Bacterial ribonuclease binase exerts an intra-cellular anti-viral mode of action targeting viral RNAs in influenza a virus-infected MDCK-II cells

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.Electronic supplementary materialThe online version of this article (10.1186/s12985-017-0915-1) contains supplementary material, which is available to authorized users.

Host Defence RNases as Antiviral Agents against Enveloped Single Stranded RNA Viruses

“…Ilinskaya and coworkers have extensively explored the potential of the RNase from Bacillus pumilus , also named Binase, on influenza A virus. The authors observed a direct action on the viral mRNA in both in vitro and in vivo models [ 265 , 266 ]. Binase was also effectively tested against CoVs (MERS-CoV and the human low pathogenic CoV-229E strain)[ 267 ].…”

Structural and Functional Differences between Homologous Bacterial Ribonucleases

“…The catalytic activity of RNases was measured against yeast high-polymeric and transfer RNA, and double-stranded RNA extracted from E. coli (Vector-Best, Novosibirsk, Russia) using a SmartSpec Plus Spectrophotometer (Bio-Rad, Hercules, CA, USA), as described earlier [7]. One unit was defined as the amount of enzyme that increases the extinction of acid-soluble products of RNA hydrolysis at 260 nm per min at 37 • C. The specific activity was calculated as the enzymatic activity per 1 mg of protein.…”

“…Catalyzing the degradation of RNA polymers, RNases indirectly participate in many cellular processes such as gene expression, cell growth and differentiation, and immune response [4][5][6]. The ability of RNases to inhibit the growth of tumor cells and the reproduction of viruses in infected cells was also shown [7,8]. The application of RNases as antitumor and antiviral agents implies their penetration into mammalian cells which contain a cytosolic RNase inhibitor (RI).…”

Close Encounters of the Viral Kind: Cross‐Kingdom Synergies at the Host–Pathogen Interface

“…Signals from commensal bacteria can alter the threshold for immune activation in either direction, either magnifying or diminishing them, which can impact the interferon response to viral infection . Bacterial ribonucleases can be internalized into host cells to specifically degrade influenza virus RNAs, reducing both viral burden and pro‐inflammatory signaling from activation of cytosolic toll‐like receptors recognizing viral RNA. The S‐layer of Lactobacillus acidophilus prevents alphaviruses and flaviviruses from interacting with dendritic cells, key cells for initiation of adaptive immune responses.…”