Intracellular localization of the viral polymerase proteins in cells infected with influenza virus and cells expressing PB₁ protein from cloned cDNA

The biosynthesis, nuclear transport, and formation of a complex among the influenza polymerase proteins were studied in influenza virus-infected MDBK cells by using monospecific antisera. To obtain these monospecific antisera, portions of cloned cDNAs endocing the individual polymerase proteins (PB₁, PB₂, or PA) of A/WSN/33 influenza virus were expressed as fusion proteins in Escherichia coli, and the purified fusion protein were injected into rabbits. Studies using indirect immunofluorescence showed that early in the infectious cycle (4 h postinfection) of influenza virus, PB₁ and PB₂ are present mainly in the nucleus, whereas PA is predominantly present in the cytoplasm of the virus-infected cells. Later, at 6 to 8 h postinfection, all three polymerase proteins are apparent both in the cytoplasm as well as the nucleus. Radiolabeling and immunoprecipitation analyses showed that the three polymerase proteins remain physically associated as a complex in either the presence or the absence of ribonucleoproteins. In the cytoplasm, the majority of the polymerase proteins remain unassociated, whereas in the nucleus they are present as a complex of three polymerase proteins. To determine whether a polymerase protein is transported into the nucleus individually, PB₁ was expressed from the cloned cDNA by using the simian virus 40 late promoter expression vector. PB₁ alone, in the absence of the other polymerase proteins or the nucleoprotein, accumulates in the nucleus. This suggests that the formation of a complex with other viral protein(s) is not required for either nuclear transport or nuclear accumulation of PB₁ protein and that the PB₁ protein may contain an intrinsic signal(s) for nuclear transport.