Abstract:
African horse sickness virus (AHSV) is transmitted by Culicoides spp. biting midges to
horses, causing serious effusion and haemorrhage in various organs and tissues, but is
asymptomatic in the insect host. Likewise, AHSV causes dramatic cytopathic effect (CPE) in
infected mammalian cells in culture, but no CPE is observed in infected insect cell cultures
despite productive virus replication. The basis for this differential host response has not yet
been investigated, but is suggestive of the induction of apoptosis in mammalian cells
following virus infection. Consequently, the aims of this investigation were essentially to
determine whether AHSV infection induces apoptosis in cultured mammalian cells, and to
subsequently identify the initiators and effectors of AHSV-induced apoptosis in mammalian
cells.
To determine whether apoptosis is induced in BHK-21 mammalian cells, the cells were
infected with AHSV-4 and the key apoptotic indicators of cell morphology, chromosomal
DNA fragmentation and caspase-3 activation were monitored. Results were obtained
providing evidence that in vitro infection of BHK-21 cells with AHSV-4 results in apoptosis
at 12 h post-infection with maximal levels of apoptosis at 24 h post-infection. By making use
of inhibitors of endosomal acidification and UV-inactivated AHSV-4, it was demonstrated
that virus disassembly, but not productive virus replication, is necessary for AHSV-4 to
trigger apoptosis in BHK-21 cells. Subsequent studies indicated that extracellular coadministration
of VP2 and VP5, which likely results in the uptake of VP2-VP5 complexes
into the endosomes, induces apoptosis. These findings therefore suggest that the outer capsid proteins are sufficient to trigger apoptosis and that they exert their effect during the early
events in AHSV cell entry, where cell binding and endosomal membrane penetration is
required.
To identify apoptotic pathways triggered during AHSV-4 infection of BHK-21 cells, the
enzymatic activity of different cellular caspases in cytoplasmic extracts of infected cells was
measured by proteolytic cleavage of caspase-specific chromogenic substrates. Results were
obtained indicating the activation of caspases-8 and -9, whereas flow cytometry analyses,
following staining of the cells with the lipophilic cation DePsipher™, revealed the loss of
mitochondrial membrane potential. This data therefore indicated that both the extrinsic and
intrinsic apoptotic pathways are activated in AHSV-infected mammalian cells. Moreover,
AHSV-4 infection of BHK-21 cells led to the nuclear translocation of nuclear factor B (NF-
B) complexes containing the Rel family members p50 and p65, thus suggesting that NF- B
may also play a role in the AHSV apoptotic machinery.
Collectively, the results obtained during the course of this investigation provide evidence for
apoptosis induction following AHSV infection of mammalian cells, and are the first to
delineate the role of early events in the virus replication cycle in the induction of apoptosis
and to demonstrate that both the death receptor and mitochondrial pathways can play an
essential role in AHSV-induced apoptosis. These results therefore add an important new
dimension to AHSV-host cell interactions and provide a foundation for the future study of
apoptosis and the role thereof in viral pathogenesis.
