Several viruses trigger apoptosis in infected cells at an early stage of infection when virus particles interact with receptors on the cell surface or at the time of fusion with cell membrane and disassembly (8
). In addition, certain viruses induce apoptosis at late stages of replication, providing a mechanism for dissemination of progeny virus. The use of apoptosis as an exit strategy for the virus has also been suggested for caliciviruses (3
). Apoptotic changes in calicivirus-infected cells had been documented for FCV and RHDV (2
), and we examined whether infection with another calicivirus, MNV-1, triggered apoptosis in RAW264.7 cells. Changes were observed in nuclear chromatin, DNA fragmentation, and caspase activation consistent with apoptosis induced by MNV-1. Moreover, the induction of apoptosis was dependent on viral replication, as evidenced by the lack of DNA fragmentation in cells infected with the UV-inactivated virus. The finding of apoptosis in the NVs suggests that this process may be part of a conserved strategy among the caliciviruses to release viral progeny.
Triggering of apoptosis and downstream caspase activation can be initiated through two main pathways. The “intrinsic or mitochondrial pathway” involves changes in mitochondrial membrane permeability that are regulated by a family of Bcl-2-related proteins. Activated proapoptotic members of this group (Bax, Bak) can homo-oligomerize, forming pores in the mitochondrial membrane and promoting leakage of cytochrome c
into the cytoplasm (7
). Cytochrome c
release from the mitochondria results in apoptosome formation and processing of procaspase-9 (27
). The “extrinsic pathway” is a sensor for extracellular signals and activates caspase-8 (18
). Caspase-8 and caspase-9 converge on caspase-3 and caspase-7, the executioner caspases, which cleave proteins involved in maintaining the filamentous network of the cytoplasm and in retaining the structural organization of nuclear membranes and chromatin as well as proteins involved in DNA replication and repair (17
). The marked increase in processed caspase-3 levels in MNV-1-infected cells was noteworthy, because caspase-3 is directly involved in the execution of cellular disassembly, which would likely facilitate the release of viral progeny from an infected cell. In addition, cleavage of caspase-3 was preceded by the appearance of the processed form of caspase-9, indicating involvement of the mitochondrial or intrinsic pathway, which has also been suggested for another calicivirus, FCV (35
Several RNA viruses (measles virus, vesicular stomatitis virus, Sindbis virus, and vaccinia virus, among others) induce apoptosis via mechanisms involving receptor binding and internalization or other replication events (8
). Alternatively, some DNA viruses (Epstein-Barr virus and papillomavirus 16) inhibit programmed cell death by inducing the expression of cellular IAP (1
). MNV-1 inhibits expression of the antiapoptotic protein survivin, which may be a previously unrecognized mechanism used by viruses to actively induce programmed cell death. Our finding of downregulation of survivin by a replicating virus is the first evidence for a pathogen-induced mechanism to reduce the levels of this antiapoptotic protein. Decreased levels of survivin have previously been observed only in the presence of molecular antagonists of survivin such as antisense compounds, ribozymes, and small interfering RNA sequences or with dominant-negative mutants at the BIRC5 gene level. Downregulation of survivin always results in typical caspase-dependent cell death accompanied by cytochrome c
release and caspase-9 activation (16
). The cellular changes (e.g., cytochrome c
release and caspase-9 activation) resulting from the nonviral downregulation of survivin expression are consistent with the apoptotic effects observed during replication of MNV-1 in RAW264.7 cells. It will be of interest to examine whether other viruses that induce apoptosis are associated with the downregulation of BIRC5 transcription. Ultimately, MNV-1 might prove to cause apoptosis by the use of a complex mechanism that involves the regulation of several genes. Because of the similarities observed between the apoptosis triggered by induced survivin inhibition and cell death caused by MNV-1 infection of RAW264.7 cells, we propose that cell death triggered by MNV-1 might be a direct consequence of BIRC5 transcription inhibition during virus replication.
Although we showed an association between MNV-1 infection and downregulation of survivin, it is not yet clear how MNV-1 affects other steps of the apoptotic pathway. We identified eight different genes (including the gene encoding survivin) in MNV-1-infected RAW264.7 cells that differed significantly in their transcription levels compared to those in mock-infected cells. Some of these genes may also contribute to the signs observed in apoptotic MNV-1-infected cells. Pycard, which was upregulated 25-fold in virus-infected cells, has been shown to associate with Bax, a key protein in the apoptotic cascade that induces cytochrome c
release from the mitochondria (36
). This effect ultimately leads to activation of caspase-9 and caspase-3, a result that was also observed in MNV-1-infected cells. In addition, the importance of caspase-11 for the activation of caspase-3 and caspase-1 has been demonstrated in experiments using caspase-11-knockout mice (30
). Therefore, the increase (8.6-fold) in transcription levels of caspase-11 in MNV-1-infected cells, together with the downregulation of survivin, might have also contributed to the more than 40-fold increase in caspase-3 activation due to MNV-1 infection.
A number of viral proteins have been reported to be directly involved in the modulation of apoptotic pathways, in particular, in the control of the mitochondrial apoptosis pathway (23
). We attempted to identify an MNV-1 protein that would individually induce downregulation of survivin. However, we have not been able to associate a single MNV-1 protein with induction of apoptosis through regulation of BIRC5. The downregulation of survivin in MNV-1-infected cells might involve multiple factors, such as the interaction of several proteins, the presence of viral protein precursors, a cascade of events, or simply active RNA replication. Alternatively, it is also possible that downregulation of survivin is mediated via an indirect effect of other cellular changes induced by MNV-1 infection.
This report shows that, in contrast to viruses that increase survivin levels during replication, MNV-1 is the first agent found to decrease the levels of survivin in cells. We demonstrated that MNV-1 replication induces apoptosis in RAW264.7 cells, activating caspases through the mitochondrial pathway, possibly as a result of downregulation of survivin. There is a consensus that the gene encoding survivin is an essential cancer gene and an appropriate target for anticancer drug discovery (5
), and the implications of our finding that certain viruses may naturally target this gene will need to be assessed in the future.