Viral components involved in influenza virus-induced apoptosis.
Two influenza viruses, the virulent clone 7a (H3N2) and the attenuated A/Fiji/15899/83
(H1NI) (A/Fiji), induce different level ofapoptosis in both MDCK and HeLa cells. Apoptosis
induced by these two viruses could be partially inhibited by two NA inhibitors (GGI67 and
DANA), when present during virus entry, but not subsequently. GGI67, which cannot enter
cells, did not affect the level of infection. These results suggest that NA plays a role in
influenza virus-induced apoptosis and that it acts early in the replication cycle. However,
ammonium chloride, which prevents virus entry reduced the level of apoptosis induced by
both viruses. In addition, amantadine, which prevents virus uncoating, inhibited apoptosis
induced by the amantadine-sensitive strain A/Udorn/307/72 (H3N2). Therefore, other viral
components acting intracellularly may also be involved in influenza virus-induced apoptosis.
Clone 7a, A/Fiji or A/WSN/33 (H1N1)(A/WSN) proteins were expressed in HeLa cells,
fused to the Herpes simplex tegument coat protein VP22. This protein has the ability to
translocate from the cell in which it is synthesised to surrounding cells. Therefore, VP22-
fusion proteins are delivered to a high number of cells, regardless of the level of transfection.
A/WSN PB1, PB2 and HA induced significant levels ofapoptosis as did the NA, M1, M2 and
NS1 proteins from clone 7a and the M1, M2, NSI and NS2 proteins from A/Fiji. Conversely,
clone 7a and A/Fiji NP and A/Fiji PB2 and NA did not induce apoptosis. Confocal
microscopy revealed that apoptosis was associated with fusion protein expression and that the
location of the proteins corresponded with that expected during influenza virus infection.
In addition, the mechanism of clone 7a NA-induced apoptosis was investigated. GGI67
inhibited apoptosis induced by the expression of clone 7a NA. Hence, NA activity is required
for the induction of apoptosis. In addition, apoptosis was completely abrogated in the
presence of an anti-transforming growth factor (TGF)-B antibody suggesting the activation of
TGF-B during the translocation of the fusion protein from one cell to another is involved in
clone 7a NA-induced apoptosis.
Furthermore, clone 7a and A/Fiji NS1 proteins induced apoptosis when expressed alone, but
they inhibited double-stranded (ds) RNA-induced apoptosis. However, the level of apoptosis
observed in the presence of dsRNA was not reduced to background levels suggesting a pro
and anti-apoptotic action of NS1 in influenza virus-infected cells.
The potential mechanisms involved in the induction of apoptosis by each protein and the
relevance to infection is discussed.