Intracellular stability of the interfering activity of a defective interfering influenza virus in the absence of virus multiplication

Half-lives of the interfering activity of a human defective interfering (DI) influenza virus and of viral RNA in MDCK cells inoculated with noninfectious DI virus were 25 and 17 days, respectively, and of viral RNA in cells inoculated with noninfectious uv-irradiated standard virus was 21 days. In neither case was there evidence of virus replication (de novo synthesis of viral proteins, haemagglutinin, or infectivity). The half-life in BHK cells was shorter, although still considerable, showing evidence of a host contribution to stability. Implications of the putative persistence of influenza virus genes in vivo to the natural history of the virus are discussed.     

Replication in vitro of the influenza virus genome: selective dissociation of RNA replicase from virus-infected cell ribonucleoprotein complexes

Summary Replication of the influenza virus genome involves two discrete step reactions: vRNA-directed primer-independent (unprimed) synthesis of cRNA; and cRNA-directed unprimed synthesis of vRNA. Nuclear extracts from both MDCK and HeLa cells infected with influenza virus A/PR8/34 exhibited unprimed synthesis of both cRNA and vRNA strands (a parameter of RNA replication). Ribonucleoprotein (RNP) complexes with the replication activity were isolated from these nuclear extracts by glycerol gradient centrifugation in the presence of 0.1 M KCl. At 0.5 M KCl, however, these complexes were dissociated into stripped RNP and soluble protein fractions. The soluble fraction contained the activity of exogenous template-dependent unprimed RNA synthesis, indicating that the RNA replicase is dissociated from RNP upon exposure to high salt concentrations. On the other hand, the high salt-treated RNP catalyzed only primer-dependent RNA synthesis, but regained a low level activity of exogenous template-dependent unprimed RNA synthesis by adding nuclear extracts from uninfected cells, suggesting that host factor(s) is involved in the functional interconversion of influenza virus RNA polymerase.     

The effect of actinomycin D on the synthesis of influenza virus-specific RNA

Summary The effect of actinomycin D on the replication of CAM strain of influenza A1 virus was studied in the human conjunctival cell line, clone 1-5C-4. Actinomycin D in a concentration of 0.5 mcg/ml suppressed the virus yield to less than l/1000th of that from the uninhibited culture. When the addition of actinomycin D was delayed, the resistance of the production of virus and viral components to actinomycin D developed progressively in the order of the soluble antigen, hemagglutinin, and the infective virus. The synthesis of virus-specific RNA was demonstrable by the incorporation of tritiated uridine when actinomycin D was added to the culture at 6 hours after infection, but not when it was added at 1 hour after infection. These findings indicated that actinomycin D illhibits influenza virus replication because it inhibits the synthesis of virusspecific RNA and that, however, it does not directly block virus-specific RNA synthesisper se but exerts the inhibitory effect on some event(s) preceding virus-specific RNA synthesis. In FL cells, in which CAM virus undergoes an abortive multiplication cycle, virus-specific RNA was synthesized in the same period after infection and in a similar quantity as in clone 1-5C-4 cells in which complete multiplication occurs.     

Isolation of a novel type of interfering influenza B virus defective in the function of M gene

Summary A novel type of interfering influenza B virus which is defective in the function of M gene has been reported. Clone 301, a B type virus clone obtained by successive back-crosses of A/Aichi/2/68 (H 3 N 2) with B/Yamagata/1/73, grew normally in MDCK cells when inoculated at a low multiplicity, but was easily converted to a hemagglutinating but non-infectious form by one cycle of high multiplicity infection. Within MDCK cells infected with infectious clone 301 at a high multiplicity, synthesis of M protein was greatly reduced. The virus particle produced by a high multiplicity infection was devoid of RNA segment 7 (M gene), contained less amount of M protein compared with the standard virus, and interfered with the replication of wild type B/Yamagata, again accompanied by a selective suppression of M protein synthesis within the co-infected cells.With 9 Figures