Most eukaryotic and viral mRNAs possess a 5Ј cap that is important for mRNA stability and efficient translation (11). The cap consists of an inverted guanosine, methylated at the N-7 position, linked to the first transcribed RNA nucleotide by a unique 5Ј-5Ј triphosphate bridge (m 7 GpppN; cap 0 structure) (32). The process of RNA capping generally consists of three steps, in which the 5Ј triphosphate end of the nascent RNA transcript is first hydrolyzed to a 5Ј diphosphate by an RNA triphosphatase, then capped with GMP by an RNA guanylyltransferase, and finally methylated at the N-7 position of guanine by an RNA guanine-methyltransferase (N-7 MTase) (13). Additionally, 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 to form cap 1 (m 7 GpppNm) and cap 2 (m 7 GpppNmNm) structures, respectively (11). Both N-7 and 2Ј-O MTases use S-adenosyl-Lmethionine (AdoMet) as a methyl donor and generate Sadenosyl-L-homocysteine (AdoHcy) as a by-product. The order of the capping and methylation steps is variable among cellular and viral RNAs (11).Many members of the Flavivirus genus are arthropod-borne human pathogens, including West Nile virus (WNV), Yellow fever virus, four serotypes of Dengue virus (DENV), Japanese encephalitis virus, St. Louis encephalitis virus, Murray Valley encephalitis virus, and Tick-borne encephalitis virus (4). The flavivirus genome is a single-stranded, plus-sense RNA of about 11,000 nucleotides that contains a type 1 cap at its 5Ј end (5, 35) and terminates with 5Ј-CU OH -3Ј (35) (see Fig. 1A). 5Ј and 3Ј untranslated regions flank a single open reading frame which encodes a polyprotein that is co-and posttranslationally processed by viral and cellular proteases into three structural proteins (capsid [C], premembrane [prM] or membrane [M], and envelope [E]) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (4). Since flaviviruses replicate in the cytoplasm, they are expected to encode their own capping enzymes, rather than to use the host's capping apparatus located in the nucleus. Alternatively, since all host proteins have to be synthesized in the cytoplasm, it is possible that cellular capping components could be retained in the cytoplasm for viral RNA capping through specific interactions with a viral protein. Of the four enzymes required for flavivirus m 7 GpppAm-cap formation, only the RNA triphosphatase and 2Ј-O MTase have been mapped to NS3 (19,36) and NS5 (8), respectively, whereas the guanylyltransferase and N-7 MTase remain to be identified. The crystal structure of a ternary complex comprising the DENV-2 MTase domain, AdoHcy, and a GTP analogue suggested that, during 2Ј-O methylation, a specific cap-binding site holds the guanine cap to register the ribose 2Ј-OH of the first transcribed adenosine in close proximity to the AdoMet CH 3 donor (2, 8). Structure and sequence alignments of DENV, vaccinia virus VP39, and other 2Ј-O MTases indicate that a conserved K-D-K-E ...

RNA elements within flavivirus genomes are potential targets for antiviral therapy. A panel of phosphorodiamidate morpholino oligomers (PMOs), whose sequences are complementary to RNA elements located in the 5-and 3-termini of the West Nile (WN) virus genome, were designed to anneal to important cis-acting elements and potentially to inhibit WN infection. A novel Arg-rich peptide was conjugated to each PMO for efficient cellular delivery. These PMOs exhibited various degrees of antiviral activity upon incubation with a WN virus luciferase-replicon-containing cell line. Among them, PMOs targeting the 5-terminal 20 nucleotides (5End) or targeting the 3-terminal element involved in a potential genome cyclizing interaction (3CSI) exhibited the greatest potency. When cells infected with an epidemic strain of WN virus were treated with the 5End or 3CSI PMO, virus titers were reduced by approximately 5 to 6 logs at a 5 M concentration without apparent cytotoxicity. The 3CSI PMO also inhibited mosquito-borne flaviviruses other than WN virus, and the antiviral potency correlated with the conservation of the targeted 3CSI sequences of specific viruses. Modeof-action analyses showed that the 5End and 3CSI PMOs suppressed viral infection through two distinct mechanisms. The 5End PMO inhibited viral translation, whereas the 3CSI PMO did not significantly affect viral translation but suppressed RNA replication. The results suggest that antisense PMO-mediated blocking of cis-acting elements of flavivirus genomes can potentially be developed into an anti-flavivirus therapy. In addition, we report that although a full-length WN virus containing a luciferase reporter (engineered at the 3 untranslated region of the genome) is not stable, an early passage of this reporting virus can be used to screen for inhibitors against any step of the virus life cycle.Many members of the Flavivirus genus, a group of arthropod-borne viruses in the family Flaviridae, cause significant human diseases; among these, West Nile (WN), dengue (DEN), Japanese encephalitis (JE), yellow fever (YF), Murray Valley encephalitis, and tick-borne encephalitis (TBE) viruses are emerging and reemerging pathogens (7). Approximately 50 to 100 million human cases of DEN virus infection occur annually (29). The recent epidemics of WN virus have caused significant morbidity and mortality in the United States (9). Vaccines for humans are available only for YF, JE, and TBE viruses (7). No drug therapy is currently available to treat flavivirus infections. It is therefore of great importance to public health to develop an efficacious drug therapy against flaviviruses.Flavivirus virions are spherical in shape, with a diameter of approximately 50 nm (21). The flavivirus genome is a singlestranded, plus-sense RNA of approximately 11 kb in length. The genomic RNA consists of a 5Ј untranslated region (UTR), a single long open reading frame (ORF), and a 3Ј UTR (43). The single ORF encodes a polyprotein that is co-and posttranslationally processed by viral and cellular proteases...

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.