Characterization of influenza virus NS1 protein by using a novel helper-virus-free reverse genetic system

We have developed a novel helper-virus-free reverse genetic system to genet ically manipulate influenza A viruses. The RNPs, which were purified from t he influenza A/WSN/33 (WSN) virus, were treated with RNase H in the presenc e of NS (nonstructural) cDNA fragments. This specifically digested the NS R NP, The NS-digested RNPs thus obtained were transfected into cells together with the in vitro-reconstituted NS RNP, The NS digested RNPs alone did not rescue viruses; however, cotransfection with the NS RNP did. This protocol was also used to rescue the NP transfectant, We obtained two NS1 mutants, dl12 and N110, using this protocol. The dl12 NS gene contains a deletion of 12 amino acids at positions 66 to 77 near the N terminus. This virus was t emperature sensitive in Madin-Darby bovine kidney (MDBK) cells as well as i n Vero cells. The translation of all viral proteins as well as cellular pro teins was significantly disrupted during a later time of infection at the n onpermissive temperature of 39 degrees C. The N110 mutant consists of 110 a mino acids which are the N-terminal 48% of the WSN virus NS1 protein, Growt h of this virus was significantly reduced at any temperature. In the virus- infected cells, translation of the M1 protein was reduced to 10 to 20% of t hat of the wild-type virus; however, the translation of neither the nucleop rotein nor NS1 was significantly interfered with, indicating the important role of NS1 in translational stimulation of the M1 protein.