Rescue of influenza virus expressing GFP from the NS1 reading frame.
In this study, several influenza NS1 mutants were examined for their growth ability in interferon (IFN)-deficient Vero cells treated with human interferon alpha (IFN-alpha). Mutants with an intact RNA binding domain showed similar growth properties as the wild-type virus, whereas viruses carrying an impaired RNA binding domain were dramatically attenuated. Relying on the ability of the first half of the NS1 protein to antagonize the IFN action, we established a rescue system for the NS gene based on the transfection of one plasmid expressing recombinant NS vRNA and subsequent coinfection with an IFN sensitive helper virus followed by adding of human IFN-alpha as a selection drug. Using this method, a recombinant influenza A virus expressing green fluorescence protein (GFP) from the NS1 reading frame was rescued. To ensure the posttranslational cleavage of GFP from the N-terminal 125 amino acids (aa) of NS1 protein, a peptide sequence comprising a caspase recognition site (CRS) was inserted upstream the GFP protein. Although a rather long sequence of 275 aa was inserted into the NS1 reading frame, the rescued recombinant vector appeared to be genetically stable while passaging in Vero cells and was able to replicate in PKR knockout mice.
Kittel
Christian
C
Institute of Applied Microbiology, University of Natural Resources and Applied Life Sciences, A-1190 Vienna, Austria.
Sereinig
Sabine
S
Ferko
Boris
B
Stasakova
Jana
J
Romanova
Julia
J
Wolkerstorfer
Andrea
A
Katinger
Hermann
H
Egorov
Andrej
A
D000818:Animals; D002522:Chlorocebus aethiops; D004285:Dogs; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006801:Humans; D009980:Influenza A virus / Q000502:physiology*; D016898:Interferon-alpha / Q000494:pharmacology; D008164:Luminescent Proteins / Q000235:genetics*; D051379:Mice; D008810:Mice, Inbred C57BL; D014709:Vero Cells; D017361:Viral Nonstructural Proteins / Q000235:genetics*; D014779:Virus Replication
C085820:INS1 protein, influenza virus; D016898:Interferon-alpha; D008164:Luminescent Proteins; D017361:Viral Nonstructural Proteins; D049452:Green Fluorescent Proteins