As shown in Fig.9B, LEPR chemical probing of this region of the wild-type YFV-17D genome essentially confirmed the previously predicted RNA structure model (41). not one but two sfRNAs were detected in YFV-infected mammalian cells. The smaller of these two sfRNAs was not observed in infected mosquito cells. The larger sfRNA could also be Naproxen sodium producedin vitroby incubation with purified XRN1. These two YFV sfRNAs formed a 5-nested set. The 5 ends of the YFV sfRNAs were found to be just upstream of the previously predicted RNA pseudoknot PSK3. RNA structure probing and mutagenesis studies provided strong evidence that this pseudoknot structure was formed and served as the molecular signal to stall XRN1. The sequence involved in PSK3 formation was cloned into the Sinrep5 expression vector and shown to direct the production of an sfRNA-like RNA. These results underscore the importance of the RNA pseudoknot in stalling XRN1 and also demonstrate that it is the sole viral requirement for sfRNA production. TheFlavivirusgenus contains nearly 80 viruses distributed worldwide and includes important human pathogens such as dengue computer virus (DENV), yellow fever computer virus (YFV), Japanese encephalitis computer virus (JEV), West Nile computer virus (WNV), and tick-borne encephalitis computer virus (TBEV). Phylogenetic analysis clustered flaviviruses into the following three major groups, based on the vector of transmission: (i) mosquito-borne viruses, (ii) Naproxen sodium tick-borne viruses, and (iii) viruses with no known vector (NKV) (13,26). Flaviviruses are small enveloped viruses containing a positive-sense single-stranded RNA genome of approximately 11 kb in length, with a 5 cap structure and a 3 nonpolyadenylated terminus. The genomic RNA is usually flanked by 5- and 3-untranslated regions (UTRs) and encodes a single polyprotein that is co- and posttranslationally processed by viral and cellular proteases into three structural proteins (C, prM, and E) Naproxen sodium and seven nonstructural proteins (NSs) (reviewed in reference30). Apart from the viral genomic RNA and the replication-related replicative-form and intermediate RNAs (11,12), an additional small flavivirus RNA (sfRNA) has been detected in mice and both mammalian and insect cells infected with flaviviruses belonging to the JEV serogroup (29,49,56). Recently, it was shown that production of sfRNA is not unique to JEV and closely related viruses but that all arthropod-borne flaviviruses generate an sfRNA upon contamination of mammalian cells (31,44). The lengths of these sfRNAs vary from 0.3 kb to 0.5 kb and are related to the length of the viral 3 UTR. Surprisingly, these sfRNAs are not direct products of the viral transcription mechanism but result from incomplete degradation of the viral genomic RNA by the host 5-3 exonuclease XRN1, as shown for Kunjin computer virus (KUNV) byin vitroassays and RNA interference (RNAi) experiments. Although the exact role of the sfRNA in the viral life cycle is still elusive, production of sfRNA was shown to be essential for KUNV cytopathogenicity in cell culture and for viral pathogenicity in infected mice (44). XRN1 is usually well conserved among eukaryotes and is the main cytoplasmic RNase associated with 5-3 mRNA decay that takes place in cytoplasmic processing bodies (P bodies), where the mRNA is usually decapped by the enzymes DCP1 and -2 and subsequently degraded 5 to 3 by XRN1 (reviewed in references2,19,21, and50). XRN1 acts in a processive manner by hydrolyzing RNA with 5-monophosphate end groups to 5-mononucleotides (53,54). Based on fluorescencein situhybridization (FISH) analysis of KUNV-infected cells, the sfRNA was reported to colocalize with XRN1 in P bodies (44). Interestingly, the role of XRN1 in a viral life cycle is not limited to flaviviruses. XRN1 has also been shown to have an antiviral activity by virtue of its exonuclease activity (18) and to act as a potent suppressor of viral RNA Naproxen sodium recombination in viruses such as tomato bushy stunt computer virus (10). Studies have shown.