedure similar to the one we used to identify the sequence in nsP4 that bound to the SG promoter (6). A 45-nt RNA representing the 3' terminus of the minus-strand RNA in which the U at position 39 from the 3' terminus was replaced by a thiouracil was purchased. This probe was labeled at its 5' terminus with ³²P, incubated with a P15 fraction containing the four SV nsPs, and UV irradiated. As expected, a mobility shift was observed upon electrophoresis (not shown). The labeled complex was excised from the gel and digested with trypsin. A second electrophoresis of the peptide-probe complex again showed a mobility shift relative to the probe alone; the labeled peptide-RNA probe complex was excised from the gel and sent to the Keck Biotechnology Resource Laboratory at Yale University for sequencing of the peptide. The sequence identified was LGKPLPAD, which corresponds to amino acids 531 to 538 in nsP4.

Recently, we described a cell-free system for the synthesis of an SV RNA transcript under the control of the SG promoter. The critical components of this system were a P15 fraction from cells expressing the four SV nsPs and a minus-strand RNA molecule extending from nt –157 to nt +175, relative to the initiation site for the SG RNA, which served as promoter-template (P-T) (5).

We have now set up a similar cell-free system but one which directs the synthesis of both G RNA and SG RNA. As with the system that makes SG RNA, we made use of a P15 fraction containing the four SV nsPs, but this time, for the P-T, we used a minus-strand RNA that contained both the G and SG promoters (Fig. 2A). This P-T is predicted to make two RNA molecules, a 5,413-nt sequence under the control of the G promoter and a 1,943-nt sequence under the control of the SG promoter. A reaction mixture similar to that described earlier (5), but with the P-T shown in Fig. 2A, was set up and incubated for 60 min at 37°C. The labeled RNA was fractionated by gel electrophoresis and stained with ethidium bromide. Figure 2B demonstrates that two products were made, with the expected sizes close to those predicted above. As in virus-infected cells, SG RNA was made in excess of G RNA. Why the G RNA migrates slightly more slowly than expected is not clear. The G and SG transcripts were purified using an oligo(dT) column, and the sequences of their 5' termini were determined (5). The 5'-terminal sequences obtained were those expected for G and SG RNA, respectively.

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FIG. 2. (A) Structure of the RNA P-T used for the in vitro synthesis of G and SG RNAs. The full-length pToto plasmid was digested with EcoRV to delete nt 2751 to 6878, ligated, digested with StuI to delete nt 8572 to 10769, and then ligated to generate the deleted form of pToto. PCR was carried out to amplify the cDNA of the deleted form of pToto by using upstream and downstream primers with added T7 and SP6 promoters, respectively. RNA was transcribed from the PCR product using the AmpliScribe SP6 high-yield transcription kit (Epicenter), as described previously (5). An arrow indicates the initiation site for the synthesis of SG RNA. The dotted lines represent sequences that are deleted. (B) In vitro synthesis of plus-strand RNAs from the G and SG promoters. The minus-strand RNA promoter template shown in panel A was incubated with a P15 fraction from cells expressing the four SV nsPs as described earlier (5), except that the concentration of each of the nucleoside triphosphates was 4.5 mM. The RNAs were fractionated by electrophoresis through an agarose gel and stained with ethidium bromide. Left lane, RNA markers; right lane, in vitro-synthesized SV RNAs.

As noted above, we have identified a sequence in nsP4, 329-LVRRLT-334, that is involved in the binding of nsP4 to the SG promoter (6); we also showed that changing either R331 or R332 to an Ala prevented such binding. Changing a nearby Arg, at position 327, to Ala did not affect binding. Since the mutant nsP4s with the amino acid changes at position 331 or 332 did not bind to the SG promoter, we considered it unlikely that they would support the synthesis of an SG RNA transcript. To test whether these mutant nsP4s would support the synthesis of G RNA, we set up a reaction mixture that would make both G and SG RNA, as shown in Fig. 2B. However, we used four different P15 preparations: all preparations contained wild-type nsP1, nsP2, and nsP3, but the four reaction mixtures contained either wild-type nsP4 or nsP4 with an alanine substitution at position 327, 331, or 332. Both G and SG RNA were made when wild-type nsP4 was used (Fig. 3) or when nsP4 with the R-to-A change at position 327 was used. However, when nsP4 with an R-to-A change at either position 331 or 332 was used, although G RNA was made normally, there was no synthesis of SG RNA. As reported earlier, when the changes at position 331or 332 were introduced into pToto, the infectious clone of SV, the RNA transcripts were not infectious (6).

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FIG. 3. In vitro synthesis of SV RNAs using wild-type nsP4 (Wt) or nsP4 with the indicated amino acid change (R327A, R331A, or R332A). The reaction mixture (25 µl) contained 5 µl of 5x reaction buffer (200 mM Tris-HCl, pH 7.9, 30 mM MgCl2, and 50 mM NaCl), 10 mM dithithreitol, 40 units of RNase inhibitor, 2 µg P-T, and 11.25 µl of P15 extract (concentrations were adjusted to 8.95 µg protein/µl) prepared from cells infected with vaccinia viruses expressing the T7 RNA polymerase, the SV polyprotein, P123, and the SV nsP4. The nucleoside triphosphate concentrations were as follows: 3 mM ATP, 2 mM UTP, 2 mM GTP, and 0.5 mM CTP. [32P]CTP (800 Ci/mM, 10 µCi/ml) was included to label the transcripts. Incubation was at 37°C for 1 hour. After electrophoresis, the gel was dried, exposed to a storage phosphor screen, and analyzed on a phosphorimager scanner.

It has been suggested that viral RDRPs may have different sites for the recognition of different promoters (3). If so, it would follow that the RDRPs of RNA viruses that make an SG RNA via internal initiation, such as SV, should have different sites for the recognition of the G and SG promoters. Our identification of different sites on SV nsP4 for the recognition of the SG and G promoters and the demonstration that, by altering the amino acid residues at positions 331 and 332 in nsP4, we could interfere with the synthesis of SG RNA, but not G RNA, lend strong experimental support to the idea that alphavirus RDRPs have different and distinct sites for the recognition of the promoters for the synthesis of SG and G RNA. It is likely that such will be found to be the case for other RNA viruses that make an SG RNA via internal initiation.

 
This work was funded by U.S. Public Health Service grants AI-49273 and AI-070728.

 
* Corresponding author. Mailing address: Department of Molecular Genetics, Microbiology, and Immunology, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ 08854. Phone: (732) 235-4596. Fax: (908) 235-5223. E-mail: stollar{at}umdnj.edu

Published ahead of print on 7 February 2007.

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Journal of Virology, April 2007, p. 4371-4373, Vol. 81, No. 8
0022-538X/07/$08.00+0     doi:10.1128/JVI.02672-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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