Arch Virol (2002) 147: 961–979
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
S. J. Morris
, H. Smith
, and C. Sweet
School of Biosciences, The University of Birmingham, Birmingham, U.K.
The Medical School, The University of Birmingham, Birmingham, U.K.
Received June 27, 2001; accepted December 10, 2001
Published online March 18, 2002 © Springer-Verlag 2002
Summary. 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 deter-
minant of apoptosis acting during fusion protein translocation between cells.
Furthermore, the induction of apoptosis was abrogated by antibody to transform-
ing growth factor-␤, which is activated by NA. This approach also showed that
VP22/NS1 proteins of both clone 7a andA/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.
The nucleotide sequences of the M and NSgenes of clone 7a and A/Fiji have been
submitted to the EMBL nucleotide sequence database and designated the following accession
numbers: clone 7a M, AJ298948; A/Fiji M, AJ298947; clone 7a NS, AJ298949; A/Fiji NS,