RNA interference (RNAi) is originally regarded as a mechanism of eukaryotic posttranscriptional gene regulation mediated by small interfering RNA (siRNA)-induced sequence-specific RNA degradation (1). It is also well known to exert as an important antiviral defense mechanism in a wide range of organisms, from plants to invertebrates (2). During the virus infection, the virus-derived long double-stranded RNA (dsRNA) is cleaved by RNAIII-like endonuclease (named Dicer) into approximately 21-to 23-nucleotide (nt) siRNA, which is incorporated into the RNA-induced silencing complex (RISC) and activates the antiviral RNAi for viral RNA degradation. In mammalian cells, although the activation of RNAi by synthetic siRNA or short hairpin RNA (shRNA) is widely used as a tool for gene knockdown and antiviral treatment, the RNAi-mediated antiviral mechanism has been debated for a long time (3), because the interferon (IFN) response of the innate immune system is well known as the dominant antiviral mechanism (4). However, more and more evidence has provided strong support for the existence of a natural RNAi-mediated antiviral response in mammals (5). Moreover, recent studies showed that in undifferentiated cells and immature mice, the RNAi-mediated antiviral response is essential (6-8).To overcome the RNAi-mediated antiviral defense, viruses have evolved to encode a viral suppressor of RNA silencing (VSR) (9, 10). For example, in plant viruses, rice hoja blancavirus NS3, tombusvirus P19, and tomato aspermy virus 2b bind to long dsRNA or siRNA to block RNAi (11-13). Turnip crinkle virus P38 and cauliflower mosaic virus P6 disrupt the components of RNAi machinery (14,15). In insect viruses, flock house virus (FHV) B2 blocks RNAi by dsRNA binding (16,17), and Wuhan nodavirus (WhNV) B2 was identified as a VSR by targeting both dsRNAs and 19). Although the majority of VSRs have been identified in plant and invertebrate viruses, several mammalian viruses were shown to encode VSRs. For instance, Ebola virus VP35, influenza A virus NS1, vaccinia virus E3L, and Nodamura virus (NoV) B2 act as VSRs by binding dsRNA (20-23). Hepatitis C virus core and HIV-1 Tat block RNAi by inhibiting the activity of

The 5'-cap structure is a distinct feature of eukaryotic mRNAs and is important for RNA stability and protein translation by providing a molecular signature for the distinction of self or non-self mRNA. Eukaryotic viruses generally modify the 5'-end of their RNAs to mimic the cellular mRNA structure, thereby facilitating viral replication in host cells. However, the molecular organization and biochemical mechanisms of the viral capping apparatus typically differ from its cellular counterpart, which makes viral capping enzymes attractive targets for drug discovery. Our previous work showed that SARS coronavirus (SARS-CoV) non-structural protein 14 represents a structurally novel and unique guanine-N7-methyltransferase (N7-MTase) that is able to functionally complement yeast cellular N7-MTase. In the present study, we developed a yeast-based system for identifying and screening inhibitors against coronavirus N7-MTase using both 96-well and 384-well microtiter plates. The MTase inhibitors previously identified by in vitro biochemical assays were tested, and some, such as sinefungin, effectively suppressed N7-MTase in the yeast system. However, other compounds, such as ATA and AdoHcy, did not exert an inhibitory effect within a cellular context. These results validated the yeast assay system for inhibitor screening yet also demonstrated the difference between cell-based and in vitro biochemical assays. The yeast system was applied to the screening of 3000 natural product extracts, and three were observed to more potently inhibit the activity of coronavirus than human N7-MTase.