Expression and characterisation of the influenza A virus non-structural protein NS1 in yeast.
The influenza A virus non-structural protein NS1 was produced using a copper-inducible expression system in the yeast Saccharomyces cerevisiae. The protein produced had a molecular weight of 26 kDa by SDS-PAGE and was reactive with anti-NS1 antisera. The recombinant NS1 protein was targetted to the nucleolus/nuclear envelope fraction of the yeast cell nucleus, showing that its localisation signals remain functional in yeast. In addition, immune-electron microscopy detected cytoplasmic inclusions reminiscent of those seen in cells infected with some influenza strains. The NS1 protein was shown to be capable of in vivo self-interaction which probably forms the basis of its propensity to form inclusions. Expression of the protein was found to be toxic to yeast cells expressing it, supporting a role for the protein in the shutdown of influenza virus-infected cells. Deletion mapping of NS1 pointed to 2 regions of the molecule being important for this toxicity: a basic C-terminal stretch which has been shown to act as a nuclear localisation signal, and an N-terminal region implicated in RNA binding.
Ward
A C
AC
Biomolecular Research Institute, Parkville, Victoria, Australia.
Azad
A A
AA
Macreadie
I G
IG
D000595:Amino Acid Sequence; D001483:Base Sequence; D002467:Cell Nucleus / Q000737:chemistry; D003001:Cloning, Molecular; D007181:Inclusion Bodies, Viral / Q000648:ultrastructure; D009980:Influenza A virus / Q000235:genetics*; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development; D014170:Transformation, Genetic; D017361:Viral Nonstructural Proteins / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics* / Q000502:physiology*
C085820:INS1 protein, influenza virus; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins