Kinetics of Synthesis of Influenza Virus Ribonucleoprotein Structures

Abstract

SUMMARYThe synthesis of influenza virus ribonucleoprotein structures (RNPs) in infected chick embryo cells was analysed by polyacrylamide gel electrophoresis (PAGE) in the presence of sodium deoxycholate which resolves the RNPs into five size classes. A relatively small proportion of total RNPs accumulated in the nucleus but free NP protein was found there in large amounts over the period 1.5 to 4 h post-infection. In contrast, by 4 h post-infection, all cytoplasmic NP was complexed into RNP structures. At early times, during a 15 min pulse of [aSSlmethionine, nearly all the newly synthesized NP was incorporated into RNPs but by 4 h the majority of pulse-labelled NP was present as free protein. However, the proportion of free NP:NP in RNPs remained constant over the 1.5 to 4 h post-infection period, indicating that there was a delay before the NP synthesized later in infection was assembled into RNP structures. Individual RNP size classes were predominantly cytoplasmic and accumulated at similar rates but were not produced in equimolar amounts. The rates of synthesis of individual RNPs were in general agreement with their rates of accumulation with the remarkable exception of RNP d (containing RNA 7, the matrix protein gene). This was synthesized nearly 10-fold faster but accumulated at the same rate as the other RNPs. Possibly RNP d is more rapidly degraded than the other RNPs.The influenza virus genome consists of eight separate segments of single-stranded negative-sense RNA (RNA-, McGeoch et al., 1976;Palese & Schulman, 1976). In the infected cell these RNA segments are transcribed into two forms of complementary plus-sense RNA (RNA+): a truncated copy which serves as messenger RNA (mRNA) to direct the synthesis of virus proteins and a complete transcript (cRNA) which serves as template for the synthesis of negative-sense virus RNA (Hay et al., 1977a, b). Where the various forms of virus-coded RNA are synthesized is unclear, but there is evidence that the cell nucleus is the probable site of transcription of virus mRNA (Armstrong & Barry, 1974;Taylor et al., 1977;Mark et al., 1978Mark et al., , 1979 Barrett et al., 1979).Within the virion the RNA segments are complexed with nucleoprotein (NP; Pons et al., 1969) to form separate ribonucleoprotein (RNP) complexes (Duesberg, 1969; Compans et al., 1972). In extracts from infected cells, RNPs containing both RNA + and RNA-have been found (Pons, 1971(Pons, , 1975 and it seems reasonable to suppose that all intracellular virus-induced RNA may be present in RNP complexes. Apart from trace amounts of other virus proteins the principal component of intracellular RNPs is the NP protein (Caliguiri & Gerstein, 1978; Rees & Dimmock, 1981 b).A number of studies has been made of the kinetics of synthesis and accumulation of virus-specific RNA (Scholtissek & Rott, 1970; Hay et al., 1977a, Barrett et al., 1978Mark et al., 1978) and proteins (Skehel, 1972(Skehel, , 1973 Meier-Ewert & Compans, 1974;Lamb & Choppin, 1976; Inglis et al., 1976 Inglis et al., , 1978 Inglis & Mahy,...

Basic Residues of the Helix Six Domain of Influenza Virus M1 Involved in Nuclear Translocation of M1 Can Be Replaced by PTAP and YPDL Late Assembly Domain Motifs

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