The Sedimentation of Influenza Virus and its RNA in Sucrose Density Gradients

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Abstract
Summary In 15 to 60% sucrose density gradients the distribution of standard preparations of influenza viruses measured by haemagglutination or infectivity was always bimodal. Approximately 17% of virus particles sedimented in a diffuse leading peak (fraction 1), while the rest was confined to a second homogeneous peak (fraction 2). Virus particles comprising fraction 1 were less dense (1.250 g./cm.3) than those of fraction 2 (1.257 g./cm.3). RNA labelled with 32P was extracted by phenol + sodium dodecyl sulphate from four strains of influenza A virus (pr 8, bel, a 2/singapore/1/57 and fowl plague) and from one strain of influenza B virus (lee). The sedimentation characteristics of each preparation of RNA on sucrose density gradients varied under different conditions of ionic strength. In the presence of 0.1 m-NaCl + 0.001 m-EDTA, RNA from fraction 2 could be resolved into at least two species. The predominant species had a sedimentation coefficient of 18 to 21 S and (with the exception of the a2 RNA) about 10 to 15% of the total RNA sedimented as a diffuse band with a sedimentation coefficient of 32 to 42 S. The bel and lee strains contained a third species of RNA, with a sedimentation coefficient of 7 to 9 S. In 0.005 m-EDTA without NaCl the RNA from fraction 2 (pr 8) sedimented principally as a 14 S component, and the 32 to 42 S component was not found. Fraction 1 virus particles also contained several RNA components when analysed under conditions of high ionic strength. A 7 to 9 S RNA species was found in amounts equal to, or greater than, the 18 to 21 S RNA component, together with a small amount of ‘38’ S RNA. In the presence of 0.005 m-EDTA the 38 S component was not found, but three components with sedimentation coefficients of 16 S, 9 S and 5 S were present in approximately equivalent amounts. From these findings it is concluded that the RNA of influenza viruses consists largely of 18 S molecules, under defined conditions of ionic strength, and that the 32 to 42 S component is an artifact caused by aggregation. It is further concluded that sucrose gradients containing 0.1 m-NaCl are unsatisfactory for studying the sedimentation characteristics of influenza RNA.

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