Exploitation of the Herpes simplex virus translocating protein VP22 to carry influenza virus proteins into cells for studies of apoptosis: direct confirmation that neuraminidase induces apoptosis and indications that other proteins may have a role.
Previously, we have shown that apoptosis induced by influenza virus was inhibited by an anti-neuraminidase compound [4-guanidino-2, 3-dehydro-N-acetylneuraminic acid (GG167; Relenza; Zanamivir)], which does not enter cells, and acts at the attachment/entry phase of virus replication. Furthermore, a virulent virus, clone 7a, induced greater levels of apoptosis than the attenuated A/Fiji and had greater neuraminidase (NA) activity. To confirm more directly that NA induces apoptosis, the NA of clone 7a and A/Fiji was expressed fused to the Herpes simplex virus tegument coat protein VP22, transfected into HeLa cells and the level of apoptosis determined. VP22 translocates between cells via the medium thus allowing expressed proteins to transfer to a larger number of cells than those originally transfected. Clone 7a NA fused to VP22 induced a significant level of apoptosis whereas A/Fiji NA/VP22 did not, confirming that NA activity is an important determinant of apoptosis acting during fusion protein translocation between cells. Furthermore, the induction of apoptosis was abrogated by antibody to transforming growth factor-beta, which is activated by NA. This approach also showed that VP22/NS1 proteins of both clone 7a and A/Fiji induced apoptosis when expressed alone but inhibited double stranded RNA-induced apoptosis suggesting that this protein may have a dual mode of action. Also, the M1 and M2 proteins of both viruses induced apoptosis but their NP proteins did not.
Morris
S J
SJ
School of Biosciences, The University of Birmingham, Edgbaston, Birmingham, UK.
Smith
H
H
Sweet
C
C
D017209:Apoptosis; D003001:Cloning, Molecular; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D009980:Influenza A virus / Q000235:genetics / Q000502:physiology; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D009439:Neuraminidase / Q000235:genetics / Q000502:physiology*; D009975:Orthomyxoviridae / Q000201:enzymology*; D010750:Phosphoproteins / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000502:physiology*; D018139:Simplexvirus / Q000737:chemistry*; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000378:metabolism; D014763:Viral Matrix Proteins / Q000235:genetics / Q000502:physiology; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000502:physiology*; D015678:Viral Structural Proteins / Q000235:genetics*; D000086082:Viroporin Proteins
D009439:Neuraminidase; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D016212:Transforming Growth Factor beta; D014763:Viral Matrix Proteins; D017361:Viral Nonstructural Proteins; D015678:Viral Structural Proteins; C085820:INS1 protein, influenza virus; C031540:M-protein, influenza virus; C488936:M1 protein, Influenza A virus; C488937:M2 protein, Influenza A virus; D000086082:Viroporin Proteins; C073413:herpes simplex virus type 1 protein VP22; C114208:protein VP22, Herpes simplex virus type 2