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Hydroxytyrosol (HT), a small-molecule phenolic compound, inactivated influenza A viruses including H1N1, H3N2, H5N1, and H9N2 subtypes. HT also inactivated Newcastle disease virus but not bovine rotavirus, and fowl adenovirus, suggesting that the mechanism of the antiviral effect of HT might require the presence of a viral envelope. Pretreatment of MDCK cells with HT did not affect the propagation of H9N2 virus subsequently inoculated onto the cells, implying that HT targets the virus but not the host cell. H9N2 virus inactivated with HT retained unaltered hemagglutinating activity and bound to MDCK cells in a manner similar to untreated virus. Neuraminidase activity in the HT-treated virus also remained unchanged. However, in the cells inoculated with HT-inactivated H9N2 virus, neither viral mRNA nor viral protein was detected. Electron microscopic analysis revealed morphological abnormalities in the HT-treated H9N2 virus. Most structures found in the HT-treated virus were atypical of influenza virions, and localization of hemagglutinin was not necessarily confined on the virion surface. These observations suggest that the structure of H9N2 virus could be disrupted by HT.

The 2-5A system is one of the major pathways for antiviral and antitumor functions that can be induced by interferons (IFNs). The 2-5A system is modulated by 5′-triphosphorylated, 2′,5′-phosphodiester-linked oligoadenylates (2-5A), which are synthesized by 2′,5′-oligoadenylate synthetases (2′,5′-OASs), inactivated by 5,- phosphatase and completely degraded by 2′-phosphodiesterase (2′-PDE). Generated 2-5A activates 2-5A-dependent endoribonuclease, RNase L, which induces RNA degradation in cells and finally apoptosis. Although 2′,5′-OASs and RNase L have been molecularly cloned and studied well, the identification of 2′-PDE has remained elusive. Here, we describe the first identification of 2′-PDE, the third key enzyme of the 2-5A system. We found a putative 2′-PDE band on SDS-PAGE by successive six-step chromatographies from ammonium sulfate precipitates of bovine liver and identified a partial amino acid sequence of the human 2′-PDE by mass spectrometry. Based on the full-length sequence of the human 2′-PDE obtained by in silico expressed sequence tag assembly, the gene was cloned by reverse transcription-PCR. The recombinant human 2′-PDE expressed in mammalian cells certainly cleaved the 2′,5′-phosphodiester bond of 2-5A trimer and 2-5A analogs. Because no sequences with high homology to this human 2′-PDE were found, the human 2′-PDE was considered to be a unique enzyme without isoform. Suppression of 2′-PDE by a small interfering RNA and a 2′-PDE inhibitor resulted in significant reduction of viral replication, whereas overexpression of 2′-PDE protected cells from IFN-induced antiproliferative activity. These observations identify 2′-PDE as a key regulator of the 2-5A system and as a potential novel target for antiviral and antitumor treatments. [ABSTRACT FROM AUTHOR]