Ribonucleases are considered as promising tools for anticancer treatment due to their selective cytotoxicity against tumor cells. We investigated a new RNase from B. altitudinis termed BALNASE (Bacillus altitudinis RNase). Balnase is a close homolog of the well-known cytotoxic binase, differing by only one amino acid residue: nonpolar hydrophobic alanine at position 106 in the balnase molecule is replaced by a polar uncharged threonine in binase. The most exciting question is how the physico-chemical properties and biological effects of RNase might be changed by A106T substitution. Here, we have developed a chromatography-based rapid and modern technique for the purification of this new RNase which allowed us to get a protein sample of high quality with specific activity of 1.2×106 units in preparative amounts, suitable for further investigation of its biological properties.

Supported by crystallography studies, secreted ribonuclease ofBacillus pumilus(binase) has long been considered to be monomeric in form. Recent evidence obtained using native polyacrylamide gel electrophoresis and size-exclusion chromatography suggests that binase is in fact dimeric. To eliminate ambiguity and contradictions in the data we have measured conformational changes, hypochromic effect, and hydrodynamic radius of binase. The immutability of binase secondary structure upon transition from low to high protein concentration was registered, suggesting the binase dimerization immediately after translocation through the cell membrane and leading to detection of binase dimers only in the culture fluid regardless of ribonuclease concentration. Our results made it necessary to take a fresh look at the binase stability and cytotoxicity towards virus-infected or tumor cells.

RNA viruses cause most of the dangerous communicable diseases. Due to their high mutation rates, RNA viruses quickly evade selective pressures and can adapt to a new host. Therefore, new antiviral approaches are urgently needed, which target more than one specific virus variant and which would optimally prevent development of viral resistance. Among the family of coronaviruses (CoV), several human pathogenic strains (HCoV) are known to cause respiratory diseases and are implied in enteric diseases. While most strains contribute to common cold-like illnesses, others lead to severe infections. One of these viruses is the newly emerged (2012), highly pathogenic Middle East respiratory syndrome coronavirus (MERS-CoV) of zoonotic origin. MERS-CoV causes a severe respiratory infection with a high mortality rate of 35 %. There is no specific treatment or infection prevention available. Here, we show that the bacterial ribonuclease Binase is able to inhibit the replication of MERS-CoV and of the low-pathogenic human coronavirus 229E (HCoV-229E) in cell culture. We demonstrate that at non-toxic concentrations, Binase decreased the titers of MERS-CoV and HCoV-229E. On a molecular level, Binase treatment reduced (i) the viral subgenomic RNAs and (ii) the viral nucleocapsidprotein (N) and non-structural protein 13 (nsp13) accumulation. Furthermore, we show that the quantity of the replication/transcription complexes within the infected cells is diminished. Thus, the data obtained might allow further development of new anti-coronaviral approaches affecting viral replication, independent of the specific virus strain.

The biological effects of ribonucleases (RNases), such as the control of the blood vessels growth, the toxicity towards tumour cells and antiviral activity, require a detailed explanation. One of the most intriguing properties of RNases which can contribute to their biological effects is the ability to form dimers, which facilitates efficient RNA hydrolysis and the evasion of ribonuclease inhibitor. Dimeric forms of microbial RNase binase secreted by Bacillus pumilus (former B. intermedius) have only been found in crystals to date. Our study is the first report directly confirming the existence of binase dimers in solution and under natural conditions of enzyme biosynthesis and secretion by bacilli. Using different variants of gel electrophoresis, immunoblotting, size-exclusion chromatography and mass-spectrometry, we revealed that binase is a stable natural dimer with high catalytic activity.