Apoptosis induction by IAVs is a multifactorial process,32
as many virus and host factors contribute to it in a complex manner.1
In the present study, we show that IAV NP induces host cell death through the mitochondrial apoptosis pathway. We also report a novel interaction between IAV NP and host protein CLU. Our results indicate that CLU has a cytoprotective role in IAV infection, and IAV NP targets CLU to induce cell death. The question, whether apoptosis induction favors IAV or host, is still under debate.1
However, there are many reports that suggest that apoptosis induction by IAV facilitates virus replication, dissemination and killing of the host immune effectors cells.32, 33, 34, 35, 36
Cell death induced by IAV may depend on the host cell type and the stage of infection.37
We validated apoptotic phenotype of NP in the human tracheal/bronchial as well as alveolar epithelial cells, which are the primary site of IAV infection and replication in humans.38
We compared the apoptotic effect of IAV NP with other IAV proteins and found that NP was a major contributor of IAV-induced cell death. The PB1F2 protein of influenza viruses is also known to induce cell death through the mitochondrial pathway,4
therefore we used A/California/2009 (H1N1) influenza virus that allowed us to study IAV-induced apoptosis in the absence of PB1F2.24
We also observed that although M2 expression did not induce cell death, M2 knockdown in IAV-infected cells mitigated apoptosis. This suggests that M2 may require additional viral factors to contribute to host cell death. Our results showed that NS1 and M1 proteins promote cell death in overexpression experiments, however, their knockdown has no effect on IAV-induced apoptosis, which is contrary to previous reports.6, 7
This can be attributed to the difference in experimental conditions such as virus strains and cell type used in our study.
CLU protein prevents the initiation of intrinsic apoptotic pathway,23
and conversely IAV NP was found to activate the same. IAV NP expression was associated with Bax movement into mitochondria, release of Cyt c from the mitochondria and activation of Casp 3, all of which are events involved in the intrinsic apoptosis pathway.30
Further, NP and CLU colocalized in the cytoplasm of IAV-infected cells during late stages of infection. We also observed that IAV NP-induced apoptosis in a dose-dependent manner (data not shown). Taken together, our data clearly suggest that during the late stages of the viral life cycle, when NP accumulates in the host cell, its apoptotic effect is more prominent and is mediated by the NP–CLU interaction. We also observed that CLU overexpression attenuated IAV replication in mammalian cells. It has been reported that the presence of Bax and activation of Casp 3 is essential for efficient IAV replication,34, 35
and CLU is known to inhibit these molecules.23
This explains why CLU overexpression may impede IAV replication. CLU is known to interact with Bax through its α
whereas in the present study CLU β
-chain was found to be responsible for the IAV NP–CLU interaction. Thus NP and Bax interaction sites on CLU are mutually exclusive. Moreover, we found that in the presence of NP, the CLU–Bax association was weakened in mammalian cells. Collectively, based on our data, we proposed a model for the regulation of apoptosis in IAV-infected cells by NP and propose a possible role of CLU (). According to this model, NP interacts with CLU and either prevents its association with Bax or dissociates the Bax–CLU complex. This will lead to Bax movement into the mitochondria, release of Cyt c, activation of Casp 3 and eventually cell death. Furthermore, we found that CLU interacts exclusively with NP among IAV proteins known to promote cell death. Also the IAV NP–CLU interaction was well preserved in IAV strains of different subtypes, pathogenicity and host specificity. This suggests that IAV NP induces cell death through a specific and conserved mechanism, which is mediated by its interaction with CLU.
Figure 8 Model for regulation of cell death by IAV NP–CLU interaction. IAV NP interacts with human CLU protein inside the cells. Through this interaction CLU association with Bax is prevented or Clu–Bax complex is dissociated. This leads to Bax (more ...)
Few other viruses also target CLU to modulate host responses. The secreted form of CLU is exploited by the NS1 protein of Dengue viruses to interfere with the complement pathway.39
The intracellular form of CLU is known to be upregulated during Hepatitis δ virus infection, as a strategy to promote cell survival.40
CLU is also known to have an important role in the regulation of PI3K signaling, NFkB pathway and cell cycle.41, 42
Implications of the IAV NP interaction with CLU on these cellular processes remains to be studied. We observed that ectopic expression of CLU reduces NP or IAV-induced apoptosis, however, it does not abrogate it completely. This indicates that NP-induced cell death may not be entirely dependent on its interaction with CLU. NP is known to modulate PKR and NFk
B signaling pathways,27, 43
which may also have a role in NP-induced cell death.
Apoptosis induction in IAV-infected cells is closely linked to the induction of inflammatory response and viral pathogenesis.3
Exchange of gene segments between high and low pathogenic IAVs is known to alter their ability to induce cell death.32
Not surprisingly, IAV proteins such as NS1 and PB1F2, which are known to cause cytokine dysregulation,44, 45, 46
are also involved in apoptosis modulation.4, 5, 6
NP is also known to interfere with the host IFN response,27
and in the current study we have shown that it contributes to host cell death also. Taken together, these facts indicate that NP could be an important virulence factor and contributor in IAV pathogenesis. It will be interesting to investigate the role of apoptotic phenotype of IAV NP in viral pathogenesis using appropriate animal models and selected IAV strains. Finally, the ubiquitous expression of CLU and the conserved nature of the IAV NP–CLU interaction makes it a promising target to develop antiviral modalities.