Functional analysis of the influenza A virus cRNA promoter and construction of an ambisense transcription system

While influenza A viral RNA is known to act as a template for the synthesis of both viral mRNA and complementary cRNA, the latter has been observed so far only to function as an intermediate in replication and give rise to progeny vRNA molecules. Here it is shown that the cRNA promoter is also capable of initiating viral mRNA synthesis, similar to vRNA-promoted transcription adhering to the cap-snatching mode of primer recruitment. Detection of cRNA promoted transcription required an inversion of the reporter gene coding sequence plus relocation of the viral polyadenylation signal. Construction of cRNA promoter variants through RNA polymerase I reverse genetics allowed us to determine the RNA polymerase-associated, base-paired conformation in a reporter gene read-out system. It again turned out to adhere to the "corkscrew" model, similar, but slightly different in its binding interactions from the corresponding vRNA conformation. The observation of two transcription reactions, initiated in either direction from influenza vRNA and cRNA template molecules, allowed us to construct bicistronic, ambisense RNA molecules for simultaneous expression of two proteins from a single segment of viral RNA.