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...

Flavivirus NS1 is a nonstructural protein involved in virus replication and regulation of the innate immune response. Interestingly, a larger NS1-related protein, NS1, is often detected during infection with the members of the Japanese encephalitis virus serogroup of flaviviruses. However, how NS1 is made and what role it performs in the viral life cycle have not been determined. Here we provide experimental evidence that NS1 is the product of a ؊1 ribosomal frameshift event that occurs at a conserved slippery heptanucleotide motif located near the beginning of the NS2A gene and is stimulated by a downstream RNA pseudoknot structure. Using site-directed mutagenesis of these sequence elements in an infectious clone of the Kunjin subtype of West Nile virus, we demonstrate that NS1 plays a role in viral neuroinvasiveness.

West Nile virus (WNV) is a human pathogen of significant medical importance with close to 40,000 cases of encephalitis and more than 1,600 deaths reported in the US alone since its first emergence in New York in 1999. Previous studies identified a motif in the beginning of non-structural gene NS2A of encephalitic flaviviruses including WNV which induces programmed −1 ribosomal frameshift (PRF) resulting in production of an additional NS protein NS1′. We have previously demonstrated that mutant WNV with abolished PRF was attenuated in mice. Here we have extended our previous observations by showing that PRF does not appear to have a significant role in virus replication, virion formation, and viral spread in several cell lines in vitro. However, we have also shown that PRF induces an over production of structural proteins over non-structural proteins in virus-infected cells and that mutation abolishing PRF is present in ∼11% of the wild type virus population. In vivo experiments in house sparrows using wild type and PRF mutant of New York 99 strain of WNV viruses showed some attenuation for the PRF mutant virus. Moreover, PRF mutant of Kunjin strain of WNV showed significant decrease compared to wild type virus infection in dissemination of the virus from the midgut through the haemocoel, and ultimately the capacity of infected mosquitoes to transmit virus. Thus our results demonstrate an important role for PRF in regulating expression of viral genes and consequently virus replication in avian and mosquito hosts.

The flavivirus NS2A protein is a small, multifunctional protein, involved in replication, virion formation and regulation of the innate immune response. Using the Kunjin strain of West Nile virus (WNV KUN ) we previously demonstrated that a single amino acid change from alanine to proline at position 30 of the NS2A protein (A30P) reduced viral cytopathicity in cells and virulence in mice. To further investigate functions of the NS2A protein we have substituted alanine at position 30 with different amino acids (A30 mutants) in a WNV KUN infectious clone. The virulence of mutant viruses in wild-type (WT) and IRF3/IRF7 double-knockout mice was influenced by the amino acid change and ranged from high to low in the order of WT.A30L.A30E.A30P/A30G. Moreover, infection of beta interferon (IFN-b)-deficient Vero cells with A30P virus showed less pronounced chromosomal DNA degradation and lower percentage of cells with positive TUNEL labelling than in WT virus infection, indicating a role for the WT NS2A protein in IFN-independent apoptotic cell death.

NS1= is a C-terminally extended form of the NS1 protein produced only by encephalitic flaviviruses from the Japanese encephalitis virus serogroup. Here we show that West Nile virus (WNV) NS1= and NS1 localize to the same cellular compartments when expressed from plasmid DNAs and also colocalize to viral RNA replication sites in infected cells. Using complementation analysis with NS1-deleted WNV cDNA, we demonstrated that NS1= is able to substitute for the crucial function of NS1 in virus replication. West Nile virus (WNV) is a mosquito-borne flavivirus within the Japanese encephalitis virus (JEV) serogroup. This serogroup also includes other encephalitic flaviviruses, such as JEV, Murray Valley encephalitis virus, and St. Louis encephalitis virus (1). The natural transmission cycle of WNV is between birds and mosquitoes, primarily the Culex species; however, it can cause incidental infections in humans. Since the outbreak of the more pathogenic WNV NY99 strain in New York in 1999 (2), WNV has emerged as a major cause of arboviral encephalitis in the United States (3). WNV strains can be divided into two distinct lineages, lineage 1 and lineage 2. Lineage 1 includes both WNV NY99 and Kunjin (WNV KUN ) (4), the prevalent strain within Australia (5). Despite high sequence similarity to the WNV NY99 strain (ϳ98% on the amino acid level) (6), most WNV KUN strains are highly attenuated, with only a small number of human infections and no fatalities reported (5, 7). Since its isolation in early 1960s, WNV KUN has been used extensively as a model for WNV infection (8, 9).The WNV KUN genome is a single-stranded positive-sense RNA of 11,022 nucleotides (10-12). After translation as a single polyprotein, it is cleaved by host and viral proteases to produce 3 structural (C, prM, and E) and 7 nonstructural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) proteins (11,12). NS1 is a multifunctional glycoprotein that is involved in viral replication (13-16) and modulation of the immune response (17-22). The key role NS1 plays in RNA replication has been shown previously, with mutations or deletions in the NS1 gene resulting in a lack of detectable RNA replication. This function can be complemented in trans by the expression of NS1 (14, 15).An additional nonstructural protein, NS1=, is produced exclusively by the members of the JEV serogroup due to the presence of a Ϫ1 programmed ribosomal frameshift at the beginning of the adjacent NS2A gene (23)(24)(25). This frameshift, occurring in 30 to 50% of translation events (23,26), results in the formation of a 52-amino-acid C-terminally extended form of NS1. Although a role in neurovirulence has previously been demonstrated (23), no specific functions for NS1= in viral replication or virus-host interactions have been identified. In the present study, we show that the NS1= protein colocalizes with NS1 in viral RNA replication sites in the endoplasmic reticulum (ER) of infected cells and can substitute for the function of NS1 in viral replication.Plasmid DNA-derived expression of NS1 and NS...