Identifying Caspases and their Motifs that Cleave Proteins During Influenza A Virus Infection

Influenza A virus (IAV) infection activates the caspases that cleave host and viral proteins, which, in turn, have pro- and antiviral functions. By employing inhibitors, RNA interference, site-directed mutagenesis, and western blotting and RT-qPCR techniques, caspases in infected mammalian cells that cleave host cortactin and histone deacetylases were identified.

Caspases, a family of cysteine proteases, orchestrate programmed cell death in response to various stimuli, including microbial infections. Initially described to occur by apoptosis, programmed cell death is now known to encompass three interconnected pathways: pyroptosis, apoptosis, and necroptosis, together coined as one process, PANoptosis. Influence A virus (IAV) infection induces PANoptosis in mammalian cells by inducing the activation of different caspases, which, in turn, cleave various host as well as viral proteins, leading to processes like the activation of the host innate antiviral response or the degradation of antagonistic host proteins. In this regard, caspase 3-mediated cleavage of host cortactin, histone deacetylase 4 (HDAC4), and histone deacetylase 6 (HDAC6) has been discovered in both animal and human epithelial cells in response to the IAV infection. To demonstrate this, inhibitors, RNA interference, and site-directed mutagenesis were employed, and, subsequently, the cleavage or resistance to cleavage and the recovery of cortactin, HDAC4, and HDAC6 polypeptides were measured by western blotting. These methods, in conjunction with RT-qPCR, form a simple yet effective strategy to identify the host as well as viral proteins undergoing caspase-mediated cleavage during an infection of IAV or other human and animal viruses. The present protocol elaborates the representative results of this strategy, and the ways to make it more effective are also discussed.

Influenza A virus (IAV) is the prototypic member of the Orthomyxoviridae family and is known to cause global epidemics and unpredictable pandemics. IAV causes human respiratory disease, influenza, commonly known as "flu". The flu is an acute disease that results in the induction of host pro- and anti-inflammatory innate immune responses and the death of epithelial cells in the human respiratory tract. Both processes are governed by a phenomenon called programmed cell death¹. The signaling for programmed cell death is induced as soon as various pathogen recognition receptors sense the incoming virus particles in host cells. This....

Regulatory approvals were obtained from the University of Otago Institutional Biological Safety Committee to work with the IAV and mammalian cells. Madin-Darby Canine Kidney (MDCK) or human lung alveolar epithelial A549 cells and IAV H1N1 subtypes were used for the present study. IAV was grown in chicken eggs, as described elsewhere¹⁷. Sterile and aseptic conditions were used to work with mammalian cells, and a Biosafety Level 2 (or Physical Containment 2) facility and Class II biosafety cabinet w.......

Treatment with caspase 3 inhibitor
It has been discovered that host cortactin, HDAC4, and HDAC6 polypeptides undergo degradation in response to IAV infection in both canine (MDCK) and human (A549, NHBE) cells^7,8,9. By using the above approaches, it was uncovered that IAV-induced host caspases, particularly caspase 3, cause their degradation^7,8.......

It is established that viruses tailor the host factors and pathways to their benefit. In turn, the host cells resist that by employing various strategies. One of those strategies is PANoptosis, which host cells use as an antiviral strategy against virus infections. However, viruses like IAV have evolved their own strategies to counter PANoptosis and exploit it to their advantage^1,3,6. This interplay involves the cleavage of vari.......

The author acknowledges Jennifer Tipper, Bilan Li, Jesse vanWestrienen, Kevin Harrod, Da-Yuan Chen, Farjana Ahmed, Sonya Mros, Kenneth Yamada, Richard Webby, the BEI Resources (NIAID), the Health Research Council of New Zealand, the Maurice and Phyllis Paykel Trust (New Zealand), the H.S. and J.C. Anderson Trust (Dunedin), and the Department of Microbiology and Immunology and School of Biomedical Sciences (University of Otago).