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• W2065843293 abstract "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. 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. Arthropod-borne Flaviviruses such as West Nile (WNV), dengue (DENV), Yellow fever (YFV), Tick-borne encephalitis (TBEV), and Japanese encephalitis (JEV) cause major outbreaks of potentially fatal diseases and affect more than 50 million people every year. The highly pathogenic North American strain of WNV (WNVNY99) has already claimed more than 1,000 lives with more than 27,000 cases reported since its emergence in New York in 1999. In contrast, the closely related Australian strain of WNV, Kunjin (WNVKUN), is highly attenuated and does not cause overt disease in humans and animals (Hall et al., 2002Hall R.A. Broom A.K. Smith D.W. Mackenzie J.S. The ecology and epidemiology of Kunjin virus.Curr. Top. Microbiol. Immunol. 2002; 267: 253-269Google Scholar). The ∼11 kb positive-stranded flavivirus RNA genome consists of 5′ and 3′ untranslated regions (UTRs) and one open reading frame, which encodes 10 viral proteins required for the viral life cycle (Liu et al., 2002Liu W.J. Sedlak P.L. Kondratieva N. Khromykh A.A. Complementation analysis of the flavivirus Kunjin NS3 and NS5 proteins defines the minimal regions essential for formation of a replication complex and shows a requirement of NS3 in cis for virus assembly.J. Virol. 2002; 76: 10766-10775Crossref Scopus (78) Google Scholar, Liu et al., 2003Liu W.J. Chen H.B. Khromykh A.A. Molecular and functional analyses of Kunjin virus infectious cDNA clones demonstrate the essential roles for NS2A in virus assembly and for a nonconservative residue in NS3 in RNA replication.J. Virol. 2003; 77: 7804-7813Crossref Scopus (133) Google Scholar, Liu et al., 2004Liu W.J. Chen H.B. Wang X.J. Huang H. Khromykh A.A. Analysis of adaptive mutations in Kunjin virus replicon RNA reveals a novel role for the flavivirus nonstructural protein NS2A in inhibition of beta interferon promoter-driven transcription.J. Virol. 2004; 78: 12225-12235Crossref Scopus (127) Google Scholar, Liu et al., 2005Liu W.J. Wang X.J. Mokhonov V.V. Shi P.Y. Randall R. Khromykh A.A. Inhibition of interferon signaling by the New York 99 strain and Kunjin subtype of West Nile virus involves blockage of STAT1 and STAT2 activation by nonstructural proteins.J. Virol. 2005; 79: 1934-1942Crossref PubMed Scopus (230) Google Scholar, Liu et al., 2006Liu W.J. Wang X.J. Clark D.C. Lobigs M. Hall R.A. Khromykh A.A. A single amino acid substitution in the West Nile virus nonstructural protein NS2A disables its ability to inhibit alpha/beta interferon induction and attenuates virus virulence in mice.J. Virol. 2006; 80: 2396-2404Crossref PubMed Scopus (197) Google Scholar, Westaway et al., 2003Westaway E.G. Mackenzie J.M. Khromykh A.A. Kunjin RNA replication and applications of Kunjin replicons.Adv. Virus Res. 2003; 59: 99-140Crossref Scopus (97) Google Scholar). Flavivirus UTRs are involved in translation and initiation of RNA replication and likely determine genome packaging (Markoff, 2003Markoff L. 5′- and 3′-noncoding regions in flavivirus RNA.Adv. Virus Res. 2003; 59: 177-228Crossref PubMed Scopus (142) Google Scholar, Westaway et al., 2002Westaway E.G. Mackenzie J.M. Khromykh A.A. Replication and gene function in Kunjin virus.Curr. Top. Microbiol. Immunol. 2002; 267: 323-351Google Scholar). In addition to full-length genomic RNA (gRNA), an abundant RNA species of about 0.5 kb derived from the 3′UTR has been detected in cells infected with mosquito-borne encephalitic flaviviruses Murray Valley encephalitis (MVE) (Urosevic et al., 1997Urosevic N. van Maanen M. Mansfield J.P. Mackenzie J.S. Shellam G.R. Molecular characterization of virus-specific RNA produced in the brains of flavivirus-susceptible and -resistant mice after challenge with Murray Valley encephalitis virus.J. Gen. Virol. 1997; 78: 23-29Crossref Scopus (57) Google Scholar), JEV (Lin et al., 2004Lin K.C. Chang H.L. Chang R.Y. Accumulation of a 3′-terminal genome fragment in Japanese encephalitis virus-infected mammalian and mosquito cells.J. Virol. 2004; 78: 5133-5138Crossref Scopus (58) Google Scholar), and WNV (Scherbik et al., 2006Scherbik S.V. Paranjape J.M. Stockman B.M. Silverman R.H. Brinton M.A. RNase L plays a role in the antiviral response to West Nile virus.J. Virol. 2006; 80: 2987-2999Crossref PubMed Scopus (111) Google Scholar). However, the mechanism of its generation and function in the viral replication cycle remained unknown. Processing bodies (PBs, also known as GW bodies) are discrete cytoplasmic granules in which mRNA degradation, mRNA surveillance, translational repression, and RNA-mediated gene silencing take place (Eulalio et al., 2007Eulalio A. Behm-Ansmant I. Izaurralde E. P bodies: at the crossroads of post-transcriptional pathways.Nat. Rev. Mol. Cell Biol. 2007; 8: 9-22Crossref PubMed Scopus (716) Google Scholar). mRNA degradation can occur through two distinct pathways: (1) deadenylation followed by degradation through 3′-5′decay mediated by the exosome and (2) decapping and degradation by a 5′-to-3′ exonucleolytic process (Tourriere et al., 2002Tourriere H. Chebli K. Tazi J. mRNA degradation machines in eukaryotic cells.Biochimie. 2002; 84: 821-837Crossref PubMed Scopus (105) Google Scholar, Wilusz et al., 2001Wilusz C.J. Wormington M. Peltz S.W. The cap-to-tail guide to mRNA turnover.Nat. Rev. Mol. Cell Biol. 2001; 2: 237-246Crossref PubMed Scopus (612) Google Scholar). The latter occurs primarily in PBs in which mRNAs are decapped by Dcp1/2 and subsequently degraded by the 5′-3′ exoribonuclease XRN1 (Sheth and Parker, 2003Sheth U. Parker R. Decapping and decay of messenger RNA occur in cytoplasmic processing bodies.Science. 2003; 300: 805-808Crossref PubMed Scopus (921) Google Scholar). XRN1 is a processive enzyme hydrolyzing RNA from the 5′ to 3′ end and an essential component of cellular mRNA decay machinery (Sheth and Parker, 2003Sheth U. Parker R. Decapping and decay of messenger RNA occur in cytoplasmic processing bodies.Science. 2003; 300: 805-808Crossref PubMed Scopus (921) Google Scholar, Stevens, 2001Stevens A. 5′-exoribonuclease 1: Xrn1.Methods Enzymol. 2001; 342: 251-259Crossref Scopus (33) Google Scholar). Interestingly, flavivirus gRNA has no 3′poly(A) tail but a cap at the 5′ end that may target it to PBs for decapping and XRN1-mediated decay. For the yeast homolog Xrn1p, it has been shown that certain structures in the target RNA influence efficiency of 5′-3′ exoribonucleolytic hydrolysis (Poole and Stevens, 1997Poole T.L. Stevens A. Structural modifications of RNA influence the 5′ exoribonucleolytic hydrolysis by XRN1 and HKE1 of Saccharomyces cerevisiae.Biochem. Biophys. Res. Commun. 1997; 235: 799-805Crossref Scopus (35) Google Scholar). The 3′UTR of flaviviruses contains a number of highly structured regions, for some of which functions have been assigned (Markoff, 2003Markoff L. 5′- and 3′-noncoding regions in flavivirus RNA.Adv. Virus Res. 2003; 59: 177-228Crossref PubMed Scopus (142) Google Scholar, Proutski et al., 1999Proutski V. Gritsun T.S. Gould E.A. Holmes E.C. Biological consequences of deletions within the 3′-untranslated region of flaviviruses may be due to rearrangements of RNA secondary structure.Virus Res. 1999; 64: 107-123Crossref Scopus (78) Google Scholar), while no functions have been determined for other highly structured regions. Here, we show that the accumulation of a subgenomic 3′UTR-derived RNA is a specific feature of members of the flavivirus genus, and, using West Nile virus as a model, we demonstrate that this subgenomic flavivirus RNA (sfRNA) is generated as a product of gRNA degradation presumably by XRN1 exoribonuclease. We also provide evidence that the production of sfRNA is likely to be a result of XRN1 stalling mediated by rigid, conserved RNA structures in the 5′ end of the 3′UTR. Importantly, we demonstrate that sfRNA has a role in facilitating efficient virus replication and virus-induced cytopathicity in cell culture and in determining viral pathogenicity in mice. To investigate whether the production of subgenomic RNA species is common for all flaviviruses, cells were either infected with different viruses or electroporated with their replicon RNAs. Northern hybridizations with the corresponding 3′UTR-specific probes showed that cells infected with mosquito-borne WNVKUN, WNVNY99, MVEV, Alfuy (ALFV), and tick-borne Samaurez Reef virus (SREV) all produced a subgenomic RNA of similar size (∼0.5 kb), while YFV-infected cells and a replicon of DENV type 2 (DENV2) produced slightly smaller RNAs (∼0.3 and ∼0.4 kb, respectively), correlating with the corresponding 3′UTR sizes (Figures 1A and 1B). In contrast, cells infected with viruses from other Flaviviridae genera, bovine viral diarrhea virus (BVDV; Pestiviruses) and (a replicon of) hepatitis C virus (HCV; Hepaciviruses), did not produce a subgenomic, 3′UTR-derived RNA (Figures 1C and 1D). Production of small subgenomic RNA was also not detected for a replicon of the unrelated Semliki Forest virus (SFV; Togaviridae, Alphaviruses) (Figure 1E). Taken together, the results show that generation of the subgenomic, 3′UTR-derived RNA is restricted to and conserved in the Flavivirus genus of the Flaviviridae family; thus, we designated it as subgenomic flavivirus RNA (sfRNA). Further analysis of sfRNA production by WNVKUN showed that it is produced in abundant amounts in all cell types tested, including those of vertebrate (human, primate, and rodent) and invertebrate (mosquito) origin (Figure S1A available online). In addition, sfRNA was shown to be produced in WNVKUN-infected mouse brain but was not packaged into virions and not a product of RNA self-cleavage in vitro under the conditions used (Figure S1B), clearly indicating the involvement of host or viral proteins in sfRNA generation. To further elucidate the mechanism of sfRNA generation, we investigated whether the production of sfRNA was dependent on viral RNA replication, expression of viral proteins, or presence of the 5′UTR. Experiments with WNVKUN RNA or cDNA clones producing RNAs containing various deletions in the viral genome from a CMV promoter (Figure 2A and Supplemental Data) showed that sfRNA could be readily detected in cells transfected with all deletion constructs, replicating (KUNrep and pKUNrep2-βgal) or nonreplicating (all remaining constructs) (Figures 2B, 2C, and 2D and Supplemental Data), thus demonstrating that RNA replication, viral proteins, or the 5′UTR are not essential for sfRNA generation. The results clearly showed that cellular proteins/factors rather than viral proteins are responsible for sfRNA production. Having excluded the role of viral factors in sfRNA generation and taking into account that sfRNA represents the 3′-terminal product of genomic RNA, we hypothesized that a cellular ribonuclease with 5′-3′ RNA hydrolyzing activity may be responsible for its generation. As XRN1 is the only known enzyme with such activity in the cytoplasm of eukaryotic cells, it was logical to assume that it may be involved in sfRNA generation. To test this hypothesis, we analyzed the effect of XRN1 depletion by siRNA on sfRNA generation in virus-infected cells. Depletion of XRN1 in two different cell lines, A549 and SVGA (by about 90% and 67%, respectively), resulted in a significant decrease in the amount of sfRNA (by 56% and 47%, respectively) compared to cells transfected with control siRNA (Figure 3A). In another XRN1 depletion experiment in A549 cells, sfRNA was reduced by 52%. Statistical analysis of sfRNA production after XRN1 knockdown in these three independent experiments showed that sfRNA was reduced by 51.7% ± 4.5%. Thus, the results demonstrate reproducible downregulation of sfRNA production in XRN1-depleted cells. Combined FISH and immunofluorescence analysis showed that viral 3′UTR-containing RNA partially colocalized with XRN1 in PBs of WNVKUN-infected cells (Figure 3B). Later (30 hr) in infection, 22% ± 7% of RNA-labeled foci overlapped with XRN1-containing foci (right panels in Figure 3B). In contrast, staining of SFV-infected cells for SFV RNA and XRN1 showed no SFV RNA in PBs (Figure 3C). The siRNA depletion and colocalization results suggest that XRN1 is likely to be involved in sfRNA generation and that this process may occur in PBs. To determine whether sfRNA is indeed resistant to XRN1 degradation, we performed in vitro digestion of total RNA isolated from WNVKUN-infected cells with recombinant XRN1. This enzyme was able to completely degrade RNA substrates from SFV and HCV (data not shown). In contrast, sfRNA was shown to be resistant to XRN1 treatment (Figure 3D), while it was readily degraded by treatment with other RNases, namely RNaseA and RNaseONE (Figure 3D). The results indicate that XRN1 is unable to degrade sfRNA and further implicate that sfRNA may be a product of XRN1 stalling. Northern hybridization mapping of RNA isolated from WNVKUN-infected cells with radiolabeled probes specific for different regions of the 3′UTR demonstrated that sfRNA corresponds to the last part of the 3′UTR (Figures S2A and S2B). Primer extension analysis showed that sfRNA isolated from WNVKUN and WNVNY99-infected cells or isolated from infected mouse brain had a size of 525 nt (Figure S2C). Flavivirus 3′UTR has a number of characteristic features (Figure 4A), including an AU-rich region at the 5′ end, a number of conserved (repeated) sequences (CS1/2/3 and RCS2/3), a cyclization sequence, putative RNA pseudoknots (PKs), a pentanucleotide, and a conserved stem-loop structure at the 3′ terminus (3′SL) (Markoff, 2003Markoff L. 5′- and 3′-noncoding regions in flavivirus RNA.Adv. Virus Res. 2003; 59: 177-228Crossref PubMed Scopus (142) Google Scholar). Using RNA structure prediction on a 3′UTR sequence alignment of different flaviviruses, we predicted a common RNA structure (designated SL-II) with remarkable similarity in overall architecture between the different viruses (Figure 4B). The high conservation of this complex RNA structure and location of the sfRNA 5′ end at its base suggest that this structure may be involved in protecting downstream 3′UTR RNA from degradation. Indeed, sfRNA sizes of 525 nt for WNVKUN and WNVNY99 (Figure 1A), 521–523 nt for JEV (Lin et al., 2004Lin K.C. Chang H.L. Chang R.Y. Accumulation of a 3′-terminal genome fragment in Japanese encephalitis virus-infected mammalian and mosquito cells.J. Virol. 2004; 78: 5133-5138Crossref Scopus (58) Google Scholar), 0.5 kb for MVEV (Urosevic et al., 1997Urosevic N. van Maanen M. Mansfield J.P. Mackenzie J.S. Shellam G.R. Molecular characterization of virus-specific RNA produced in the brains of flavivirus-susceptible and -resistant mice after challenge with Murray Valley encephalitis virus.J. Gen. Virol. 1997; 78: 23-29Crossref Scopus (57) Google Scholar) and ALFV (Figure 1A), 0.4 kb for DENV2 (Figure 1A), and 0.3 kb for YFV (Figure 1A) correspond well to the respective locations of SL-II (SL-E in YFV) in the 3′UTRs (Figure 4B). The SL-II structure is remarkably similar to the previously predicted SL-IV structure (Proutski et al., 1997Proutski V. Gould E.A. Holmes E.C. Secondary structure of the 3′ untranslated region of flaviviruses: similarities and differences.Nucleic Acids Res. 1997; 25: 1194-1202Crossref Scopus (162) Google Scholar) located ∼160 nt downstream of the beginning of the SL-II structure. Both WNV SL-II and SL-IV are followed by short conserved hairpins previously designated RCS3 and CS3, respectively (Figure 4A) (Khromykh and Westaway, 1994Khromykh A.A. Westaway E.G. Completion of Kunjin virus RNA sequence and recovery of an infectious RNA transcribed from stably cloned full-length cDNA.J. Virol. 1994; 68: 4580-4588PubMed Google Scholar). Sequence alignment of SL-II and SL-IV of WNVKUN, WNVNY99, JEV, MVEV, ALFV, DENV2, and SL-E of YFV (Proutski et al., 1997Proutski V. Gould E.A. Holmes E.C. Secondary structure of the 3′ untranslated region of flaviviruses: similarities and differences.Nucleic Acids Res. 1997; 25: 1194-1202Crossref Scopus (162) Google Scholar) showed conservation of a number of regions (Figure S3) that appear to form the backbone of the predicted SL-II (SL-E) and SL-IV RNA structures (Figure 4B). To experimentally determine the RNA structure responsible for protection of 3′UTR from degradation, we generated an extensive series of recombinant viruses with various deletions and mutations in the 3′UTR (Figure 4C) and determined their effect on sfRNA generation in infected cells (see Supplemental Data for more detailed description). Northern blot analysis showed that deletions/mutations in SL-II, as expected, resulted in the loss of full-length sfRNA (termed sfRNA1) (Figure 4D), supporting its essential role in sfRNA production. Additional deletions downstream of SL-II led to the production of two smaller, less abundant sfRNA species termed sfRNA2 and sfRNA3 (Figure 4D), suggesting that two additional rigid secondary/tertiary RNA structures downstream of SL-II, likely to be SL-IV and DB1 (Figure 4A), can protect WNVKUN RNA from complete degradation. Importantly, plaque size and virus growth in mammalian and mosquito cells correlated with the generation and amount of full-length sfRNA1 (Figures 4E and 4F), showing that production of abundant amounts of full-length sfRNA1 is essential for efficient viral replication. To confirm the uniqueness of flavivirus 3′UTR RNA structure(s) in the ability to protect from degradation, another highly structured RNA, internal ribosomal entry site (IRES) from encephalomyocarditis virus (EMCV) followed by neomycin transferase sequence (neo), was inserted upstream of SL-II in the 3′UTR of the WNV replicon WN-NeoRep (Shi et al., 2002Shi P.Y. Tilgner M. Lo M.K. Kent K.A. Bernard K.A. Infectious cDNA clone of the epidemic west nile virus from New York City.J. Virol. 2002; 76: 5847-5856Crossref PubMed Scopus (177) Google Scholar) (Figure S4A), and cells stably expressing this replicon were generated by selection with G418. When total RNA isolated from these cells was subjected to northern blot analysis with 3′UTR-specific probe, only sfRNA1, but no other larger RNA-encompassing IRES-neo insertion, was detected (Figure S4B), demonstrating that the 5′ end of sfRNA1, but not the IRES structure, prevents degradation. This provides additional evidence for the uniqueness of flavivirus 3′UTR structure(s) in their ability to protect downstream RNA sequences from degradation. To further characterize sfRNA function, a mutant virus not capable of producing sfRNA1 and sfRNA2 with minimal changes in RNA structure, FL-IRAΔCS3 was constructed. The mutations consisted of a 3 nt substitution in the IRA within SL-II and a 10 nt deletion of the CS3 sequence (ΔCS3) downstream of SL-IV (Figure 4C). As shown above, IRA mutation abolished sfRNA1 production (Figure 4D, lane 15), while ΔCS3 mutation abolished sfRNA2 production (Figure 4D, lane 9). The mutant virus with combined IRAΔCS3 mutations showed a severe defect in the production of sfRNA1 (Figure 5A, lanes 5 and 6), no visible plaque formation in Vero cells (Figure 5B), and decreased replication efficiency in mammalian (Vero) and mosquito (C6/36) cells (Figures 5C and 5D). However, maximum differences in virus titers were only about 5- to 10-fold. The mutant FL-IRA containing only 3 nt substitution in SL-II abolishing sfRNA1 production was also compromised in the ability to form clearly visible plaques (Figure 5B), but not so much in replication efficiency (Figures 5C and 5D). The mutations/deletions did not have a major effect on RNA translation, replication, or packaging efficiencies when introduced into replicon RNAs (Figure S5). To analyze the apparent discrepancy between dramatic differences in plaque morphology and only moderate differences in virus growth kinetics, mutant viruses were investigated for induction of cytopathicity. The number of viable cells was significantly higher in wells infected with FL-IRA and FL-IRAΔCS3 compared to FLSDX and FLΔCS3 (Figure 6A). Quantification analysis using released crystal violet stain showed clear differences in cytopathic effect (CPE) with about 70% of cells dead at 6 days postinfection (d.p.i.) with FLSDX and only 10% or less of cells dead after infection with either FL-IRA or FL-IRAΔCS3 mutants (Figure 6B). This difference in viral cytopathicity was confirmed by measuring the release of lactate dehydrogenase (LDH) by dying cells into the cell culture supernatant (Figure 6C).Figure 6sfRNA1 Production Is Essential for Virus-Induced CytopathicityShow full caption(A and B) Vero cells were infected and fixed at indicated time points after infection.(A) Cells were stained 6 days after infection.(B) Crystal violet was released from cells by methanol and OD 620 nm measured. Percentage of dead cells was calculated. CPE, cytopathic effect.(C) LDH release into cell culture fluid of Vero cells infected with mutant viruses was measured 4 d.p.i. by colorimetric assay. Background from negative cells was subtracted. Percentage of dead cells was calculated.(D) LDH release from Vero cells transfected with plasmid DNA producing sfRNA (pCMVβgal3′).(E) Partial rescue of virus-induced cytopathicity by complementation with sfRNA. Vero cells were transfected with sfRNA-producing plasmid pCMVβgal3′ and control pCMVβgal. At 24 hr posttransfection, cells were infected with MOI = 1 FLSDX and FL-IRAΔCS3. LDH release was measured 8 d.p.i.As a positive control for (C), (D), and (E), mock cells were lysed with 0.1% Triton X-100. Data are represented as average + SD.(F) Partial rescue of viral plaque formation by sfRNA complementation. Vero cells were transfected with plasmids. At 24 hr later, cells were infected with corresponding viruses, overlayed with agarose, and stained 6 d.p.i.(G) Partial rescue of virus production by sfRNA complementation.Viral titers in culture fluid of DNA-transfected and virus-infected cells (as in [E]) were determined 48 hr after infection by plaque assay on BHK cells. P values were calculated by unpaired Student's t test.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A and B) Vero cells were infected and fixed at indicated time points after infection. (A) Cells were stained 6 days after infection. (B) Crystal violet was released from cells by methanol and OD 620 nm measured. Percentage of dead cells was calculated. CPE, cytopathic effect. (C) LDH release into cell culture fluid of Vero cells infected with mutant viruses was measured 4 d.p.i. by colorimetric assay. Background from negative cells was subtracted. Percentage of dead cells was calculated. (D) LDH release from Vero cells transfected with plasmid DNA producing sfRNA (pCMVβgal3′). (E) Partial rescue of virus-induced cytopathicity by complementation with sfRNA. Vero cells were transfected with sfRNA-producing plasmid pCMVβgal3′ and control pCMVβgal. At 24 hr posttransfection, cells were infected with MOI = 1 FLSDX and FL-IRAΔCS3. LDH release was measured 8 d.p.i. As a positive control for (C), (D), and (E), mock cells were lysed with 0.1% Triton X-100. Data are represented as average + SD. (F) Partial rescue of viral plaque formation by sfRNA complementation. Vero cells were transfected with plasmids. At 24 hr later, cells were infected with corresponding viruses, overlayed with agarose, and stained 6 d.p.i. (G) Partial rescue of virus production by sfRNA complementation. Viral titers in culture fluid of DNA-transfected and virus-infected cells (as in [E]) were determined 48 hr after infection by plaque assay on BHK cells. P values were calculated by unpaired Student's t test. To investigate whether sfRNA production alone induces cell death, the plasmid pCMVβgal3′ producing sfRNA without other viral components was transfected, and cell death was assessed by LDH release. sfRNA production alone did not lead to significant cell death (Figure 6D), indicating that sfRNA must act in the context of viral infection to promote virus-induced cytopathicity. In order to confirm the role of sfRNA in virus-induced cytopathicity, the defect in sfRNA production was complemented by providing sfRNA1 in trans from transfected pCMVβgal3′. The cytopathicity of FL-IRAΔCS3 was, indeed, partially rescued in cells producing sfRNA from the transfected plasmid as determined by LDH release (Figure 6E). This was confirmed by the detection of more clear plaques in cells transfected with pCMVβgal3′ and infected with FL-IRAΔCS3 than in infected cells transfected with the control pCMVβgal (Figure 6F). In addition, viral titers were significantly increased in cells transfected with pCMVβgal3′ and infected with FL-IRAΔCS3 compared to those in infected cells transfected with the control plasmid (Figure 6G). In these experiments, transfection efficiency was between 50% and 60%, while 100% of cells were infected as determined by X-gal staining and immunofluorescence analyses, respectively (data not shown). Thus, the relatively inefficient but significant complementation can be explained by the limited transfection efficiency of the plasmid supplying the complementing sfRNA. Overall, the results demonstrate that production of abundant amounts of sfRNA1 during virus infection is a determinant of viral cytopathicity in cell culture. To investigate the effect of altered sfRNA production on viral pathogenicity in vivo, FLSDX, FL-IRA, FLΔCS3, and FL-IRAΔCS3 were injected in 3-week-old mice, a highly sensitive animal model for WNVKUN infection. Mice infected with the wild-type virus FLSDX started to develop severe symptoms as early as day 6 after injection, and all animals had to be sacrificed 9 days after injection (Figure 7A). Mice injected with FLΔCS3, which produces less sfRNA1, developed symptoms after 7 days, with a mortality rate of 60% throughout the observation period (Figure 7A). In contrast, mice injected with either FL-IRA or FL-IRAΔCS3 showed no signs of WNVKUN-induced encephalitis during the observation period (Figure 7A). Kinetic analysis of virus accumulation in the spleen and brain of infected mice demonstrated that all four viruses replicated in vivo with the sfRNA mutant viruses being detected in the brain later in infection at levels similar to corresponding parental viruses (i.e., FLSDX for FL-IRA and FLΔCS3 for FL-IRAΔCS3; Figures 7B and 7C). The results show that the production of sfRNA1 is not essential for viral replication and spread in vivo, while it has a crucial role in determining viral pathogenicity. We demonstrated that all representative viruses from the genus Flaviviruses, but not from other genera of the Flaviviridae family, produce an abundant, subgenomic, noncoding RNA derived from the 3′UTR of gRNA. We have shown that RNA replication, viral proteins, or 5′UTR are not essential for generation of this subgenomic flavivirus RNA (sfRNA). We have provided evidence that sfRNA is a product of incomplete degradation of gRNA, which is likely to involve cellular 5′-3′ exoribonuclease XRN1, one of the key enzymes in the cellular mRNA decay pathway. In agreement with the high level of conservation between factors and pathways involved in mRNA decay of mosquito and human cells (Opyrchal et al., 2005Opyrchal M. Anderson J.R. Sokoloski K.J. Wilusz C.J. Wilusz J. A cell-free mRNA stability assay reveals conservation of the enzymes and mechanisms of mRNA decay between mosquito and mammalian cell lines.Insect Biochem. Mol. Biol. 2005; 35: 1321-1334Crossref Scopus (24) Google Scholar), sfRNA was produced in cells of different origin, including mosquito cells. In general, mRNA decay in the cell can follow different pathways. It can be regulated by decapping, deadenylation, translation, cis-acting elements, and “nonsense” sequences and has been shown to be a critical control point for determination of transcript abundance (Wilusz et al., 2001Wilusz C.J. Wormington M. Peltz S.W. The cap-to-tail guide to mRNA turnover.Nat. Rev. Mol. Cell Biol. 2001; 2: 237-246Crossref PubMed Scopus (612) Google Scholar). 5′-3′ mRNA degradation takes place in PBs, which contain most enzymes and proteins needed for" @default.
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• W2065843293 date "2008-12-01" @default.
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• W2065843293 title "A Highly Structured, Nuclease-Resistant, Noncoding RNA Produced by Flaviviruses Is Required for Pathogenicity" @default.
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