In cell-free extracts derived from rabbit reticulocytes, encephalomyocarditis RNA can be translated completely, and the products can be processed extensively to give encephalomyocarditis virion proteins and several nonvirion proteins, including a genome-coded protein required for processing. The latter is probably a protease. Translation is very efficient. Under typical conditions, each EMC RNA is translated approximately eight times during a 3-h period. Kinetic analyses (time-course experiments, pulse-chase experiments, and pulse-stop experiments) have been used to determine the time of appearance of major products, and these times have been correlated with map positions. The gene for the putative protease is located near the middle of the genome downstream from the virion protein genes. Ribosomes can travel the length of encephalomycarditis RNA within 30 min, but there is a delay in their progress along the RNA at some point soon after they traverse the region coding for virion protein precursors. This delay results in the accumulation of precursors for a period of about 10 min before the putative protease is made and virion proteins (epsilon, alpha, and gamma) are released by proteolysis.

The nucleotide sequence has been determined for the first 53 bases of brome mosaic virus RNA4, the monocistronic messenger for brome mosaic virus coat protein. The sequence includes the binding site for wheat embryo ribosomes. The 5'-terminal base is a modified guanosine attached to the penultimate base through a 5' p-p-p 5' link. The initiating AUG codon is only 10 nucleotides from the 5'-terminus. The triplets following the AUG codon correspond to the known sequence of brome mosaic virus coat protein.

Poliovirus RNA can be translated completely and accurately in rabbit reticulocyte lysates; the nascent polyprotein is processed to give primary products la, X, and lb indistinuishable from those made in poliovirus-infected HeLa cells. The capsid precursor protein la is processed to form the capsid proteins VPO, VP1, and VP3, while the noncapsid precursor lb is processed to form protein 2.

Depolarized dynamic light scattering was used to measure the translational and rotational diffusion of tobacco mosaic virus, TMV, in aqueous solutions of dextran (M ∼505 000). TMV is an electrically charged, nucleoprotein assembly with the shape of a stiff, rigid rod. Dextran is an uncharged, flexible carbohydrate polymer. The TMV was held at a fixed, dilute concentration (0.5 mg/mL), while the concentration of dextran spanned both dilute and semidilute regimes (0−14.5% by weight). There was no evidence of phase separation or strong aggregation of the TMV particles in the presence of the dextran. The TMV particles dominated the depolarized scattering at all dextran concentrations. The angular variation of the decay rates of the autocorrelation functions always followed the form expected for symmetric top molecules in the absence of translational−rotational coupling. Nevertheless, translational and rotational motions are almost surely coupled in most dextran-containing solutions. The apparent translational and rotational diffusion rates decreased with added dextran, but not exactly according to the rise in macroscopic solution viscosity. A transition occurred at about 6.5% dextran. Beyond this concentration, pronounced failures of the continuum (Stokes−Einstein) relation between diffusion and viscosity were found. Translational diffusion continued more rapidly than expected on the basis of the macroscopic viscosity, while rotational diffusion fell sharply below expectation. The quotient D r/D t of rotational and translational diffusion, which presumably cancels effects due to viscosity, also dropped suddenly above the transition point. These findings are consistent with a sudden onset of topological constraints to rotational motion of the TMV, without onset of severe constraints to translational motion. Temperature dependent studies showed that either the solution or the solvent viscosity can describe translation and rotation fairly well, at least at concentrations below the transition. Energies of activation for translational and rotational diffusion of TMV were similar and not strongly dependent on dextran concentration in this regime.

The four RNAs of brome mosaic virus induce substantial incorporation of amino acids into protein when used as messengers in a cell-free proteinsynthesizing system derived from wheat embryo. RNA showed, quite unequivocally, that viral coat protein can be synthesized in a cell-free system from wheat embryo programmed with the RNA of satellite tobacco necrosis virus (STNV). The in vitro product has nearly the same spectrum of tryptic peptides as authentic STNV coat protein, although it is slightly lower in molecular weight, presumably as a consequence of an aberrant post-translational proteolytic cleavage. Klein et al. presented evidence that translation is initiated correctly and that synthesis of the single product is substantial. The exceptional success of this in vitro synthesis is attributable to the careful definition, by these investigators and also by Marcus and his colleagues (4, 5), of several of the important variables of the wheat system.The efficacy of the wheat-embryo system, the partial success of brome mosaic virus (BMV) RNA and STNV RNA translation in Escherichia coli extracts (6-8), and the fact that wheat is a natural host of BMV (although not of STNV) has prompted us to test the BMV RNAs as messengers in a wheat-embryo system. We find that the BMV RNAs serve as excellent messengers-with an efficiency comparable to that reported for bacteriophage RNA in E. coli extracts.BMV is one of several RNA-containing plant and animal viruses whose genetic complement is divided among several nucleic acids (9 MATERIALS AND METHODSPreparation of BMV, BMV RNA, and BMV Coat Protein. BMV (Russian strain) was grown and isolated by procedures described (10). BMV RNA was isolated from purified virus by phenol extraction (11). RNA 4, RNA 3, and a mixture of RNA 1 and 2 were obtained by fractionation of whole BMV RNA on 5-20% sucrose density gradients centrifuged in a Spinco SW25 rotor at 25,000 rpm for 18 hr followed by a second separation of appropriately combined fractions (6).Coat protein was prepared by dialyzing the virus against 1 M CaCI2. This degrades the virus and quantitatively precipitates the viral RNA, leaving the protein in solution (12).Isolation of Wheat Embryo and Preparation of an S23 CellFree Extract. Wheat embryos were isolated from seeds of Kenosha winter wheat (Triticum vulgare L, cultivar kenosha, 1799 Abbreviations: STNV, satellite tobacco necrosis virus; BMV, brome mosaic virus.