Template-dependent initiation of Sindbis virus RNA replication in vitro

Abstract

Recent insights into the early events in Sindbis virus RNA replication suggest a requirement for either the P123 or P23 polyprotein, as well as mature nsP4, the RNA-dependent RNA polymerase, for initiation of minus-strand RNA synthesis. Based on this observation, we have succeeded in reconstituting an in vitro system for template-dependent initiation of SIN RNA replication. Extracts were isolated from cells infected with vaccinia virus recombinants expressing various SIN proteins and assayed by the addition of exogenous template RNAs. Extracts from cells expressing P123C>S, a protease-defective P123 polyprotein, and nsP4 synthesized a genome-length minus-sense RNA product. Replicase activity was dependent upon addition of exogenous RNA and was specific for alphavirus plus-strand RNA templates. RNA synthesis was also obtained by coexpression of nsP1, P23C>S, and nsP4. However, extracts from cells expressing nsP4 and P123, a cleavage-competent P123 polyprotein, had much less replicase activity. In addition, a P123 polyprotein containing a mutation in the nsP2 protease which increased the efficiency of processing exhibited very little, if any, replicase activity. These results provide further evidence that processing of the polyprotein inactivates the minus-strand initiation complex. Finally, RNA synthesis was detected when soluble nsP4 was added to a membrane fraction containing P123C>S, thus providing a functional assay for purification of the nsP4 RNA polymerase.

FIG. 1

In vitro synthesis of SIN RNA. P15 fractions were prepared from BHK-21 cells infected with the indicated vaccinia virus-SIN recombinants and vTF7-3 (lanes 1 to 3). Reaction mixtures were incubated with JNTSCATX template RNA at 30°C for 60 min under standard conditions. Denatured products were separated on an agarose gel and visualized by autoradiography. Lanes 4 and 5 are radiolabeled RNA transcript markers corresponding to JNTSCATX subgenomic (S) and genomic (G) RNAs, respectively.

FIG. 2

Time course of RNA accumulation. P15 fractions were prepared from BHK-21 cells infected with vTF7-3 and vaccinia virus recombinants expressing P123_C>S and Ub-nsP4 (Tyr) (nsP4) as indicated above each lane. Reactions with JNTSCATX template RNA were incubated at 30°C for the indicated times (in minutes) under standard conditions. Denatured (A) or nondenatured (B) RNAs were separated on agarose gels and visualized by autoradiography (A) or by staining with ethidium bromide (B). In panels A and B, the position of genome-length JNTSCATX RNA is indicated. In panel B, 28S and 18S rRNAs are indicated.

FIG. 3

RNase H analysis of in vitro-synthesized RNA. (A) Diagram indicating where the minus-strand-specific primers anneal to the JNTSCATX template and the lengths (in bases) of the fragments expected after complete digestion with RNase H. (B) P15 fractions were prepared from BHK-21 cells infected with vaccinia virus recombinants expressing P123_C>S, Ub-nsP4 (Tyr), and vTF7-3. Reaction mixtures were incubated with JNTSCATX template RNA under standard conditions, and the products were denatured, annealed to specific primers, and digested with RNase H (+) or incubated without added enzyme (−). The resulting fragments were separated on a 3.5% polyacrylamide–urea gel and visualized by autoradiography. As a control, a minus-sense transcript from pJNTSCATX(−) was analyzed in parallel. To the right, the positions of various radiolabeled RNA size markers are indicated (bases).

FIG. 4

In vitro replication of full-length SIN RNA and heterologous Togaviridae templates by the SIN replicase. In vitro transcripts from the full-length or subgenomic replicon cDNAs were used as templates in P15 extracts containing P123_C>S and Ub-nsP4 (Tyr). Reaction mixtures were incubated at 30°C for 60 min under standard conditions. The products were denatured, separated on an agarose gel, and visualized by autoradiography. −, no added template RNA; SFV, Semliki Forest virus; VEE rep, Venezuelan equine encephalitis virus subgenomic RNA replicon; RRV, Ross River virus; RUB, rubella virus. The positions of genome-length SIN RNA (11.7 kb) and 28S and 18S rRNAs are indicated to the right.

FIG. 5

In vitro replication of SIN RNA templates with different 3′ termini. (A) Diagram representing the JNTSCATX(+) cDNA with the positions of the various runoff sites indicated. Shown below are the predicted RNA substrates generated after transcription. Letters to the left are abbreviations of the restriction enzymes used to linearize pJNTSCATX(+); F, FspI; Bs, BsgI; X, XhoI; E, EarI; Bg, BglI. The numbers to the right indicate the transcript length (in bases). (B) As diagrammed in panel A, plus-sense SIN RNAs with different 3′ termini were tested in the in vitro replication assay. After pJNTSCATX(+) was linearized with various restriction enzymes, 3′-protruding ends were removed with Klenow enzyme, and RNA templates were synthesized with SP6 polymerase. In vitro replication assays were performed with P15 extracts containing P123_C>S and Ub-nsP4 (Tyr) under standard conditions, and the products were analyzed by gel electrophoresis. The size of genome-length RNA produced from pJNTSCATX(+) linearized with BsgI is indicated by the bar to the right.

FIG. 6

Comparison of the in vitro activities of replication complexes containing cleaved and uncleaved SIN nonstructural components. (A) P15 fractions were prepared from BHK-21 cells infected with vTF7-3, vUb-nsP4 (Tyr), and vaccinia virus-SIN recombinants expressing the indicated SIN polyproteins. In vitro assays were performed with SIN-specific JNTSCATX RNA substrate (+) or a heterologous HCVΔpoly(A) RNA substrate (−) under standard conditions. Denatured products were separated on an agarose gel and visualized by autoradiography. (B) SIN-specific proteins in the P15 fraction were separated on an SDS–8% polyacrylamide gel and detected by Western blotting with antisera (α) specific for nsP1, nsP2, nsP3, or nsP4. A lysate from mock-infected cells (mock) which had only been infected with vTF7-3 was included as a control. The positions of the SIN polyproteins and nsPs are indicated. Note that SIN nsP3 migrates as multiple species because of differential phosphorylation (19). (C) Experimental procedures were as described for panel A. In panels A and C, the bar on the left indicates the position of genome-length JNTSCATX RNA.

FIG. 7

Distribution of SIN nsPs in P15 and S15 fractions. P15 and S15 fractions were prepared from BHK-21 cells infected with the indicated vaccinia virus-SIN recombinants and vTF7-3. Material from equal numbers of cells was separated by SDS-PAGE on 8% polyacrylamide gels, and SIN-specific proteins were detected by Western blotting and probing with antiserum specific for nsP2 or nsP4. A lysate from cells which had only been infected with vTF7-3 (mock) was included as a control. The levels of SIN-specific protein were quantified with a Betagen Betascope, and the percentage of a particular SIN nsP present in the P15 and S15 fractions is indicated at the bottom.

FIG. 8

Extracts containing soluble nsP4 allow initiation of SIN RNA replication in vitro. P15 fractions were prepared from BHK-21 cells infected with vaccinia virus-SIN recombinant v123_C>S and vTF7-3 (lanes 2 to 5). The S15 fraction was prepared from cells coinfected with vTF7-3 and vUb-nsP4 (Tyr) or from cells infected with vTF7-3 alone (mock). In vitro reaction mixtures containing the P15 fraction and increasing amounts of nsP4-containing (5, 10, and 18 μl in lanes 2, 3, and 4, respectively) or mock (18 μl in lane 5) S15 fraction were incubated under standard conditions with JNTSCATX template RNA, and the denatured products were separated on an agarose gel. A P15 fraction from cells coinfected with vTF7-3, v123_C>S, and vUb-nsP4 (Tyr) was assayed in parallel as a positive control (lane 1).