Bo Öberg

Binding of histidine to tobacco mosaic virus RNA.

Abstract Tobacco mosaic virus (TMV) can be aminoacylated with the amino acid histidine in the presence of ATP, magnesium ions and a crude ligase preparation from yeast or mammalian cells.

Replicative structures of poliovirus RNA in vivo.

Abstract A new approach to characterize the replicative structures of poliovirus RNA in vivo is described. The replicative intermediate and the replicative form could be isolated by phenol extraction from poliovirus infected cells. However, cell homogenates treated with diethylpyrocarbonate prior to phenol extraction contained mainly single-stranded RNA and most of the replicative intermediate and 20 to 40% of the replicative form disappeared. This effect was probably due to a separation of the strands of an essentially single-stranded replicative structure prior to phenol extraction. Diethylpyrocarbonate did not appear to cause a selective loss or melting of double-stranded structures. Free minus strands corresponding to the reduced level of replicative intermediate and replicative form could be detected by annealing with poliovirus RNA. Poliovirus polymerase in vitro was inhibited effectively by diethylpyrocarbonate. The results indicate that poliovirus RNA is synthesized in a replicative structure which is mainly single-stranded and kept together by the polymerase and very short regions of base-pairing.

Biochemical and biological characteristics of carbethoxylated poliovirus and viral RNA

Abstract Diethylpyrocarbonate inactivates the infectivity of single-stranded poliovirus RNA but not that of the double-stranded replicative form. The inactivation is probably due to carbethoxylation of amino groups which are not hydrogen-bonded and shielded by the phosphate-sugar backbone in a double helix. The electrophoretic mobility of either single- or double-stranded RNA in polyacrylamide-agarose gels was unchanged after treatment with diethyl pyrocarbonate. The rate of ribonuclease inactivation by diethyl pyrocarbonate was determined using the infectivity of poliovirus RNA as an assay system. Poliovirions are inactivated by diethyl pyrocarbonate. The inactivated virus has a higher density and a higher electrophoretic mobility than untreated virus. Isolated RNA from diethyl pyrocarbonate-inactivated virions is still infectious and the virions are sensitive to ribonuclease. Poliovirus is partially degraded in saturated solutions of diethyl pyrocarbonate. The loss of infectivity might be caused by a change of the protein coat since carbethoxylated virus adsorbs abnormally to cells.

Partition studies on nucleic acids II. Countercurrent distribution of virus RNA

Abstract 1. 1. Partition coefficients for some polynucleotides, poliovirus RNA and tobacco mosaic virus (TMV)RNA as well as intact poliovirus have been determined in an aqueous polymer two-phase system with varying ionic composition (dextran 500, 5%, w w-polyethylene glycol 6000, 4%, w w ). 2. Countercurrent distribution of poliovirus, poliovirus RNA and TMV-RNA has been performed in these phase systems. A new type of apparatus for counter-current distribution was used in these model experiments. 3. The distribution curve for poliovirus RNA is in good agreement with the theoretical distribution, and 10–60% of the infectivity was recovered. 4. Intact poliovirus and poliovirus RNA are well separated with 60 transfers. This number of transfers also gives a minor separation of poliovirus RNA and TMV-RNA.

Gel filtration of nucleic acids on pearl-condensed agarose

Abstract The use of gel filtration to separate molecules with molecular weights above 2 · 105 was made possible by the introduction of granulated agar gels by Polson (1961) . These gels might allow separation of high molecular nucleic acids according to size. An artificial mixture of T2 DNA and E. coli RNA has been separated on granulated 1,5% agarose gel ( Boman and Hjerten, 1962 ). The low flow rates in gels with agar concentrations less than 3% made them difficult to use but this was overcome by the introduction of pearl-condensed agar or agarose ( Bengtsson and Philipson, 1964 , Hjerten, 1964 ). The present study describes gel filtration of nucleic acids from KB cells and poliovirus RNA on pearl-condensed 2% agarose. It is also shown that the composition of the buffer influences the elution pattern of poliovirus RNA.

Interaction of formate esters with viral RNA and viral polymerase

Abstract Esters of chloroformic acid inactivate the infectivity of single-stranded poliovirus RNA, but not that of the double-stranded replicative form. Diethyl pyrocarbonate, a similar structure does not inactivate the infectivity of the replicative form nor that of the replicative intermediate. The chloroformate esters are hydrolyzed with half-lives of 0.8 to 72 min at pH 7 and 0°, and the rate of inactivation of the RNA infectivity occurs at a comparable rate under the same conditions. The efficiency of inactivation of infectivity varied between the esters, and was probably not due to breakage of the phosphodiester bonds since no reduction in size could be demonstrated. Inactivation of poliovirus polymerase in vitro was also observed for the chloroformate esters. The inactivation of the polymerase is probably due to a reaction with the protein moiety and not with the RNA, with the exception of methyl chloroformate which in vitro inactivates RNA infectivity in the polymerase complex.