Distinct sites on the Sindbis virus RNA-dependent RNA polymerase for binding to the promoters for the synthesis of genomic and subgenomic RNA

Abstract

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.

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.

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.

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 5× 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.