Cell-free synthesis of encephalomyocarditis virus

Abstract

We developed a system for complete replication of encephalomyocarditis virus (EMCV) in a test tube by using an in vitro translation extract from Krebs-2 cells. Efficient virus synthesis occurred in a narrow range of Mg(2+) and EMCV RNA concentrations. Excess input RNA impaired RNA replication and virus production but not translation. This suggests the existence of a negative-feedback mechanism for regulation of RNA replication by the viral plus-strand RNA or proteins.

FIG. 1.

(A) Time course of synthesis and processing of EMCV-specific proteins in EMCV RNA-programmed Krebs-2 S10. EMCV RNA (20 μg/ml) was translated in the presence of [³⁵S]methionine under standard reaction conditions. At the times indicated, 5-μl aliquots of the reaction mixture were withdrawn, supplemented with SDS sample buffer, and subjected to SDS-15% PAGE. An autoradiogram of the dried gel is shown. The assignment of polypeptides is based on a comparison with proteins synthesized in EMCV-infected BHK-21 cells (34). An asterisk indicates the position of 1B. (B) EMCV RNA dose response of protein synthesis. Reaction mixtures (12.5 μl) were programmed by the indicated concentrations of EMCV RNA at 32°C for 2 h. Conditions for translation and PAGE were as described for panel A.

FIG. 2.

EMCV RNA replication. (A) Kinetics of EMCV RNA and protein synthesis. EMCV RNA (10 μg/ml)-programmed reaction mixtures (50 μl) were pulse-labeled with 1.5 μl of [α-³²P]CTP (10 mCi/ml [3,000 Ci/mmol]) for 1 h (the label was added 30 min before the times indicated in the figure) (4). Samples were treated with proteinase K, and RNA was extracted with a mixture of phenol and chloroform. Viral proteins were pulse-labeled with [³⁵S]methionine for 30 min (15 min before the times indicated in the figure) and dissolved in an SDS sample buffer. Portions of each sample were assayed for trichloroacetic acid-insoluble radioactivity. Data (average of two independent determinations) are given as percentages of maximum incorporation. (B) Products of RNA synthesis (from the reaction described for panel A) analyzed by native Tris-borate-EDTA-1% agarose gel electrophoresis and autoradiography (16). The positions of single-stranded (vRNA) and double-stranded (dsRNA) EMCV RNA are indicated. (C) Mg²⁺ concentration dependence of RNA synthesis. Reaction mixtures contained 10 μg of EMCV RNA/ml and the indicated concentrations of MgCl₂. (D) EMCV RNA dose response of RNA synthesis. Reaction mixtures contained 3 mM MgCl₂ and the indicated concentrations of EMCV RNA. For panels C and D, RNA was labeled with [α-³²P]CTP between 4 and 5 h of incubation and analyzed as described above.

FIG. 3.

Synthesis of EMCV in vitro. (A) Krebs-2 S10 extracts were incubated for 20 h either at 32°C (incubations a and c) or 0°C (incubation b) in the absence (incubation a) or presence (incubations b and c) of EMCV RNA (20 μg/ml). The samples were treated with a mixture of RNase A and T1 (21). Using serial dilutions of samples, a plaque assay was carried out on confluent BHK-21 cells (30). Plaques characteristic for EMCV, which was propagated in Krebs-2 cells in vivo, are shown for comparison (incubation d). (B) Isopycnic banding of in vitro-synthesized EMCV in a cesium chloride density gradient. A 250-μl translation reaction mixture was programmed with EMCV RNA for 20 h at 32°C and treated with the RNase A/T1 cocktail as described above. Triton X-100 was added to 0.5%, and the mixture was diluted to 5 ml with CsCl/phosphate-buffered saline (1.34 g/cm³ solution density) (30). The mixture was subjected to centrifugation at 45,000 rpm for 18 h at 6°C in a SW55 rotor. Fractions (0.25 ml) were collected and assayed for infectivity following serial dilutions. The arrow indicates the location of the virus formed in EMCV-infected Krebs-2 cells. (C) Time course of the infectivity titer of in vitro-synthesized EMCV. (D) Effect of creatine phosphate concentrations on EMCV yields. Reaction mixtures containing the indicated concentrations of creatine phosphate and 3.4 mM MgCl₂ were programmed with EMCV RNA for 20 h and assayed for infectivity as described above. In panels B, C, and D, programming was with 10 μg of EMCV RNA/ml. (E) EMCV RNA dose response of virus yield. Reaction mixtures were programmed with the indicated concentrations of EMCV RNA for 20 h at 32°C, treated with RNase A/T1, and assayed for infectivity following serial dilutions. In panels C, D, and E, the data are averages (with standard deviations from the means) of three independent titer determinations.