Abstract
Influenza virus remains a major health concern worldwide, and there have been continuous efforts to develop effective antivirals despite the use of annual vaccination programs. The purpose of this study was to determine the anti-influenza activity of Bax inhibitor-1 (BI-1). Madin-Darby Canine Kidney (MDCK) cells expressing wild type BI-1 and a non-functional BI-1 mutant, BI-1 ∆C (with the C-terminal 14 amino acids deleted) were prepared and infected with A/PR/8/34 influenza virus. BI-1 overexpression led to the suppression of virus-induced cell death and virus production compared to control Mock or BI-1 ∆C overexpression. In contrast to BI-1 ∆C-overexpressing cells, BI-1-overexpressing cells exhibited markedly reduced virus-induced expression of several viral genes, accompanied by a substantial decrease in ROS production. We found that treatment with a ROS scavenging agent, N-acetyl cysteine (NAC), led to a dramatic decrease in virus production and viral gene expression in control MDCK and BI-1 ∆C-overexpressing cells. In contrast, NAC treatment resulted in the slight additional suppression of virus production and viral gene expression in BI-1-overexpressing cells but was statistically significant. Moreover, the expression of heme oxygenase-1 (HO-1) was also significantly increased following virus infection in BI-1-overexpressing cells compared to control cells. Taken together, our data suggest that BI-1 may act as an anti-influenza protein through the suppression of ROS mediated cell death and upregulation of HO-1 expression in influenza virus infected MDCK cells.
Highlights
Influenza viruses, which are enveloped RNA viruses belonging to the family Orthomyxoviridae, are a significant cause of respiratory infections, causing millions of deaths annually and imposing significant economic losses [1]
After preparing Madin-Darby Canine Kidney (MDCK) cells expressing the lentiviral construct containing wild type Bax inhibitor-1 (BI-1) or the non-functional deletion mutant form (BI-1 ∆C; deletion of 9 amino acids (EKDKKKEKK) from the C-terminal region of BI-1) (Figure 1A) [19,29], we confirmed the endogenous or exogenous expression of BI-1 from the control, wild type BI-1, and non-functional BI-1 ∆C overexpressing MDCK cells by RT-PCR analysis using endogenous or exogenous specific primer set and overexpression was confirmed by western blot analysis (Figure 1B,C)
The anti-influenza activity of BI-1 overexpressing cells was determined using cell viability assays after influenza virus administration, revealing that the overexpression of BI-1 in MDCK cells led to the significant suppression of virus-induced cell death compared to that in mock or BI-1 ∆C-overexpressing cells (Figure 2A)
Summary
Influenza viruses, which are enveloped RNA viruses belonging to the family Orthomyxoviridae, are a significant cause of respiratory infections, causing millions of deaths annually and imposing significant economic losses [1]. Three classes of anti-influenza virus drugs are available for influenza management with different modes of action: the M2 channel blockers such as amantadine and rimantadine, neuraminidase (NA) inhibitors (e.g., oseltamivir) [4], and RNA polymerase inhibitors (e.g., ribavirin) [5]. All of these drugs are effective against virus infection if administered quickly, but concerns have been raised because of their adverse effects and the emergence of drug-resistant influenza A/H1N1 viruses [6,7]. The development of new therapeutic agents is urgently needed to treat influenza infections
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