Influenza (flu) is a highly contagious respiratory disease that affects millions of people each year with significant cost to the economy. Currently, influenza A virus (IAV) of the subtype H1N1 is responsible for seasonal epidemics that may result in severe respiratory illness and deaths worldwide. The annual Influenza virus epidemics are estimated to cost the U.S. 10.4 billion in direct medical expenses and 16.4 billion in lost potential earnings. The frequent appearances of new strains of flu made the generation of effective vaccines difficult. Therefore, the detailed molecular mechanisms how IAV replicates and propagates need to be investigated to design effective antiviral drugs and vaccines. Influenza induces cell death in infected cells to facilitate virus replication. Our preliminary studies identified the Bcl-2 interacting killer (Bik) as a crucial cellular protein promoting IAV infection in human airway epithelial cells (AECs) and in mice. We found that IAV replication was attenuated in bik-/- compared to bik+/+ cells, as indicated by reduced viral titers. Following infection, bik-deficient mouse airway epithelial cells (MAECs) showed more stable trans-epithelial resistance following infection, were less sensitive to infection-induced cell death, and had reduced levels of viral RNA compared to bik-sufficient MAECs. Knockdown of Bik reduced viral protein expression levels and the sensitivity of human airway epithelial cells (HAECs) to IAV-induced cell death. Survival analysis of mice infected with IAV showed that bik-/- mice survived for significantly longer days after infection and were 10-fold less likely to die from infection compared to bik+/+ mice. Additionally, the bik+/+ mice lost significantly more weight compared to the bik-/- mice. IAV activated caspase 3 in a Bik-dependent manner and cleavage of viral NP and M2 proteins were inhibited when Bik was knocked down, implying that Bik-mediated caspase cleavage of viral proteins is linked to virus replication. Thus, we hypothesize that Bik is crucial for intracellular cleavage of components of viral protein(s) to promote virus replication. Although cleavage of viral proteins has been shown to promote virus infectivity, littl is known about relevant host cell proteases. This study will investigate the role of caspases in enhancing virus infectivity and tests whether targeted inhibition of caspases mitigate IAV infectivity. This hypothesis will be tested in Specific Aims 1 and 2.
Aim 1 will investigate whethe Bik mediates caspase-dependent cleavage of viral proteins in IAV infected cells.
Aim 2 will determine whether modulating the expression of Bik or inhibition of caspase activations mitigate viral replication and susceptibility to IAV- induced mortality in vivo. A better understanding of te molecular mechanisms how IAV manipulates host cellular proteins to facilitate virus replication will provide insight into new therapeutic approaches that target cellular and/or viral proteins to reduce infection and disease progression in humans.

Public Health Relevance

Flu infection claims thousands of lives every year despite the currently available vaccinations. Frequent appearances of new strains of flu made the generation of effective vaccine difficult. Therefore, to formulate more effective treatment, understanding the molecular mechanisms how influenza A viruses manipulate host cellular proteins to facilitate virus replication is crucial. This study can lead to new therapeutic approaches that target cellular or viral protein(s) to reduce influenza A virus infection and disease progression in humans.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Exploratory/Developmental Grants (R21)
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Lung Cellular, Molecular, and Immunobiology Study Section (LCMI)
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Hauguel, Teresa M
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Lovelace Biomedical & Environmental Research
United States
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Chand, Hitendra S; Mebratu, Yohannes A; Kuehl, Philip J et al. (2017) Blocking Bcl-2 resolves IL-13-mediated mucous cell hyperplasia in a Bik-dependent manner. J Allergy Clin Immunol 140:1456-1459.e9
Mebratu, Yohannes A; Tipper, Jennifer; Chand, Hitendra S et al. (2016) Bik Mediates Caspase-Dependent Cleavage of Viral Proteins to Promote Influenza A Virus Infection. Am J Respir Cell Mol Biol 54:664-73
Chand, Hitendra S; Mebratu, Yohannes A; Montera, Marena et al. (2016) T cells suppress memory-dependent rapid mucous cell metaplasia in mouse airways. Respir Res 17:132