title: Electroporation of influenza virus ribonucleoprotein complexes for rescue of the nucleoprotein and matrix genes.
abstract: Reverse genetics has been successfully used for the generation of recombinant influenza virus with altered biological properties. The standard method is based on DEAE-dextran transfection of in vitro reconstituted influenza virus ribonucleoprotein complex (RNP) into helper virus infected cells with subsequent selection of the recombinant viruses. Here we report the utilization of electroporation for reverse genetics of influenza virus as an improvement over the standard method. In a neuraminidase (NA) gene rescue system, we were able to demonstrate that electroporation of in vitro reconstituted NA RNP of influenza A/WSN/33 (H1N1) virus into WSN/HK virus infected cells allows the rescue of the transfectant WSN virus. The titer of transfectant virus obtained using electroporation is comparable to that generated using the DEAE-dextran transfection method. More significantly, the ratio of transfectant virus to helper virus is as much as 20-fold greater than that achieved using the DEAE-dextran system. We have also used electroporation to generate recombinant influenza virus carrying cDNA-derived matrix (M) gene or nucleoprotein (NP) gene of the WSN virus by using the temperature-sensitive (ts) mutants ts51 and ts56 as helper viruses. In the case of electroporation of M gene RNP, 88% of the viruses isolated after selection at 39 degrees C were transfectants. In contrast, the majority of viruses obtained using the DEAE-dextran transfection method were revertants of the helper virus. The NP-gene transfectant was only generated by the electroporation method. Our results suggest that electroporation of influenza virus RNP may be a useful method for generation of recombinant influenza viruses, especially in a system in which a ts mutant is used as helper virus.
mesh_terms: D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D018274:Electroporation; D005814:Genes, Viral; D006378:Helper Viruses / Q000235:genetics; D006801:Humans; D009980:Influenza A virus / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D019590:Nucleocapsid Proteins; D009698:Nucleoproteins / Q000235:genetics*; D016601:RNA-Binding Proteins; D014758:Viral Core Proteins / Q000235:genetics*; D014763:Viral Matrix Proteins / Q000235:genetics*
chemical_list: D017931:DNA Primers; C031540:M-protein, influenza virus; C048842:NP protein, Influenza A virus; D019590:Nucleocapsid Proteins; D009698:Nucleoproteins; D016601:RNA-Binding Proteins; D014758:Viral Core Proteins; D014763:Viral Matrix Proteins