In Vitro Synthesis of Influenza Viral RNA: Characterization of an Isolated Nuclear System That Supports Transcription of Influenza Viral RNA1

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Abstract
An in vitro system for the synthesis of influenza viral RNA was developed using isolated nuclei prepared from influenza virus-infected HeLa cells. In this system, two species of positive-sense RNA, i.e. , mRNA and cRNA (complementary RNA to vRNA), were found to be synthesized when analyzed by RNA-RNA hybridization using a minus-strand RNA probe and high resolution gel electrophoresis. The in vitro RNA synthesis required Mg 2+ , GTP, CTP, UTP, a high concentration of ATP, and an ATP regenerating system. Neither actinomycin D nor α-amanitin, potent inhibitors for cellular DNA-dependent RNA polymerases, inhibited the RNA synthesis. Addition of ApG or capped RNA, well-known primers for virion-associated RNA polymerase, markedly enhanced the extent of RNA synthesis. The maximum activity was observed with nuclei isolated from cells at 5 h after infection. This system is useful for the purification and characterization of factors involved in the transcription of these two species positive-sense RNA.