Influenza Virus Induces Apoptosis via BAD-mediated Mitochondria Dysregulation

Journal of Virology 2013 Jan;87(2):1049-1060

Tran AT, Cortens JP, Du Q, Wilkins JA, Coombs KM

Departments of Medical Microbiology and Internal Medicine, University of Manitoba, Winnipeg R3E 3P4 Canada

 

Abstract:

Influenza virus infection results in host cell death and major tissue damage. Specific components of the apoptotic pathway, a signaling cascade that ultimately leads to cell death, are implicated in promoting influenza virus replication. BAD is a cell death regulator that constitutes a critical control point in the intrinsic apoptosis pathway, which occurs through the dysregulation of mitochondrial outer membrane permeabilization and the subsequent activation of downstream apoptogenic factors. Here we report a novel proviral role for the proapoptotic protein BAD in influenza virus replication. We show that influenza virus-induced cytopathology and cell death are considerably inhibited in BAD knockdown cells and that both virus replication and viral protein production are dramatically reduced, which suggests that virus-induced apoptosis is BAD dependent. Our data showed that influenza viruses induced phosphorylation of BAD at residues S112 and S136 in a temporal manner. Viral infection also induced BAD cleavage, late in the viral life cycle, to a truncated form that is reportedly a more potent inducer of apoptosis. We further demonstrate that knockdown of BAD resulted in reduced cytochrome c release and suppression of the intrinsic apoptotic pathway during influenza virus replication, as seen by an inhibition of caspases-3, caspase-7, and procyclic acidic repetitive protein (PARP) cleavage. Our data indicate that influenza viruses carefully modulate the activation of the apoptotic pathway that is dependent on the regulatory function of BAD and that failure of apoptosis activation resulted in unproductive viral replication.

PMID: 23135712

 

Supplement:

This study constituted part of a larger study designed to better understand some of the unusual features of highly pathogenic influenza virus infection – that host responses to the virus often cause more damage to the host than the viral infection per se. Thus, we designed a genome-wide positive selection strategy screen to identify host proteins that could be knocked down without detrimental effects upon the cell, but that importantly also would protect the cell from virus-induced cell death. The larger study, recently published in the journal Cell Death and Disease1, identified approximately 140 potential host targets. Confirmatory tests (Fig. 1) showed that each of more than a dozen genes could be knocked down with insignificant effects upon cell viability. BAD was one of these genes. The precise roles that BAD play, such that knockdown protects cells from virus-induced toxicity, are only partially understood. Based upon what is known about normal BAD functions, and alterations that occur in the state of BAD phosphorylation during influenza virus infection, a temporal model can be built (Fig. 2). This model proposes that early activation of apoptosis is detrimental to viral replication; thus the virus induces BAD phosphorylation early in influenza virus infection which inhibits BAD proapoptotic activity. However, BAD is activated later in infection, leading to induction of host cell apoptosis, which we and others show is required for efficient viral replication. Therefore, modulating various “non-essential” host targets, such as BAD, may offer an alternate means to attenuate virus-induced pathology. Thus, given that influenza virus rapidly mutates, making it necessary to often re-formulate virus-targeted anti-viral strategies and vaccines, these host genes could potentially serve as more stable therapeutic targets.

Kevin M. Coombs-F1

Figure 1. RNAi screen of influenza virus-infected A549 cells. Each of ~20,000 human genes were tested in preliminary duplicate global shRNA knockdown screens. The 138 genes identified in both duplicate screens were then re-tested here in triplicate siRNA arrays. Each of the 138 genes is represented by a circle. Note that knockdown of most genes caused reduction in virus growth (y-axis). More importantly, knockdown of any of about a dozen genes, including BAD (indicated), protected cells from influenza virus-induced cell death as indicated by cell viability values >90% (x-axis).

 

Kevin M. Coombs-F2

Figure 2. Proposed model for the role of BAD during Influenza virus infection.  Influenza virus infection early in the viral lifecycle leads to the temporal phosphorylation of BAD, first at S112 and then at S136, which inhibits BAD pro-apoptotic activity.  During late stages of viral replication, an as yet undetermined factor activates BAD through dephosposphorylation.  Active BAD blocks anti-apoptotic factors Bcl-2 and Bcl-xL, subsequently leading to the activation of caspase-3 through the dysregulation of the mitochondria and release of cytochrome c.  Activated caspase-3 has been shown to enhance the release of vRNPs and promote efficient influenza virus replication.

 

References

1.    Tran AT, Rahim MN, Ranadheera C, Kroeker A, Cortens JP, Opanubi KJ, Wilkins JA, Coombs KM 2013 Knockdown of specific host factors protects against Influenza virus-induced cell death. Cell Death & Disease. 4:e769.

 

Acknowledgements: This study was supported by grants ROP-104906 and MOP-106713 from the Canadian Institutes of Health Research awarded to Kevin Coombs.

 

Contact:

Kevin Coombs, Ph.D.

Professor and Chair

Dept of Medical Microbiology

730 William Avenue

University of Manitoba

Winnipeg, MB R3E 0J9

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