Viral noncoding RNAs have been shown to play an important role in virus-host interplay to facilitate virus replication. We report that members of the genus Flavivirus, a large group of medically important encephalitic RNA viruses, produce a unique and highly structured noncoding RNA of 0.3-0.5 kb derived from the 3' untranslated region of the viral genome. Using West Nile virus as a model, we show that this subgenomic RNA is a product of incomplete degradation of viral genomic RNA by cellular ribonucleases. Highly conserved RNA structures located at the beginning of the 3' untranslated region render this RNA resistant to nucleases, and the resulting subgenomic RNA product is essential for virus-induced cytopathicity and pathogenicity. Thus, flaviviruses evolved a unique strategy to generate a noncoding RNA product that allows them to kill the host more efficiently.

Flavivirus nonstructural (NS) proteins are involved in RNA replication and modulation of the host antiviral response; however, evidence is mounting that some NS proteins also have essential roles in virus assembly. Kunjin virus (KUN) NS2A is a small, hydrophobic, transmembrane protein that is part of the replication complex and inhibits interferon induction. Previously, we have shown that an isoleucine (I)-to-asparagine (N) substitution at position 59 of the NS2A protein blocked the production of secreted virus particles in cells electroporated with viral RNA carrying this mutation. We now show that prolonged incubation of mutant KUN NS2A-I59N replicon RNA, in an inducible BHK-derived packaging cell line (expressing KUN structural proteins C, prM, and E), generated escape mutants that rescued the secretion of infectious virus-like particles. Sequencing identified three groups of revertants that included (i) reversions to wild-type, hydrophobic Ile, (ii) pseudorevertants to more hydrophobic residues (Ser, Thr, and Tyr) at codon 59, and (iii) pseudorevertants retaining Asn at NS2A codon 59 but containing a compensatory mutation (Thr-to-Pro) at NS2A codon 149. Engineering hydrophobic residues at NS2A position 59 or the compensatory T149P mutation into NS2A-I59N replicon RNA restored the assembly of secreted virus-like particles in packaging cells. T149P mutation also rescued virus production when introduced into the full-length KUN RNA containing an NS2A-I59N mutation. Immunofluorescence and electron microscopy analyses of NS2A-I59N replicon-expressing cells showed a distinct lack of virus-induced membranes normally present in cells expressing wild-type replicon RNA. The compensatory mutation NS2A-T149P restored the induction of membrane structures to a level similar to those observed during wild-type replication. The results further confirm the role of NS2A in virus assembly, demonstrate the importance of hydrophobic residues at codon 59 in this process, implicate the involvement of NS2A in the biogenesis of virus-induced membranes, and suggest a vital role for the virus-induced membranes in virus assembly.

Kunjin virus (KUN) is an Australian flavivirus closely related to other members of the Japanese encephalitis virus subgroup. The KUN genome consists of single-stranded RNA of positive polarity comprising 11,022 nucleotides (17) with one long open reading frame encoding 3,433 amino acids in three structural proteins (C, prM, and E) and seven nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (8). NS proteins are assumed to be involved primarily in the replication of viral RNA as a part of a replication complex (RC). Extensive analysis of the KUN RC in infected cells by immunogold labeling, radioimmunoprecipitation assays, and glutathione S-transferase (GST)-pull down assays identified a consensus composition of the RC which consists of NS1, NS2A, NS3, NS4A, and NS5 proteins (29, 41). Specific cell proteins, such as elongation factor 1 alpha, which interacts with the terminal stem-loop of the 3Ј untranslated region (3ЈUTR) of several flaviviruses, may also be a part of the flavivirus RC (2, 3).NS5 protein of flaviviruses consists of 905 amino acids and contains seven motifs characteristic for RNA-dependent RNA polymerase situated in the C-terminal two-thirds of the protein (24) (Fig. 1) and two methyltransferase motifs situated in the N-terminal part of the protein (25) (Fig. 1). NS5 proteins of dengue 1 virus (34), West Nile virus (32), and KUN (13) were shown to possess nonspecific in vitro RNA-dependent RNA polymerase activity. Flavivirus NS3 is a multifunctional protein possessing protease, helicase, and RNA triphosphatase activities (31). The protease activity resides in the first 167 to 180 amino acids (5,6,10,11,40), while the C-terminal region commencing from codons 160 to 170 contains motifs for nucleoside triphosphatase, RNA helicase, and RNA-stimulated triphosphatase (4,9,12,27,38,39). C-terminally truncated NS3 product (NS3Ј or p50), resulting from an alternative cleavage (QRR2GR [arrow marks cleavage site]) have been detected in tick-borne encephalitis virus-and dengue virus-infected cells (1,30,35), but the function of this truncated protein is not known.Our previous studies demonstrated that replication of the defective KUN genomic RNA with NS5 protein truncated to retain only the first 397 amino acids was efficiently complemented by the helper RC produced from the KUN replicon (subgenomic) RNA in repBHK cells, while genomic RNA with a further truncation leaving only the first 227 amino acids in NS5 was complemented very inefficiently (20). Amino acid sequence comparisons of the N-terminal region of NS5 proteins from different flaviviruses revealed the presence of three small (10 to 20 amino acids) highly conserved domains, which we named a (KUN amino acids 141 to 151), b (KUN amino

Point mutations that resulted in a substitution of the conserved 3-penultimate cytidine in genomic RNA or the RNA negative strand of the self-amplifying replicon of the Flavivirus Kunjin virus completely blocked in vivo replication. Similarly, substitutions of the conserved 3-terminal uridine in the RNA negative or positive strand completely blocked replication or caused much-reduced replication, respectively. The same preference for cytidine in the 3-terminal dinucleotide was noted in reports of the in vitro activity of the RNA-dependent RNA polymerase (RdRp) for the other genera of Flaviviridae that also employ a double-stranded RNA (dsRNA) template to initiate asymmetric semiconservative RNA positive-strand synthesis. The Kunjin virus replicon results were interpreted in the context of a proposed model for initiation of RNA synthesis based on the solved crystal structure of the RdRp of 6 bacteriophage, which also replicates efficiently using a dsRNA template with conserved 3-penultimate cytidines and a 3-terminal pyrimidine. A previously untested substitution of the conserved pentanucleotide at the top of the 3-terminal stem-loop of all Flavivirus species also blocked detectable in vivo replication of the Kunjin virus replicon RNA.The Flaviviridae comprise three genera of positive-strand RNA viruses (Flavivirus, Pestivirus, and Hepacivirus). Their infectious genomes have a single open reading frame and no 3Ј-poly(A) tail (17). The 5Ј-and 3Ј-penultimate and/or -terminal nucleotides of genomic RNA are conserved within each genus, and they are each adjacent to one or more conserved terminal stem-loops: e.g., for Hepacivirus RNA (4, 18, 37), Flavivirus RNA (8,36,39,41,43,48), and Pestivirus RNA (3, 13, 53). Other common features are the similarities in gene order and function of the major viral enzymes in NS3 (protease and helicase) and in NS5/NS5B (RNA-dependent RNA polymerase [RdRp]) (17, 30).The Flavivirus RNA replication strategy has been established with Kunjin virus (KUN) (11,12,24,(49)(50)(51). The genomic or RNA positive strand is translated, a replication complex is formed cotranslationally on the 3Ј untranslated region (UTR) of the template RNA, and it transcribes an RNA negative strand that remains base paired with the RNA positive strand as a replicative form (RF). We found no evidence for the presence of a free RNA negative strand in KUN replicontransfected cells (22). We have defined the consensus composition of the KUN replication complex (RC) as NS1, NS3, NS5, and hydrophobic nonstructural proteins NS2A and NS4A plus the RF (24,34,50,51). The RF functions as a recycling template (shown by the kinetics of pulse-chase labeling) for asymmetric and semiconservative replication of progeny RNA positive strands by the viral replicase, and no subgenomic RNA is synthesized (11). Purified 32 P-labeled KUN RF was converted to a replicative intermediate (RI) when presented as a substrate to a lysate of KUN-infected cells (2). The Pestivirus Bovine viral diarrhea virus (BVDV) (15,47) and Hepacivirus Hepatitis ...