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 ...

We report the first full-length infectious clone of the current epidemic, lineage I, strain of West Nile virus (WNV). The full-length cDNA was constructed from reverse transcription-PCR products of viral RNA from an isolate collected during the year 2000 outbreak in New York City. It was cloned into plasmid pBR322 under the control of a T7 promoter and stably amplified in Escherichia coli HB101. RNA transcribed from the full-length cDNA clone was highly infectious upon transfection into BHK-21 cells, resulting in progeny virus with titers of 1 ؋ 10 9 to 5 ؋ 10 9 PFU/ml. The cDNA clone was engineered to contain three silent nucleotide changes to create a StyI site (C to A and A to G at nucleotides [nt] 8859 and 8862, respectively) and to knock out an EcoRI site (A to G at nt 8880). These genetic markers were retained in the recovered progeny virus. Deletion of the 3-terminal 199 nt of the cDNA transcript abolished the infectivity of the RNA. The plaque morphology, in vitro growth characteristics in mammalian and insect cells, and virulence in adult mice were indistinguishable for the parental and recombinant viruses. The stable infectious cDNA clone of the epidemic lineage I strain will provide a valuable experimental system to study the pathogenesis and replication of WNV.

West Nile virus (WNV) belongs to the genus Flavivirus, which includes significant human pathogens such as dengue virus (DEN), yellow fever virus (YF), the tick-borne encephalitis virus complex, Japanese encephalitis virus (JE), Murray Valley encephalitis virus, and WNV. WNV is widely distributed throughout Africa, the Middle East, parts of Europe, Russia, India, Indonesia, and most recently, North America (6). Since the introduction of WNV into the United States in 1999, the virus has caused more than 4,156 known human cases and at least 284 deaths (for updates, see http://www.cdc.gov/ncidod /dvbid/westnile/surv&controlCaseCount03.htm). The WNV epidemic in the United States in 2002 represents the largest meningoencephalitis outbreak in the Western hemisphere and the largest WNV outbreak ever reported (7). Prevention and treatment of WNV infection have become public health priorities.Flavivirus virions contain a single plus-sense RNA genome of approximately 11 kb in length, which consists of a 5Ј untranslated region (UTR), a single long open reading frame (ORF), and a 3Ј UTR (24). The 5Ј and 3Ј UTRs are approximately 100 nt and 400 to 700 nt in length, respectively. The encoded polyprotein is cotranslationally 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 (the glycoproteins NS1 and NS2a, the protease cofactor NS2b, the protease and helicase NS3, NS4a, and NS4b; and the methyltransferase and RNA-dependent RNA polymerase [RdRp] NS5) (8). During the flavivirus replication cycle, the synthesis of plus-and minus-sense RNAs is asymmetric; plus-sense RNA is produced in 10-to 100-fold excess over minus-sense RNA (11,23,28).The terminal nucleotides of the 3Ј UTR of flaviviruses can form highly conserved secondary and tertiary structures (4,5,24,26,31). Upstream of the 3Ј stem-loop structure, conserved sequences (CS) CS1, CS2, and repeated CS2 (RCS2) are retained among mosquito-borne flaviviruses. The JE subgroup and YF also have a distinct CS3 and repeated CS3 (RCS3). There is limited information available in regard to the functions of these CS elements. CS1 has the potential to base pair with a sequence within the conserved sequence in the 5Ј region of the capsid gene (5ЈCS) (13). Recent studies using DEN (33), Kunjin (16), and YF (10,22) replicons have shown that base pairing between 5ЈCS and CS1 is essential for flavivirus replication. For CS2, RCS2, CS3, and RCS3 elements, studies using