Flaviviruses are a group of single-stranded, positive-sense RNA viruses causing ϳ100 million infections per year. We have recently shown that flaviviruses produce a unique, small, noncoding RNA (ϳ0.5 kb) derived from the 3 untranslated region (UTR) of the genomic RNA (gRNA), which is required for flavivirus-induced cytopathicity and pathogenicity (G. P. Pijlman et al., Cell Host Microbe, 4: 579-591, 2008). This RNA (subgenomic flavivirus RNA [sfRNA]) is a product of incomplete degradation of gRNA presumably by the cellular 5-3 exoribonuclease XRN1, which stalls on the rigid secondary structure stem-loop II (SL-II) located at the beginning of the 3 UTR. Mutations or deletions of various secondary structures in the 3 UTR resulted in the loss of full-length sfRNA (sfRNA1) and production of smaller and less abundant sfRNAs (sfRNA2 and sfRNA3). Here, we investigated in detail the importance of West Nile virus Kunjin (WNV KUN ) 3 UTR secondary structures as well as tertiary interactions for sfRNA formation. We show that secondary structures SL-IV and dumbbell 1 (DB1) downstream of SL-II are able to prevent further degradation of gRNA when the SL-II structure is deleted, leading to production of sfRNA2 and sfRNA3, respectively. We also show that a number of pseudoknot (PK) interactions, in particular PK1 stabilizing SL-II and PK3 stabilizing DB1, are required for protection of gRNA from nuclease degradation and production of sfRNA. Our results show that PK interactions play a vital role in the production of nuclease-resistant sfRNA, which is essential for viral cytopathicity in cells and pathogenicity in mice.Arthropod-borne flaviviruses such as West Nile virus (WNV), dengue virus (DENV), and Japanese encephalitis virus (JEV) cause major outbreaks of potentially fatal disease and affect over 50 million people every year. The highly pathogenic North American strain of WNV (WNV NY99 ) has already claimed more than 1,000 lives with over 27,000 cases reported since its emergence in New York in 1999 and has raised global public health concerns (9). In contrast, the closely related Australian strain of WNV, WNV KUN , is highly attenuated and does not cause overt disease in humans and animals (11). WNV KUN has been used extensively as a model virus to study flavivirus replication and flavivirus-host interactions (13, 14, 16-19, 26, 38, 39).The ϳ11-kb positive-stranded, capped WNV genomic RNA (gRNA) lacks a poly(A) tail and consists of 5Ј and 3Ј untranslated regions (UTRs) flanking one open reading frame, which encodes the viral proteins required for the viral life cycle (6,15,38,39). Flavivirus UTRs are involved in translation and initiation of RNA replication and likely determine genome packaging (13,14,16,21,30,(39)(40)(41). Both the 5Ј UTR (ϳ100 nucleotides [nt] in size) and the 3Ј UTR (from ϳ400 to 700 nucleotides) can form secondary and tertiary structures which are highly conserved among mosquito-borne flaviviruses (1,8,10,14,29,32,34). More specifically, the WNV KUN 3Ј UTR consists of several conserved regions and se...