Small noncoding RNA modulates japanese encephalitis virus replication and translation in trans
© Fan et al; licensee BioMed Central Ltd. 2011
Received: 27 August 2011
Accepted: 1 November 2011
Published: 1 November 2011
Sequence and structural elements in the 3'-untranslated region (UTR) of Japanese encephalitis virus (JEV) are known to regulate translation and replication. We previously reported an abundant accumulation of small subgenomic flaviviral RNA (sfRNA) which is collinear with the highly conserved regions of the 3'-UTR in JEV-infected cells. However, function of the sfRNA in JEV life cycle remains unknown.
Northern blot and real-time RT-PCR analyses indicated that the sfRNA becomes apparent at the time point at which minus-strand RNA (antigenome) reaches a plateau suggesting a role for sfRNA in the regulation of antigenome synthesis. Transfection of minus-sense sfRNA into JEV-infected cells, in order to counter the effects of plus-sense sfRNA, resulted in higher levels of antigenome suggesting that the presence of the sfRNA inhibits antigenome synthesis. Trans-acting effect of sfRNA on JEV translation was studied using a reporter mRNA containing the luciferase gene fused to partial coding regions of JEV and flanked by the respective JEV UTRs. In vivo and in vitro translation revealed that sfRNA inhibited JEV translation.
Our results indicate that sfRNA modulates viral translation and replication in trans.
Japanese encephalitis virus (JEV), a member of the Flaviviridae family, is a major zoonotic agent. Pigs and birds are the principal viremic hosts and mosquitoes are responsible for the transmission between these vertebrates to human . In humans, JEV causes acute meningomyeloencephalitis with high mortality rate . The JEV genome is a single-stranded positive sense RNA of about 10,976-nts that encodes a single large open reading frame (ORF) flanked by a 95-nucleotide (nt) long 5' untranslated region (UTR) and a 585-nt long 3' UTR with no poly A tail. Cap-dependent translation of the JEV ORF results in a polyprotein which is co- and post-translationally processed by viral as wells as host proteases to yield three structural proteins (C, prM, and E), and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5).
As with all positive-sense RNA viruses, JEV RNA replication begins with the synthesis of negative-strand antigenome, which serves as a template for the synthesis of progeny positive-strand genomic RNA. The asymmetric RNA replication leading to 10- to 100-fold excess of positive strands over negative strands which was observed in Kunjin virus and dengue virus (DENV), and in JEV infected cells [4–7]. In addition to genome and antigenome, flaviviruses produce a small subgenomic RNA (named sfRNA) representing highly conserved regions of the 3'-UTR [8–11]. The sfRNA is more abundant in JEV-infected mosquito cells than mammalian cells and the molar ratio of sfRNA to genomic RNA can range from 0.25 to 5.14 . The abundant accumulation of this RNA suggests that it may play an important role in viral life cycle. Using West Nile virus (WNV) as a model, Pijlman et al. demonstrated that the sfRNA is a product of incomplete degradation of viral genome by cellular ribonuclease XRN1 and is essential for virus-induced pathogenicity . It was reported that a pseudoknot structure at the 3'-UTR is responsible for stalling XRN1 from degrading the RNA further, which results in sfRNA [9, 10].
The 3'-UTR of flaviviruses has been shown to serve various important functions such as translation, replication, and encapsidation [12–17]. There are several functional motifs in the 3'-UTR including the conserved sequences (CS motifs), cyclization motifs, pseudoknot structure, and the 3'-stemloop (3'-SL) motif. Two pairs of cyclization motifs were reported. Hahn et al. first reported that the 5' and 3' conserved sequences are complementary to each other and potentially form a cyclization structure . The second pair of cyclization motif is the upstream of AUG codon (5'UAR), which is complementary to the sequences located in the 3'-UTR (3'UAR). The UAR cyclization motifs have been shown to be required for replication in DENV and WNV [19–23]. In addition, Friebe and Harris identified another element located downstream of the AUG (designated 5'DAR) also involved in DENV replication and possibly genome cyclization . Although these conserved sequences were found in JEV, the functions of these motifs have not been characterized in detail. Yun et al. analyzed the 3'-UTR of JEV and defined it into six domains . By constructing serial deletion mutants, they demonstrated that the two 3'-proximal domains are sufficient for RNA replication, while the other four domains are dispensable but required for maximal replication efficiency suggesting the cis-acting sequences required for JEV replication might be slightly different from those other flaviviruses. In addition to RNA-RNA interactions, numerous studies have been shown that 3'-UTR interacts with both viral and cellular proteins, and is required for RNA synthesis and translation [15, 26–36]. In JEV, viral NS3 and NS5 proteins as well as cellular Mov 34 and La proteins have been shown to bind to the 3'-SL and play roles in viral replication [37–39]. Previously we showed that the cellular protein GAPDH binds more efficiently to the 3' end of minus-strand RNA than to the 3'-SL of plus-strand RNA, suggesting a role for promoting asymmetric RNA replication .
The presence of such essential motifs in sfRNA and its abundance in infected cells present a compelling indication of a possible function that prompted us to elucidate its role in the viral life cycle at the cellular level. We found that high levels of sfRNA accumulates in the cytoplasm during the late stages of viral life cycle suggesting that sfRNA may inhibit either viral translation or minus-strand synthesis or both. To test this, plus- or minus-strand forms of the sfRNA were separately transfected in virus-infected cells and the effects on genome and antigenome accumulation were measured. The effect of sfRNA on JEV translation was determined by co-transfecting plus or minus sense of sfRNA with a luciferase reporter RNA for in vivo translation studies in cultured cells and a rabbit reticulocyte lysate assay system was used for in vitro translation studies. Our results indicated that the sfRNA inhibits antigenome synthesis and also down regulates viral translation in trans.
sfRNA localizes to the cytoplasm along with a major proportion of genomic RNA while a small proportion of genomic RNA is localized in the nucleus
Time course study of viral genome and antigenome synthesis in JEV-infected cells
The presence of the sfRNA inhibits antigenome synthesis
The sfRNA inhibits translation
The discovery of sfRNA in flaviviruses has generated considerable interest in its generation, localization, and its possible function in flavivirus infected cells. In this study, we have shown that JEV sfRNA is localized in cytoplasm along with the genomic RNA (Figure 1A). Although the biogenesis of JEV sfRNA has not yet been studied, it has been reported that in WNV and YFV, sfRNA is a product of incomplete degradation of viral genome by cellular ribonuclease XRN1 and is co-localized to the P-body in the cytoplasm . Consistent with their report we found that JEV sfRNA is also localized to the cytoplasm along with the genomic RNA. On the other hand, we also found that a few genomic RNA is also localized to the nucleus consistent with reports on the presence of flaviviral proteins and flaviviral RNA replication in the nuclear fraction [41–43]. The presence of abundant sfRNA in the cytoplasm of infected cells hints at possible roles of the sfRNA in cytoplasmic events during infection, namely viral RNA replication and translation.
The RNA synthesis of plus-strand RNA viruses is asymmetrical meaning that positive-sense genomic RNA strands are generated in excess over minus sense antigenome and the ratio is about 10:1 to 100:1. Northern analysis from a previous study showed that the ratio of plus-to-minus strands at 8 h postinfection was 3:1 which rapidly increased thereafter to 11.7:1 by 18 h postinfection in porcine kidney cells infected with JEV at an MOI of 10 . In this study, we describe the kinetics on the synthesis of JEV genome and antigenome in BHK-21 cells at an MOI of 0.01 using strand specific oligonucleotides for real-time RT-PCR. Our results indicated that the ratio of plus-to-minus strands during 24-48 h postinfection was in the range of 29:1 to 244:1 during inspection period (Figure 2D). Interestingly, we found that the time point at which the antigenome accumulation reaches a plateau coincides with the appearance of sfRNA as shown in our results from Northern and real-time RT-PCR analyses. It would be ideal to show the time course of antigenome and sfRNA accumulation together but since it was impossible to distinguish sfRNA accumulation from genomic RNA accumulation in real-time RT-PCR experiments, we employed Northern analysis which clearly distinguishes the two (Figure 2A). The time course of antigenome, on the other hand, real-time RT-PCR is much more sensitive than Northern analysis especially during the early time points (Figure 2C). Thus, we compare the same amounts of RNA under the same condition by these two different methods.
The artificial addition of (-)sfRNA (by transfection) at 28 h postinfection countered the effects of naturally occurring sfRNA thereby increasing the accumulation of antigenome (Figure 3D and 3F) indicating that sfRNA could negatively interfere with antigenome synthesis. The addition of (-)sfRNA may not only anneal to the naturally occurring plus-sense sfRNA but also to the 3'-UTR of the genomic RNA. However, the probability of the minus-sense sfRNA to bind to the plus-sense sfRNA is more than its binding to the genomic 3'-UTR because of (i) the binding strength of same size shorter complementary RNA should be greater than the binding of a short RNA sequence to a large RNA polynucleotide akin to a highly complementary primer dimer and a PCR template. This was observed in our in vitro luciferase assays (Figure 4) where the individual addition of either only (+)sfRNA or (-)sfRNA reduced translation but the addition of both (+)sfRNA (50 ng) and (-)sfRNA (10 ng) into the reaction partially restored translation and the addition of more (-)sfRNA (50 ng) restored translation to control levels probably because the plus-sense and the minus-sense sfRNA hybridized to form duplex RNA thereby preventing sfRNA from interfering with translation; (ii) cyclization of the genome could render the 3'-UTR inaccessible to the minus-sense sfRNA. Curiously, when (-)sfRNA is transfected, the amount of naturally occurring plus-sense sfRNA does not decrease (Figure 3A and 3C) and is similar to that of mock, indicating that naturally occurring plus-sense sfRNA is either not degraded or that the rate of sfRNA generation (RNA turnover) is very high.
It has also been reported that the RNA elements in the flaviviral 3'-UTR influences viral translation efficiency [13, 15, 33, 35]. However, Alvarez et al. developed a replicon system that can be used to discriminate between translation and RNA replication. They demonstrated that deletion of individual domains of the 3'-UTR did not significantly affect viral translation but it impaired or abolished RNA synthesis . Our results showed that JEV translation efficiency in cultured cells was reduced in the presence of sfRNA (Figure 4). JEV translation efficiency in vitro was also impaired in the presence of sfRNA but was restored to control levels by the addition of equal amounts antisense sfRNA into the reaction. This clearly shows that sfRNA does impair JEV translation in tran s, as depicted in Figure 5B, proteins binding to 3'-UTR elements are essential to promote viral translation. Transfection of (+)sfRNA sequesters proteins binding to the 3'-UTR of genome and reduces translation, while if (-)sfRNA is transfected it could bind to the 3'-UTR of the genome and prevent the host proteins from binding the 3'UTR (competes with host factors binding to the 3'UTR). In addition, transfection of (-)sfRNA could also prevent the interaction of the 3' and 5' regions of the genome and that too could reduce translation. Thus, transfecting of either (+) or (-)sfRNA reduces translation. Furthermore, the sfRNA could titrate the host factors and even the newly synthesized viral proteins like RdRp thereby drastically reducing minus-strand RNA synthesis that results in the aforementioned asymmetry in RNA accumulation.
As seen in our results (Figures 2A, 3A and 3C), sfRNA is present in great abundance in the late stages of the viral replication cycle and that sfRNA interferes and impairs both antigenome synthesis and JEV translation (Figures 3 and 4). It could be thought that the JEV genomic RNA produced during the late stages of the viral replication cycle is bound for packaging and should not be used as templates for antigenome synthesis or for translation and the presence of sfRNA is suspected to compete against the translation and antigenome synthesis. From our data, we conclude that sfRNA could be the switch (a trans-acting riboswitch) that shuts down both antigenome synthesis and JEV translation thereby promoting only genomic RNA synthesis that needs to be packaged and released for the next infectious cycle.
Materials and methods
Cells and viruses
Baby hamster kidney (BHK-21) cells were grown in RPMI 1640 medium supplemented with 2% fetal bovine serum (FBS) (Gibco-BRL) at 37°C. JEV strain RP9, a variant of NT109 isolated originally from Culex tritaeniorhynchus was used in this study .
Construction of plasmids
pGEMT/JEV3642-3821 plasmid (Figure 2B) used for making RNA standard in real-time RT-PCR was generated by cloning the 180-nt PCR product amplified from JEV cDNA with the JEV3642(+) (nt 3642-3662) and JEV3821(-) (nt 3802-3821) primers into pGEMT-easy vector (Promega). To generate pGEMT/JEV10450-10976 construct, we followed the same method as for pGEMT/JEV3642-3821 plasmid, except that JEV10450(-) (nt 10450-10476) and JEV10950(+) (nt 10950-10976) primers were used for PCR. PCR products were amplified from sfRNA with primers containing T7 promoter at 5' end and a unique restriction site at the 3' end, then cloned into pUC18 plasmid to generate pUC18/JEV(+)10450-10976, and pUC18/JEV(-)10976-10454 for making (+)sfRNA and (-)sfRNA respectively. Sequences of each construct were confirmed by sequencing.
RNA preparation and Northern blot analysis
RNA extraction and Northern analyses were done as described previously . Briefly, total RNA was extracted with Trizol (Invitrogen) or REzol™ C&T reagent (Protech). Cytoplasm/Nucleus fractionation was done using Cytoplasmic & Nuclear RNA Purification kit (Norgen) according to manufacturer's instruction. Approximate 2.5 μg or 7-10 μg of cytoplasmic RNA were used per lane in formaldehyde-agarose gel electrophoresis for the detection of plus- or minus-strand, respectively. To label oligonucleotide probe, approximately 100-pmol of oligonucleotide was 3' tailed with Digoxigenin (DIG)-ddUTP using a DIG Oligonucleotide 3'-End Labeling Kit (Roche Molecular Biochemicals). For labeling sfRNA probe, 1 μg of linearized DNA was used for in vitro transcription with DIG RNA labeling mix (Roche Molecular Biochemicals). Hybridization was done at 54°C for oligonucleotide probes and 68°C for riboprobes. DIG luminescent detection of the viral specific bands was done according to the manufacturer's instructions (Roche Molecular Biochemicals).
Synthetic oligonucleotides and accession number
JEV genome nucleotide positions correspond to those for JEV RP9, GenBank accession number AF014161. 3JEV10950(-) oligonucleotide (5'-AGATCCTGTGTTCTTCCTCACCACCAG-3') detects RNA containing the very 3'-terminal 27 nts of the JEV genome. 18S rRNA(-) oligonucleotide (5'-GCACTTACTGGGAATTCCTCG-3') location corresponds to mouse 18S rRNA, GenBank accession number X00686. Mitochondria 12S rRNA(-) oligonucleotide (5'-AAGGCCAGGACCAACCT-3') was synthesized according to GenBank accession number NC_005089.
The method used for real-time RT-PCR assay was as described previously . The in vitro transcripts of positive-sense RNA were generated from T7 transcription (Promega) of the Sal I-linearized pGEMT/JEV3642-3821 and the minus-sense transcripts were transcribed from SP6 promoter of the Nco I-linearized pGEMT/JEV3642-3821 (as diagramed in Figure 2B). The amount of purified RNA was measured by spectrophotometry and the copy number was calculated based on the concentration measured and its molecular weight. The known amounts of RNAs were serially diluted 10-fold (1.78 × 1011 to 1.78 × 105 copies) and subjected to real-time RT-PCR using the one-step RT-PCR master mix reagent kit following the manufacturer's instructions (Applied Biosystems). Oligonucleotides JEV3642(+) and JEV3821(-) were used as primers for binding to minus and plus-strand RNA, respectively, during RT step carried out at 48°C for 30 min. The PCR amplification conditions were 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min with primers JEV3650(+), JEV3726(-) and TaqMan probe JEV3705(-) (sequences and binding positions are illustrated in Figure 2B). The assay was performed on an ABI 7000 Sequence Detector using TaqMan One-Step RT-PCR master mix to analyze the emitted fluorescence during amplification (Applied Biosystems). A linear equation of known amounts of RNA to CT value was determined.
The intracellular plus- or minus-strand RNAs in JEV-infected cells were determined by using strand specific primers during RT step as described above. Cytoplasmic RNAs were extracted at the indicated time points postinfection. RNA from each time point was diluted to a concentration of 100 ng/μl and 10 ng/μl, respectively, and subjected to real-time RT-PCR together with the known amount of in vitro transcripts. The amount of intracellular genome or antigenome per cell was determined by dividing the copy number by the numbers of cells counted at each time point postinfection.
For run-off transcription, Sal I-linearized pUC18/JEV10450-10976 or Xba I-linearized pUC18/JEV(-)10976-10454 were used for making plus- or minus-strand form of the sfRNA, respectively. Before RNA transfection, cells in 6-well plates at 50 to 80% confluence (approximately 2 × 106 cells) were infected with JEV RP9 at an MOI of 0.01 by incubating cells with inoculum at 37°C for 1 h, refeeding with 2 ml of growth medium containing 5% FBS, and incubating at 37°C for 27 h. For transfection, each dish of cells was rinsed three times with RPMI and treated for 10 min at 0°C with 200 μl of Opti-MEM medium (Gibco-BRL) containing 10 μl of lipofectin (Invitrogen) and 1 μg of RNA transcripts. Cells were rinsed with 2 ml of RPMI medium three times and incubated at 37°C with 2 ml of medium containing 5% FBS until cytoplasmic RNA extraction was done at the indicated time points.
JEV minicon (kindly provided by Dr. Yi-Ling Lin) contains a Renilla luciferase (Rluc) fused in-frame to the JEV coding regions as a single ORF in the following order; the core (nt 96-158), Rluc (933 nts), E (nt 2388-2477), NS1 (nt 2478-2693), and NS5 (nt 10203-10391), and this ORF unit was flanked by the authentic JEV 5'- and 3'-UTRs (Figure 4A). The Rluc-reporter plasmid was transcribed using a Megascript SP6 Transcription kit (Ambion) according to the manufacturer's instructions, in the presence or absence of m7GpppA nucleotide (New England Biolabs). Luciferase assays were performed using extracts from transfected cells and also from the in vitro translation assay. For in vivo translation in cultured cells, 1 μg of transcribed minicon RNA, together with 1 μg of plus- or minus-strand of sfRNA were transfected into BHK-21 cells using Lipofectamin 2000 (Invitrogen). Renilla luciferase activity was measured at 8 hour posttransfection. For in vitro translation, 50 ng of RNAs were translated in nuclease-treated rabbit reticulocyte lysate (Promega) in the presence of 40 units of RNasin Ribonuclease Inhibitor (Promega), 20 μM of each amino acid, and either plus-strand, minus-strand of sfRNA, or a 445-nt control RNA composed of JEV sequences (nt 2401-2689) plus 156 nts derived from vector sequences. The reactions were incubated at 30°C for 30 min and 2.5 μl of reaction sample was measured with 20/20n Single-Tube Luminometer (Promega).
For statistical analysis, one-way ANOVA Dunnet's multiple comparisons test was used to compare the control group against others (GraphPad Software, San Diego, CA, USA).
List of abbreviations
downstream of the AUG
Japanese encephalitis virus
multiplicity of infection
small flaviviral RNA
upstream of the AUG
West Nile virus.
This work was supported by grant NSC 98-2320-B-259-002-MY3 from National Science Council, Taipei, Taiwan, Republic of China. We thank Dr. David Brian for many helpful discussions and Dr. Yu-Pin Su for constructive comments on this manuscript.
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