Abstract
BackgroundInfluenza is a severe contagious disease especially in children, elderly and immunocompromised patients. Beside vaccination, the discovery of new anti-viral agents represents an important strategy to encounter seasonal and pandemic influenza A virus (IAV) strains. The bacterial extra-cellular ribonuclease binase is a well-studied RNase from Bacillus pumilus. Treatment with binase was shown to improve survival of laboratory animals infected with different RNA viruses. Although binase reduced IAV titer in vitro and in vivo, the mode of action (MOA) of binase against IAV at the molecular level has yet not been studied in depth and remains elusive.MethodsTo analyze whether binase impairs virus replication by direct interaction with the viral particle we applied a hemagglutination inhibition assay and monitored the integrity of the viral RNA within the virus particle by RT-PCR. Furthermore, we used Western blot and confocal microscopy analysis to study whether binase can internalize into MDCK-II cells. By primer extension we examined the effect of binase on the integrity of viral RNAs within the cells and using a mini-genome system we explored the effect of binase on the viral expression.ResultsWe show that (i) binase does not to attack IAV particle-protected viral RNA, (ii) internalized binase could be detected within the cytosol of MDCK-II cells and that (iii) binase impairs IAV replication by specifically degrading viral RNA species within the infected MDCK-II cells without obvious effect on cellular mRNAs.ConclusionOur data provide novel evidence suggesting that binase is a potential anti-viral agent with specific intra-cellular MOA.
Highlights
Influenza is a severe contagious disease especially in children, elderly and immunocompromised patients
We reported that binase degrades vRNA, which is not protected by the virion/RNP complex and that binase added to cells (HEK293, A549) reduces of a GFP reporter gene expression by a transient influenza A virus (IAV) mini-genome system [21, 22]
Binase does not interfere with HA/receptor binding and does not degrade viral RNA within free virus particles It was previously reported that pre-incubation of virus with binase resulted in decreased virus titers after infection [13, 21, 22], that binase could degrade free viral RNA [21], and that treatment of IAV-infected A549 cells led to titer reduction [22]
Summary
Influenza is a severe contagious disease especially in children, elderly and immunocompromised patients. The discovery of new anti-viral agents represents an important strategy to encounter seasonal and pandemic influenza A virus (IAV) strains. Influenza A virus (IAV) is an RNA virus, which poses a great health risk causing seasonal epidemics and periodically worldwide pandemics. Despite their seasonal character, influenza epidemics are unpredictable and have been recognized as a major cause of morbidity and increased mortality [1]. Influenza virus infection leads to a disease that results in 0.25–0.5 million deaths annually worldwide [2]. Based on the segmented nature of IAV genome, co-infections can lead to reassortants with completely new antigenic characteristics (antigen shift) that can cause pandemic outbreaks [6, 7].
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