Antiviral Potential of a Proteolytic Inhibitor from Streptomyces Chromofuscus 34–1

ABSTRACT

The novel proteinaceous protease inhibitor, isolated from the culture supernatants of Streptomyces chromopfuscus 34–1 (SS 34–1) demonstrated a specific and selective anti-influenza virus effect. By the use of complementary virological assays it was demonstrated that the expression of the viral haemagglutinin (HA) on the surface of infected cells, the virus-induced cytopathic effect (CPE) and the infectious virus yields (IVY), used as measures of A/Germany/34, strain Rostock (H7N1) (A/Rostock) virus growth, were all reduced at non-toxic concentrations of SS 34–1 in a dose-dependent way. In a single cycle of viral growth, the penetration of viral particles and the late stages of viral replication were the most susceptible to inhibition.

The preparation protected mice from mortality in the experimental A/Aichi/2/68 (H3N2) influenza virus infection. The SS 34–1-treatment (0.16 mg/mouse/day) of virus-infected mice led to a considerable reduction of mortality rate (index ofprotection = 71.0 %) and marked prolongation of mean survival time (+ 5.5 days). The lesions of the lungs due to infection as well as lung infectious virus titres were significantly reduced—δ log₁₀ TCID₅₀/ml up to 4.5. The application of SS 34–1 induced a continuous rise of the anti-protease activity but did not cause substantial changes in the lung protease activity of healthy mice. The infection triggered a slight reduction of protease activity in the lungs at 5 and 48 h p.i. and a marked increase at 24 h and 6 day p.i. Protease inhibition in the lungs was reduced at 24 and 48 h p.i. and an increase was observed at 5 h, 6 and 9 day p.i. SS 34–1-treatment brought both activities to normal levels. It was assumed that SS 34–1 exerted its inhibitory effect indirectly, through an increase of the protease-inhibitory activity. Histopathological examination of the lungs showed that under SS 34–1-treatment pulmonary lesions were less severe than those of untreated controls.

The isolated novel protease inhibitor was purified by anion-exchange chromatography and reversed phase-HPLC. Analysis by differential scanning calorimetry (DSC) revealed that at pH 7.5 the denaturation temperature was 75 °C and the denaturation itself was irreversible. Analysis by circular dichroism (CD) in the UV region 190–250 nm showed that at pH 7.5 the spectrum presented 2 minima at 208 and 222 nm, typical for an α-helical structure. At pH 2.5 and after heating the spectrum corresponded to an unordered structure.

Keywords:

• influenza virus
• viral inhibition
• protease inhibitor
• Streptomyces