Flaviviruses are transmitted between bird and mammalian hosts via mosquitoes or ticks. Flaviviruses, such as dengue virus, Japanese encephalitis virus, West Nile virus (WNV), St. Louis encephalitis virus, Murray Valley virus, and tick-borne encephalitis virus, can sometimes cause severe disease in infected humans (10, 25). The genomes of flaviviruses are singlestranded, positive-polarity RNAs of approximately 11 kb and encode a single large polyprotein that is posttranslationally processed by viral and cellular proteases into three structural proteins and seven nonstructural proteins (35). During the flavivirus replication cycle, which takes place in the cytoplasm of infected cells, the genomic RNA serves as the only viral mRNA and is also the template for transcription of the complementary minus-strand RNA. The minus-strand RNA in turn serves as a template for the synthesis of genomic RNA. Plusstrand synthesis is 10 to 100 times more efficient than minusstrand synthesis (35). The noncoding regions (NCRs) of the flavivirus genome contain terminal RNA structures that are conserved between divergent flaviviruses even though only short sequences in these regions are conserved (8,9,28,37,38). The terminal RNA structures located at the 3Ј ends of the genome and complementary minus-strand RNAs differ from each other in shape and size. Deletion or mutation of either 3Ј-terminal structure in flavivirus infectious clones resulted in no progeny virus production, indicating that these regions are essential for virus replication (10a, 32, 46). However, specific cis-acting signal sequences within these structures have not yet been mapped or functionally analyzed. The WNV 3Ј-terminal RNA plus-and minus-strand structures have previously been reported to bind specifically to different sets of cell proteins (3,38).Understanding the mechanisms and components involved in the initiation and regulation of nascent viral-genome RNA synthesis from the minus-strand template is the ultimate goal of ongoing studies. The formation in solution of the 3Ј-terminal stem-loop structure of the WNV minus-strand RNA [WNV 3Ј(Ϫ) SL RNA] was previously confirmed by RNase structure probing (38). Three RNA-protein complexes (RPCs) were detected by gel shift mobility assays performed with baby hamster kidney (BHK) cytoplasmic extracts and the WNV 3Ј(Ϫ) SL RNA probe (38). The same pattern of RPCs was observed when WNV-infected or uninfected BHK S100 cytoplasmic cell extracts were used, suggesting that the proteins in these complexes were cellular proteins. UV-induced cross-linking studies indicated that the molecular masses of the RNA binding proteins in these complexes were 42, 50, 60, and 108 kDa. The specificities of these RNA-protein interactions were demonstrated by competition gel mobility shift and competition UVinduced cross-linking assays (38).

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