We sought to produce persistent expression of influenza virus cloned DNA in cells permissive for virus infection. Such cells would be useful for investigation of the molecular biology of influenza virus and for isolation of specific viral mutants via 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. For selection of cells expressing transfected viral DNA sequences we employed an expression vector containing a mutant dihydrofolate reductase (DHFR) gene as a selectable marker. Alternatively, a neomycin resistance gene was used for coinfection and transformed cells were selected in the presence of G418, an analogue of neomycin. A series of recombinant DNA molecules were constructed between the DHFR expression vector and influenza nucleoprotein (NP) DNA under the control of an inducible metallothioneine promoter or a constitutive SV40 promoter. Transfection of simian CV-1 cells with these DNA recombinants followed by selection with methotrexate yielded cloned cell populations which were analyzed for NP synthesis. NP expressing cells were isolated at high frequency only when the inducible metallothioneine promoter was employed. Our failure to obtain NP expressing cells in the constitutive system suggests NP synthesis may be toxic to cell growth. Similar studies with influenza non-structural protein (NS) recombinants showed that stable synthesis of NS occurred only when the inducible system and the neomycin-resistant gene were employed. The stability of both the NP and NS producing cells is currently being analyzed. The expressed influenza virus protein in these cells will be analyzed for its ability to complement mutants that have a defect in that protein.
