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Granboulan, Nicole (Institute de Recherches sur le Cancer, Villejuif, Seine, France), and Richard M. Franklin. High-resolution autoradiography of Escherichia coli cells infected with bacteriophage R17. J. Bacteriol. 91:849-857. 1966.-The ultrastructural alterations in Escherichia coli infected with the RNA bacteriophage R17 were further investigated by means of the technique of high-resolution autoradiography. Tritiated precursors to ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and protein were employed in separate experiments. A striking inhibition of cellular RNA, DNA, and protein synthesis was noted. Whereas normal RNA synthesis occurs in the nucleoid, in infected cells RNA synthesis is predominantly cytoplasmic, but later in the latent period, and during the stage of active viral growth, the label is localized in a polar region. In the late stages of viral growth, RNA synthesis occurs only around the crystals. Protein synthesis also becomes localized in a polar region, but DNA synthesis remains confined to the nucleoid. Under conditions of chloramphenicol inhibition of viral-coat protein synthesis, RNA label is localized in the paranuclear lesion, providing further indication that RNA forms this fibrillar structure.

The ribonucleic acid (RNA) product resulting from annealing R17 RNA with denatured replicative form or replicative intermediate could be divided into two distinct types of RNA by precipitation in 1.5 m NaCl. The RNA found in the salt supernatant fluid was resistant to digestion by ribonuclease, had a sedimentation coefficient of 15S, and displayed a sharp thermal transition. The RNA in the salt supernatant fluid appeared to be identical to replicative form. The RNA found in the salt precipitate was resistant to digestion by ribonuclease, but possessed both single- and double-stranded characteristics. The RNA sedimented as a broad band in a sucrose gradient, with a sedimentation coefficient of 15S, and displayed a melting transition characteristic of a mixture of single- and double-stranded RNA. Mild ribonuclease digestion of the salt-precipitable RNA produced a ribonuclease-resistant material with sedimentation properties identical to the RNA found in the salt supernatant fluid.

Purified replicative form (RF) and replicative intermediate (RI) prepared from Escherichia coli cells infected with the ribonucleic acid (RNA) bacteriophage R17 were denatured with dimethyl sulfoxide at 37 C or in aqueous solvents of low ionic strength at 97 C. Denaturation was demonstrated for RF and RI by an increase in specific infectivity and a striking change in the hyperchromicity curves after treatment. RI denaturation was also demonstrated by a shift in the buoyant density in Cs(2)SO(4) from 1.619 to the buoyant density of single-stranded R17 RNA (1.627). Analysis of the denatured RI hyperchromicity curves and the equilibrium distributions of denatured RI in Cs(2)SO(4) gradients revealed, however, a residual double-stranded component. Velocity sedimentation of denatured RI was performed, and the weight distribution of S values was calculated. From the known relation between molecular weight and S values, it was possible to transform the weight distribution into a number distribution of chain lengths. This distribution was compared with that predicted from the steady-state hypothesis for RI. Deviations from the predicted distribution may be due to the residual double-stranded component.

NaClO(4) was employed in a technique for the rapid extraction of reovirus ribonucleic acid (RNA). The extracted RNA, which was purified in a Cs(2)SO(4) equilibrium density gradient, had a buoyant density of 1.61 g/cm(3) and a sedimentation coefficient of 15S in a 7 to 20% sucrose gradient. It was 90% resistant to ribonuclease in a solution of high ionic strength (0.1 m NaCl). The sensitivity of reovirus RNA to ribonuclease increased with decreasing ionic strength. The thermal denaturation transition of the RNA began at 78 C and was complete at 85 C. The T(m) of the transition was 81 C in 0.01 m tris(hydroxymethyl)aminomethane buffer (pH 7.2) containing 0.001 m ethylenediaminetetraacetate. Thermal denaturation of reovirus RNA resulted in the formation of three ribonuclease-sensitive fractions. Denaturation at 25 C in the presence of dimethyl sulfoxide resulted in the formation of two ribonuclease-sensitive fractions.

Escherichia coli cells were irradiated with ultraviolet light to stop ribosomal RNA synthesis. After infection of such cells with the single-stranded RNA phage R17, the RNA most rapidly labeled by a pulse of tritiated uridine sedimented in a broad band in the 16S region of sucrose gradients. The effect of ribonuclease on this material and its behavior during a "chase" period in nonradioactive medium suggest that it consists of a core of double-stranded RNA, one strand of which-the viral strand-is continually displaced by a growing strand forming single-stranded tails and ultimately free 27S viral RNA.

A correlation of cytochemical changes with virus production has been studied in L cells infected with Mengovirus. After a latent period of about 2 hours, virus was produced rapidly, reaching maximum titers of up to 12,000 particles per cell in 6 to 8 hours. The earliest cytological change was in the nucleus and consisted of a slight condensation of chromatin. There is no evidence, however, for the multiplication of either the viral RNA or protein in the nucleus. RNA, of high molecular weight, accumulated in the perinuclear area of the cytoplasm and was later found in inclusions. The perinuclear RNA was digestible with RNase and may be located in or on ribosomes. The inclusion RNA was resistant to RNase but could be removed by pepsin or potassium permanganate; it is probably in completed virus particles. Viral antigen was first observed in a perinuclear location and later in the above-mentioned inclusions. Although the viral protein contains appreciable amounts of arginine and lysine, it is not a basic protein of the histone type. Phase-contrast microscopy of living cells clearly demonstrated the role of the inclusions in release of virus from infected cells. A comparison is made between these cytological changes in Mengo-infected cells and those which have been found by other workers in polio-infected cells. There are many very similar changes.

A detailed qualitative and quantitative comparison was made of the ultrastructure of single-stranded ribonucleic acid (RNA) from bacteriophage R17 and double-stranded replicative form (RF) and replicative intermediate (RI) from cells infected with this bacteriophage. The nucleic acids were prepared for electron microscopy by the protein monolayer spreading technique of Kleinschmidt. Single-stranded RNA aggregated during spreading in the absence of urea, whereas RF and RI did not. On the other hand, RF and RI appeared to be susceptible to shear during spreading, whereas R17 RNA was not. From the maximal length of RF, a base translation of 3.14 A was calculated. This value favors a 10-fold helix model of double-stranded RNA. The same base translation was found for R17 RNA, indicating a stacked base structure for single-stranded RNA spread in the presence of urea. RI is a branched structure and the branches are removed by ribonuclease treatment. The branches are believed to be nascent single-stranded viral RNA. The contour length of the branch was equal to the contour length of the main chain up to the branch point, as predicted from theoretical analysis of the replication of viral RNA. The structure of RF and the main chain of RI was also analyzed by plotting the log (end-to-end distance squared) versus log (contour length). This demonstrated structures intermediate in stiffness between a random coil and a rigid rod.

Replicative intermediate (RI) is considered to be the double-stranded ribo-nucleic acid (RNA) template for synthesis of viral RNA, with bound nascent single-stranded viral RNA. A theoretical description of RI is based on the analysis of a steady state of biopolymerization on a template which determines not only nucleotide sequence, but also chain length. The hydrodynamic properties of RI isolated from Escherichia coli infected with bacteriophage R17 are compared with those of RNA isolated from R17 (single-stranded RNA) and of replicative form (RF) isolated from E. coli infected with R17. RF is double-stranded RNA template without any single-stranded component. Whereas S for R17 RNA is a function of the ionic strength (Gamma/2) of the solvent, S is almost invariant with Gamma/2 for RF. By contrast S for RI lies between the sedimentation constants for R17 RNA and RF and S varies with Gamma/2 as does R17 RNA. The weight distribution of S for RI demonstrates the heterogeneity of this material, and the variation in the weight distribution with ionic strength demonstrates the duality of structure in RI. Using S and [eta], the Mw for RI is estimated to be 2.6 x 10(6) daltons, as compared with the theoretical value of 2.9 x 10(6) daltons.

Purified replicative form (RF) and replicative intermediate (RI) prepared from Escherichia coli infected with R17 were denatured in 0.15 m NaCl, 0.015 m sodium citrate containing 65% dimethylsulfoxide. Denaturation of RF or RI was demonstrated spectrophotometrically, chromatographically, and by sedimentation analysis. Denatured RF or RI was annealed by carefully decreasing the temperature from 62 to 20 C. Annealing was accompanied by a decreased absorbance at 260 mmu. The decrease in absorbance during annealing appeared to be dependent upon the rate of cooling and the concentration of ribonucleic acid (RNA). Denatured RF or RI was annealed with R17 RNA which was labeled with (3)H-uridine. The annealed product was 73 to 82% resistant to 0.1 mug/ml of ribonuclease. Annealing R17 RNA with either denatured RF or RI resulted in the formation of a ribonuclease-resistant product with a sedimentation profile resembling that of native RI. Melting the annealed products in 85.7% dimethyl sulfoxide produced 27S single-stranded R17 RNA and a heterogeneous population of more slowly sedimenting RNA.