Flaviviruses encode a single methyltransferase domain that sequentially catalyzes two methylations of the viral RNA cap, GpppA-RNA3m 7 GpppA-RNA3m 7 GpppAm-RNA, by using S-adenosyl-L-methionine (SAM) as a methyl donor. Crystal structures of flavivirus methyltransferases exhibit distinct binding sites for SAM, GTP, and RNA molecules. Biochemical analysis of West Nile virus methyltransferase shows that the single SAMbinding site donates methyl groups to both N7 and 2-O positions of the viral RNA cap, the GTP-binding pocket functions only during the 2-O methylation, and two distinct sets of amino acids in the RNA-binding site are required for the N7 and 2-O methylations. These results demonstrate that flavivirus methyltransferase catalyzes two cap methylations through a substrate-repositioning mechanism. In this mechanism, guanine N7 of substrate GpppA-RNA is first positioned to SAM to generate m 7 GpppA-RNA, after which the m 7 G moiety is repositioned to the GTP-binding pocket to register the 2-OH of the adenosine with SAM, generating m 7 GpppAm-RNA. Because N7 cap methylation is essential for viral replication, inhibitors designed to block the pocket identified for the N7 cap methylation could be developed for flavivirus therapy.Eukaryotic mRNAs contain a 5Ј cap structure that is essential for RNA splicing, export, stability, and translation (12). In general, RNA capping consists of four steps. (i) The 5Ј-triphosphate end of the nascent RNA transcript is hydrolyzed to a 5Ј diphosphate by an RNA triphosphatase; (ii) the GMP moiety of GTP is transferred to the 5Ј diphosphate of RNA by an RNA guanylyltransferase; (iii) the N7 position of guanine is methylated by an RNA guanine-methyltransferase (N7 MTase), yielding a cap 0 structure (GpppN); and (iv) the first and second nucleotides of many cellular and viral mRNAs are further methylated at the ribose 2Ј-OH position by a nucleoside 2Ј-O MTase, so as to form cap 1 (m 7 GpppNm) and cap 2 (m 7 GpppNmNm) structures, respectively (12). S-Adenosyl-L-methionine (SAM) is the methyl donor for both the N7 and 2Ј-O methylations, generating Sadenosyl-L-homocysteine (SAH) as a by-product. Because host mRNA capping occurs in the nucleus, viruses replicating in the cytoplasm encode their own unique machineries for RNA capping. For example, the plus-strand RNA alphaviruses methylate GTP prior to the transfer of m 7 GMP to the 5Ј diphosphate of the RNA (2). The minus-strand RNA vesicular stomatitis virus (VSV) transfers GDP, rather than GMP, to the 5Ј monophosphate of the RNA (24). The differences between the host and viral cap formation processes could potentially be used for development of antiviral therapy.The genus Flavivirus contains a number of significant human pathogens, including the four serotypes of dengue virus (DENV), yellow fever virus (YFV), Japanese encephalitis virus, West Nile virus (WNV), and tick-borne encephalitis virus (6). Among those, DENV alone was estimated to cause 50 million human cases annually (38). Flaviviruses replicate in the cytoplasm. The viral ge...
To determine if West Nile virus (WNV) infection of insect cells induces a protective RNAi response, Drosophila melanogaster S2 and Aedes albopictus C6/36 cells were infected with WNV, and the production of WNV-homologous small RNAs was assayed as an indicator of RNAi induction. A distinct population of approximately 25 nt WNV-homologous small RNAs was detected in infected S2 cells but not C6/36 cells. RNAi knockdown of Argonaute 2 in S2 cells resulted in slightly increased susceptibility to WNV infection, suggesting that some WNV-homologous small RNAs produced in infected S2 cells are functional small interfering RNAs. WNV was shown to infect adult D. melanogaster, and adult flies containing mutations in each of four different RNAi genes (Argonaute 2, spindle-E, piwi, and Dicer-2) were significantly more susceptible to WNV infection than wildtype flies. These results combined with the analysis of WNV infection of S2 and C6/36 cells support the conclusion that WNV infection of D. melanogaster, but perhaps not Ae. albopictus, induces a protective RNAi response.
Many flaviviruses cause significant human disease worldwide. The development of flavivirus chemotherapy requires reliable high-throughput screening (HTS) assays. Although genetic systems have been developed for many flaviviruses, their usage in antiviral HTS assays has not been well explored. Here we compare three cell-based HTS assays for West Nile virus (WNV) drug discovery: (i) an assay that uses a cell line harboring a persistently replicating subgenomic replicon (containing a deletion of viral structural genes), (ii) an assay that uses packaged virus-like particles containing replicon RNA, and (iii) an assay that uses a full-length reporting virus. A Renilla luciferase gene was engineered into the replicon or into the full-length viral genome to monitor viral replication. Potential inhibitors could be identified through suppression of luciferase signals upon compound incubation. The antiviral assays were optimized in a 96-well format, validated with known WNV inhibitors, and proved useful in identifying a new inhibitor(s) through HTS of a compound library. In addition, because each assay encompasses multiple but discrete steps of the viral life cycle, the three systems could potentially be used to discriminate the mode of action of any inhibitor among viral entry (detected by assays ii and iii but not by assay i), replication (including viral translation and RNA synthesis; detected by assays i to iii), and virion assembly (detected by assay iii but not by assays i and ii). The approaches described in this study should be applicable to the development of cell-based assays for other flaviviruses.
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