J. Hal. Biol. (1967) 25, 383-405 n the Structure and Replication of Influenza Virus PETER H. DUESBERG AND WILLIAM 8. ROBINSON Department of Molecular Biology and Virus Laboratory University of California, Berkeley, California 94720, U.S.A. (Received 23 September 1966, and in revised form 18 November 1966) Influenza virus PR8 has been purified from the allantoio fluid of infected chick embryos and from tissue culture medium. Preliminary characterization indicates that the virus contains at least two major proteins and several RNA components with sedimentation coefficients (SZoZW) ranging from about 9 to 18 s. Synthesis of virus-specific RNA m influenza virus-infected chick embryo cells has been demonstrated under conditions where cell RNA synthesis is suppressed by actinomycin D. In addition to the single-stranded viral RNA components, a smaller fraction of virus-specific double-stranded RNA is labeled with [%C]- uridine in the presence of actinomycin D. This RNA has been purified after incubation of the RNA from infected cells with RNase. It has an approximate sedimentation coefficient of 8 to 10 s, shows a sharp thermal transition and has a lower buoyant density in cesium sulfate than single-stranded influenza virus -RNA. Preliminary evidence indicates that the double-stranded RNA may be an intermediate in viral RNA synthesis. 1. Introduction Because of the unusual genetic behavior of influenza virus and the failure to demonstrate infectious viral RNA, there has been considerable :interest in the size and structure of influenza virus RNA (Hirst, 1962). Recently Newcastle disease virus (Duesberg & Robinson, 1965) another myxovirus, and several structurally related RNA tumor viruses (Robinson, Pitkanen & Rubin, 1965; Robinson & Ral~da, 1965; Duesberg $ Robinson, 1966; Duesberg & Blair, 1966), have been shown to contain a singl.e-strandedRNA molecule with a higher sedimentation coefficient than the RNA’s of the small RNA viruses such as polio and the R,NA phages. Using similar methods we have purified influenza virus PR8 and isolated its RNA. Pre- liminary characterization indicates that the RNA as recovered consists of several distinct single-stra,ndedpieces, none of which has a higher sedimentation coefficient t,han 18 S.In the presence of Mg2+ and at high ionic strength, part of the RNA can be isolated as an aggregate sedimenting at 38 S. The aggregate is dissociated in the presenceof EDTA and at low ionic strength. There has also been much interest in the mechanism of nucleic acid synthesis during the replication of viruses containing single-stranded nucleic acids. The small bacteriophage 56X174, which contains a single-stranded DNA, has been shown to make double-stranded DNA during its replication (Sinsheimer, Starman, Nagler & @&brie, 1962). In an analogous fashion, at least partly double-stranded RNA is formed in cells infected with small RNA-containing animal viruses (Montagnier & Sanders, 1963; Baltimore, Becker & Darnell, 1964; Bishop, Summers & Levintow* 383