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.

A possible role in RNA replication for interactions between conserved complementary (cyclization) sequences in the 5-and 3-terminal regions of Flavivirus RNA was previously suggested but never tested in vivo. Using the M-fold program for RNA secondary-structure predictions, we examined for the first time the base-pairing interactions between the covalently linked 5 genomic region (first ϳ160 nucleotides) and the 3 untranslated region (last ϳ115 nucleotides) for a range of mosquito-borne Flavivirus species. Base-pairing occurred as predicted for the previously proposed conserved cyclization sequences. In order to obtain experimental evidence of the predicted interactions, the putative cyclization sequences (5 or 3) in the replicon RNA of the mosquito-borne Kunjin virus were mutated either separately, to destroy base-pairing, or simultaneously, to restore the complementarity. None of the RNAs with separate mutations in only the 5 or only the 3 cyclization sequences was able to replicate after transfection into BHK cells, while replicon RNA with simultaneous compensatory mutations in both cyclization sequences was replication competent. This was detected by immunofluorescence for expression of the major nonstructural protein NS3 and by Northern blot analysis for amplification and accumulation of replicon RNA. We then used the M-fold program to analyze RNA secondary structure of the covalently linked 5-and 3-terminal regions of three tick-borne virus species and identified a previously undescribed additional pair of conserved complementary sequences in locations similar to those of the mosquito-borne species. They base-paired with ⌬G values of approximately ؊20 kcal, equivalent or greater in stability than those calculated for the originally proposed cyclization sequences. The results show that the base-pairing between 5 and 3 complementary sequences, rather than the nucleotide sequence per se, is essential for the replication of mosquito-borne Kunjin virus RNA and that more than one pair of cyclization sequences might be involved in the replication of the tick-borne Flavivirus species.Despite its essential role in the virus replication cycle as a template for the synthesis of minus-strand RNA, the conformation of the genomic RNA of Flavivirus species has not been defined. Particularly important is the mode of its presentation to the RNA-dependent RNA polymerase NS5, and possibly other components of the replicase complex (RC) (28, 51), in order for copying to commence correctly from the 3Ј end. The size of the Flavivirus genome is about 11 kb, and the complete nucleotide sequence is available for a range of species, (7, 12, 14-16, 18, 25, 27, 29, 33, 40, 45, 54). All sequences share a common gene order (5Ј-C prM E NS1 NS2A NS2B NS4A NS4B NS5-3Ј), i.e., structural (C, prM, and E) followed by nonstructural (NS) genes, and are flanked by 5Ј and 3Ј untranslated regions (UTR) of about 100 and 600 nucleotides, respectively (39). Conserved complementary cyclization sequences (CS) of 8 nucleotides in the 5Ј region of the c...

In a previous study on the replication of Kunjin virus using immunoelectron microscopy (E. G. Westaway, J. M. Mackenzie, M. T. Kenney, M. K. Jones, and A. A. Khromykh, 1997, J. Virol. 71, 6650-6661), NS1 and NS3 were found associated with double-stranded RNA (dsRNA) within vesicle packets (VP) in infected Vero cells, suggesting that these induced membrane structures may be the cytoplasmic sites of RNA replication. NS2B and NS3 (comprising the virus-encoded protease) were colocalized within distinct paracrystalline (PC) or convoluted membranes (CM), also induced in the cytoplasm, suggesting that these membranes are the sites of proteolytic cleavage. In this study we found by immunofluorescence (IF) that the small hydrophobic nonstructural proteins NS2A and NS4A were located in discrete foci in the cytoplasm of infected cells at both 16 and 24 h postinfection, partially coincident with dsRNA foci. In cryosections of infected cells at 24 h, NS2A was located by immunogold labeling primarily within VP, associated with labeled dsRNA. NS2A fused to glutathione S-transferase (GST) bound strongly to the 3' untranslated region of Kunjin RNA and also to the proposed replicase components NS3 and NS5 in cell lysates. NS4A was localized by immunogold labeling within a majority of the virus-induced membranes, including VP, CM, and PC. GST-NS4A bound weakly to the 3' untranslated region of Kunjin RNA but was bound to NS4A strongly and to most of the other viral nonstructural proteins, including NS3 and NS5. Taken together the results indicate that the flavivirus replication complex includes NS2A and NS4A in the VP in addition to the previously identified NS1 and NS3.

The subcellular location of the nonstructural proteins NS1, NS2B, and NS3 in Vero cells infected with the flavivirus Kunjin was investigated using indirect immunofluorescence and cryoimmunoelectron microscopy with monospecific antibodies. Comparisons were also made by dual immunolabelling using antibodies to double-stranded RNA (dsRNA), the putative template in the flavivirus replication complex. At 8 h postinfection, the immunofluorescent patterns showed NS1, NS2B, NS3, and dsRNA located in a perinuclear rim with extensions into the peripheral cytoplasm. By 16 h, at the end of the latent period, all patterns had changed to some discrete perinuclear foci associated with a thick cytoplasmic reticulum. By 24 h, this localization in perinuclear foci was more apparent and some foci were dual labelled with antibodies to dsRNA. In immunogold-labelled cryosections of infected cells at 24 h, all antibodies were associated with clusters of induced membrane structures in the perinuclear region. Two important and novel observations were made. First, one set of induced membranes comprised vesicle packets of smooth membranes dual labelled with anti-dsRNA and anti-NS1 or anti-NS3 antibodies. Second, adjacent masses of paracrystalline arrays or of convoluted smooth membranes, which appeared to be structurally related, were strongly labelled only with anti-NS2B and anti-NS3 antibodies. Paired membranes similar in appearance to the rough endoplasmic reticulum were also labelled, but less strongly, with antibodies to the three nonstructural proteins. Other paired membranes adjacent to the structures discussed above enclosed accumulated virus particles but were not labelled with any of the four antibodies. The collection of induced membranes may represent virus factories in which translation, RNA synthesis, and virus assembly occur.