Efforts to achieve persistent expression of influenza A virus cloned DNA in cells permissive for virus infection were initiated becuase such cells should be useful for investigation of the molecular biology of influenza virus and for isolation of specific viral mutants through complementation by the expressed gene. In this manner, naturally occurring or laboratory engineered mutants containing viable deletion mutations could be isolated for evaluation of their level of attenuation. Initially, simian cells permissive for influenza A virus infection were stably transformed with a full legnth cloned influenza A nucleoprotein gene under the control of an inducible metallothionein promoter and linked to a dihydrofolate reductase gene to facilitate selection of transformed cells. The tranformed cells which were selected synthesized an influenza A viral nucleoprotein which was indistinguishable from the nucleoprotein synthesized in virus-infected cells with respect to meolecular weight and intracellular localization. It was estimated that transformed (CV1-NP) cells produced only 1% of the amount of nucleoprotein synthesized in simian cells infected with influenza A virus. Nonetheless, when these cells were infected with influenza virus mutants which synthesized temperature sensitive nucleoprotein, protein nucleoprotein, protein expressed by the cloned gene was able to complement the synthesis of plus-strand and minus-strand viral RNA for some mutants and only plus-strand synthesis for other nutants. This indicated that the nucleoprotein expressed in the transformed cells form cloned influenza A virus neucleoprotein cDNA exhibited functional activity. Furthermore, under appropriate conditions CV1-NP cells complemented the replication of ts mutant viruses. This complementation effect is currently being analysed to gain a better understanding to the biological functions of the viral nucleoprotein and to develop a strategy for isolation of viable deletion mutants.
