Mechanisms of differential induction of apoptosis by H3N2 and H1N1 influenza viruses

Abstract

Background: The abilities of seven H3N2 and four H1N1 influenza viruses to infect MDCK cells and produce apoptosis were measured and related to their neuraminidase (NA) activity and the role of individual influenza virus proteins in induction of apoptosis was also investigated by transient transfection assays in HeLa cells with expression vectors containing individual virus genes. Methods: MDCK cells were inoculated at a high multiplicity of infection (5 EID 50/cell) and apoptosis was measured by cytotoxicity and morphology while infection was quantified by fluorescence staining with anti-nucleoprotein (NP) antibody. The Invitrogen Voyager™ vector expressing influenza virus genes as VP22 fusion proteins was transfected into HeLa cells using lipofectamine. Results and discussion: H3N2 viruses with high NA activities (1.4–1.8 μmol/l/min) induced high levels of apoptosis (83–94%) and infected 91–98% of cells, while H1N1 viruses with low NA activities (0.22–0.33 μmol/l/min) were poor apoptosis inducers (11–19%) and infected few (15–21%) cells. The differences in % infected cells reflected differences in haemagglutinin (HA) receptor binding affinity. Treatment of viruses with bacterial NA to remove sialyl groups from oligosaccharides on virus HA increased their ability to infect MDCK cells and the increase correlated inversely with the number of potential glycosylation sites around the receptor binding site. Transfection experiments showed that NA (from clone 7a but not A/Fiji) and NS1 (from clone 7a and A/Fiji) induced apoptosis, while NP (clone 7a and A/Fiji) did not.