Patent Application
20150225739
The present invention is related to an expression vector comprising an internal ribosome entry site (IRES) element and the multigene expression system thereof, and more particularly, to those comprising an IRES element of a gene icp35 in White spot syndrome virus.
In eukaryotes and eukaryotic viruses, the regulation of protein synthesis is either cap-dependent or else uses a cap-independent translation mechanism. In cap-dependent translation, 5′-terminal m7GpppG cap needs to be first recognized by eIF4F, and then combines with a 43S pre-initiation complex for the following reactions such as translation initiation, elongation and termination. While cap-independent translation can initiate the translation within mRNA without any cap structure. Such special translation mechanism includes: internal ribosome entry site (IRES), leaky scanning or translation reinitiation, or the like.
IRES elements, one type of cap-independent translation, are located in the upstream of initiation codon ATG or nearby. Without the help of 5′-terminal cap of mRNA or some translation-related factors, this IRES elements can attract and combine with 40S ribosome subunit directly by its RNA sequence to initiate the translation and synthesize proteins. IRES element consists of RNA sequences that often show stable secondary and/or tertiary structure. Due to the direct attraction and combination with host translation systems by the structure itself, IRES elements can synthesize proteins within mRNA. Recent studies related to IRES translation mechanism (see Fitzgerald, K. D. and B. L. Semler, Bridging IRES elements in mRNAs to the eukaryotic translation apparatus. Biochimica Et Biophysica Acta-Gene Regulatory Mechanisms, 2009. 1789(9-10): p. 518-528.) found that many viruses use IRES translation to regulate viral protein synthesis, which RNA virus studies are in the majority. For example, some viruses in at least three RNA virus classes including: Picornaviruses, Flaviviruses and Dicistroviruses have IRES elements. However, only a very limited number of IRES elements have been reported in DNA viruses, including: Kaposi's sarcoma-associated herpesvirus (KSHV), Epstein-Barr virus (EBV), Herpes simplex virus (HSV), Murine gammaherpesvirus 68 (MHV-68), Marek's disease virus (MDV) and Simian vacuolating virus 40 (SV40).
In addition, previous studies also have shown that White spot syndrome virus (WSSV), a large dsDNA virus, has two IRES elements which are respectively located in the 5′ UTR of vp28 and the coding region of vp31/vp39b to regulate the expression of VP28 and VP39B. In Northern blotting, a wssv019 riboprobe was used to detect two transcripts: a large 5.5-kb polycistronic transcript encoding wssv023, wssv019 and other genes, and a small 1.3-kb monocistronic mRNA encoding only wssv019. (see Chen, L. L., et al., Natural and experimental infection of white spot syndrome virus (WSSV) in benthic larvae of mud crab Scylla serrata. Dis Aquat Org, 2000. 40(2): p. 157-61.) This suggests that the expression of wssv019 might be regulated by a cap-independent mechanism for protein synthesis.
On the other hand, multigene expression systems are mostly applied in research fields such as gene therapy, cancer treatment, vaccine production, or co-expression of different subunit proteins to form protein complexes, etc. Therefore, if a successful development for the multigene expression system which can easily be operated, obtain study results for a short time, and can be widely applied in various cell lines and in vivo multigene expression vectors is made, use value and market potential for the multigene expression system can thus increase significantly. In eukaryotes, co-expression strategies for expressing two or more proteins at the same time can be roughly divided into the following types, including: using two promoters, bidirectional promoter, fusion protein, IRES and 2A-peptides, or the like.
As for dual-promoter, two promoters were constructed in the same vectors and transferred into cells via transfection, but its drawback is that two promoters would mutually interfere and compete transcription materials such as transcription factors and RNA polymerases, leading to the decrease or inactivation in one of both. These results caused inconsistent expression levels between two expressed proteins and failed to achieve the expected effect. As for the use of fusion proteins, it was in doubt about whether proteins could fold accurately and whether their activities could be affected.
Previous published viral IRES elements had a length of about hundreds of nucleotides, such that multigene expression vectors had a limited cloning capacity when constructed. Also, most viral IRES had a lower activity as compared with cap-dependent translation mechanism, and thus caused inconsistent protein expression levels of co-expression. This drawback allowed rare IRES elements to be practically applied in multigene expression systems, such as IRES of Encephalomyocarditis Virus (EMCV) or hepatitis C virus (HCV), which was widely applied in mammalian cells now.
In previous studies, multigene expression for insect cells mostly used above-mentioned co-expression strategies and baculovirus expression systems. Although baculovirus expression systems had a high expression efficiency, it consumed longer time during screening and virus titer evaluation, which took about 1 to 2 weeks to screen recombinant baculoviruses having high vieulence for further researches. Thus, the development for new multigene expression vectors is still needed to accelerate the research progress.
In the light of these defects in prior art, one object of the present invention is to provide an expression vector comprising a novel internal ribosome entry site (IRES) element, which can be easily operated in insect cells, shrimp cells or Crustacean cells and has an excellent expression efficiency.
Another object provided herein is a multiple expression gene system, which can shorten the experimental process and reduce studying costs effectively as compared with the conventional baculovirus expression systems.
Thus, the present invention provides an expression vector comprising an internal ribosome entry site (IRES) element, which comprises a sequence of SEQ ID NO: 1.
In some embodiments, the sequence can be an IRES element from a gene icp35 of White spot syndrome virus (WSSV).
In some embodiments, the expression vector can be a dual-gene expression vector.
In some embodiments, the dual-gene expression vector can be a dual-luciferase reporter vector. For example, in a specific embodiment, the dual-luciferase reporter vector can comprise a sequence of SEQ ID NO: 2, but not limited to this.
In other embodiments, the dual-gene expression vector can be a dual-fluorescence reporter vector. For example, in a specific embodiment, the dual-fluorescence reporter vector can comprise a sequence of SEQ ID NO: 3, but not limited to this.
A multigene expression system is also provided, which can comprise any expression vector according to the above-mentioned embodiments.
In some embodiments, the multigene expression system refers to an insect cell expression system or a Crustacean cell expression system. For example, in a specific embodiment, the multigene expression system can be a Spodoptera frugiperda (Sf9) expression system.
Embodiments according to the inventive concept of the present invention are provided such that those skilled in the art can more completely understand the present invention. It should also be understood that the following embodiments are not limited by any of the details of the foregoing description, unless otherwise specified, but rather should be construed broadly within its spirit and scope as defined in the appended claims.
The accompanying drawings are included to provide a further understanding of the present invention, and are incorporated in and constitute a part of this specification. The drawings illustrate exemplary embodiments of the present invention and, together with the description, serve to explain principles of the present invention. In the drawings:
The accompanying drawings are included to provide a further understanding of the present invention, and are incorporated in and constitute a part of this specification. The drawings illustrate exemplary embodiments of the present invention and, together with the description, serve to explain principles of the present invention. In the drawings:
A shows Western blot analysis results for detected positive controls of purified total WSSV virions: the envelope protein VP28 and the nucleocapsid protein VP51C.
B shows the results for time course analysis of WSSV ICP35 (WSSV019) expression.
A shows RT-PCR analysis results for each experimental group at each time point, wherein: the expression levels of icp35 and ie1 in pleopod was detected at 24 and 48 hpi, and in stomach at 96 hpi were monitored. EF-1α was used as an internal control.
B shows results of cumulative mortalities which were observed and recorded every 12 h.
C shows results of cumulative mortality for the mock-infected PBS controls.
D shows results for time course study of WSSV loads after dsRNA silencing. WSSV copy number and shrimp genomic DNA were measured using the IQ REAL WSSV quantitative system (GeneReach Biotechnology Corp.). (**P<0.005 by one-way analysis of variance [ANOVA]).
A shows schematic representation of the WSSV gene clusters wssv023/wssv019 (icp35), vp31/vp39b/vp11, and vp60b/wssv478/wssv479/vp28. The 5′ UTR fragment of icp35(-468/-1) or the IRES elements of VP39B or VP28 were inserted into the intercistronic region of the bicistronic reporter plasmid ie1/pRL-FL;
B shows results for the ratio of firefly to Renilla luciferase. Bicistronic plasmids of icp35(-468/-1) or IRES123-919 or vp28-IRES were transfected into Sf9 cells, and Renilla and firefly luciferase activities were measured at 48 h post-transfection. The ratio of firefly to Renilla luciferase was used as an indicator of IRES activity. The FL/RL ratio for icp35(-468/-1) was set to 100%. Three independent transfection assays were performed, and the mean±SD was calculated.
A shows RNA MFold prediction of the secondary structure of icp35 5′ UTR at 25° C.;
B shows schematic diagram of the bicistronic vector pRL-FL with the wssv ie1 promoter and the 5′ UTR structure of icp35. Various 5′- and 3′-end deleted fragments in the 5′ UTR of icp35 were subcloned into the intercistronic region between the Renilla luciferase and firefly luciferase;
C shows that Sf9 cells were transiently transfected with the bicistronic plasmids, and at 48 h post-transfection Renilla and firefly luciferase activities were measured. The ratios of firefly to Renilla luciferase for each bicistronic plasmid are shown, and the highest IRES activity [icp35(-198/-1)] was set to 100%. The actual ratio was 1.32±0.20. Three independent transfection assays were performed and the mean±SD was calculated (*P<0.05, ****P<0.00005 by one-way analysis of variance [ANOVA]);
D and
A shows schematics of bicistronic plasmids containing icp35(-291/-1) and icp35(-198/-1) fragments with the WSSV ie1 promoter (+P) or without the WSSV ie1 promoter (−P);
B shows relative luciferase activities of firefly (shown as gray bar) and Renilla (shown as dark gray bar). Plasmids were transfected into Sf9 cells, and 48 h later FL (gray bar) and RL (dark gray bar) activities were measured. FL and RL data for the control plasmid icp35(-291/-1)(+P) were set to 100%. Three independent transfection assays were performed and the mean±SD was calculated (****P<0.00005, Student's t-test).
A shows schematic diagram of the ie1/pRL-FL-based bicistronic construct and the two primers used for reverse transcription (RT)-PCR. The primer set P1/P2 represented ie1P(+3/+31)-F/pRL-FL-R1241 (see Table 1);
B shows RT-PCR results for total RNA extracted from Sf9 cells transfected (T) or untransfected (UT) with the ie1/pRL-icp35(-198/-1)-FL bicistronic plasmid. In the negative RT(−) controls, RTase was omitted to verify that the RT-PCR products were specifically amplified from RNA and not from any contaminating plasmid DNA. The DNA lane denoted the PCR product from the ie1/pRL-icp35(-198/-1)-FL plasmid and was used as a positive size control. Lane M shows 1 kb DNA ladder I markers (LAMDA Biotech Inc.), and lane N was the negative control (no cDNA);
C shows results for mRNA expression levels of Renilla luciferase (RL) and firefly luciferase, which was measured at 48h post-transfection, and total RNA was isolated from ie1/pRL-FL-transfected Sf9 cells and from Sf9 cells transfected with the ie1/pRL-icp35(-198/-1)-FL bicistronic plasmid. Quantitative RT-PCR was performed to analyze the mRNA expression levels of Renilla luciferase (RL) and firefly luciferase was calculated as 2−[Ct(FL)−Ct(RL)] and the ratio of firefly luciferase to Renilla luciferase of ie1/pRL-FL was set to 1. The data represented the mean±SD from three independent transfection experiments.
A shows results of Northern blot analysis, wherein the arrow indicated the bicistronic RNA transcript and the open arrow indicated the position of the 18s rRNA;
A shows schematic diagrams of bicistronic plasmids containing icp35(-198/-1) with or without a stable stem-loop (SL) upstream of the RL ORF;
A shows schematic diagram of the bicistronic plasmid containing the icp35(-198/-1) IRES element;
B shows relative luciferase activities of firefly (FL) and Renilla (RL). The bicistronic plasmid ie1/pRL-icp35(-198/-1)-FL was transiently transfected to Sf9 cells, and 24 h later, the cells were treated with tunicamycin (2.5 μg/ml) or DMSO (100%). Luciferase activities were measured at 15 h post-transfection. Luciferase activities are expressed relative to the levels in untreated (control) cells, which were set to 100%.
C shows results for Western blot analysis in which the level of phosphorylated eIF2α in the Sf9 cells at 15 h post-transfection was detected by Western blot analysis using the antibodies for phospho-eIF2α and total eIF2α. (*P<0.05, ***P<0.0005 by one-way analysis of variance [ANOVA]).
A shows schematic diagram of the IRES-containing bicistronic reporter plasmid;
B shows relative luciferase activities of firefly (FL) and Renilla (RL). Bicistronic plasmids were transfected into Sf9 cells with or without QC. Luciferase activities were measured at 48 hour post-transfection. The Figure shows changes relative to the corresponding untreated mock controls (0 μM), which was set to 100%. Three independent transfection assays were performed and the mean±SD was calculated (*P<0.05, **P<0.005 by one-way analysis of variance [ANOVA]);
C shows cumulative mortality of shrimp. Shrimp (L. vannamei; 4 g mean weight; 14 shrimp per group) were first injected with 50 n1 WSSV inoculum or PBS, and then immediately injected with QC (5 μg/g) or PBS. The cumulative mortality of each group was recorded every 12 h, with dead shrimp being removed from the tank as soon as possible. Data were analyzed using a Kaplan-Meier log rank×2 test (Graphpad). Asterisks indicated significant cumulative differences between groups (*P<0.05, ***P<0.0005);
D shows the WSSV infection level in the WSSV-challenged groups. The swimming legs from some of the dead shrimp in the WSSV-challenged groups were tested with an IQ2000 kit to determine the WSSV infection level. The hpi above each lane indicated time of collection. The bands of size 296, 550, and 910 bp represen WSSV genes, and the band of size 848 bp represents shrimp genomic DNA. Samples for the marker bands (M) of size 333, 666 and 840 bp, and the positive infection controls (20, 200, and 2000 copies/reaction; right hand panel) were provided by the kit. The positive infection controls respectively represent a light (+), moderate (++), and heavy (+++) infection. Lane N is the negative control;
E shows the WSSV infection level in two other replicate groups (WSSV+PBS and WSSV+QC). Two other replicate groups (WSSV+PBS and WSSV+QC) were used to collect live shrimp samples at 1, 2, 6 and 9 days post-injection (dpi). WSSV infection levels were determined using the IQ2000 kit as described above.
A shows the expression levels of RPS10, RPS19 in Sf9 cells at 48h post-transfection which were monitored by RT-PCR analysis. EF-1α was used as an internal control;
B shows relative luciferase activities of firefly (FL) and Renilla (RL). Sf9 cells were co-transfected with 0.3 μg of bicistronic vector plus 0.1 μg dsRNA using Effectene (Qiagen) according to the manufacturer's protocol. At 72 h post-transfection the cells were assayed using dual luciferase reagents (Promega) following the manufacturer's protocol. (***P<0.0005 by one-way analysis of variance [ANOVA] with three replicates.)
Preferred embodiments of the present invention will be described below in more detail with reference to the accompanying drawings. The present invention may, however, be embodied in different forms and should not be constructed as limited to the embodiments set forth herein. Other objectives, advantages, and novel features of the invention will become more apparent from the following detailed description when taken in conjunction with the accompanying drawings.
First, one embodiments of the present invention provided an expression vector comprising an internal ribosome entry site (IRES) element having a sequence of SEQ ID NO: 1. The IRES element was from a gene icp35 of White spot syndrome virus (WSSV) encoding a highly expressed non-structural protein, which could be transcripted to a polycistronic mRNA with other genes. The expression vector was designed respectively to form a dual-luciferase reporter vector (ie1/pRL-icp35 IRES-FL, including a sequence of SEQ ID NO: 2) and a dual-fluorescence reporter vector (ie1/pIZV5-DR-icp35 IRES-GFP, including a sequence of SEQ ID NO: 3) for the following examples.
A. Preparation and Analysis for the Dual-Luciferase Reporter Vector (Ie1/Pr1-Icp 35 Ires-F1, Including a Sequence of SEQ ID NO: 2)
Materials
Experimental Animals
The Pacific white shrimp Litopenaeus vannamei used in these examples were all WSSV-free, as confirmed by using an IQ2000TM WSSV diagnostic kit (GeneReach Biotechnology Corp.). The shrimp (mean weight 4 g) were obtained from a culturefarm in Tung Kang, Taiwan, or from the Aquatic Animal Center in National Taiwan Ocean University, and were acclimatized in the laboratory in water tanks with a salinity of 30±1 ppt at 26±1° C. for at least 3-5 days before the experiments.
The virus used in this example was the WSSV Taiwan isolate WSSV-TW (GenBank accession no. AF440570), which originated from a batch of WSSV-infected Penaeus monodon shrimp collected in Taiwan in 1994. The preparation of the WSSV inoculums was followed the methods described by Tsai et al. (see Tsai, M. F., G. H. Kou, H. C. Liu, K. F. Liu, C. F. Chang, S. E. Peng, H. C. Hsu, C. H. Wang, and C. F. Lo. 1999. Long-term presence of white spot syndrome virus (WSSV) in a cultivated shrimp population without disease outbreaks. Dis Aquat Organ 38:107-114, which was incorporated herein by reference.)
Briefly, a 0.5 g frozen sample of infected P. monodon carapace was ground together with 4.5 ml of 0.9% NaCl until the mixture became homogenized. After centrifugation at 1,000×g for 10 minutes at 4° C., the supernatant was filtered with a 0.45 μm filter (Millipore), and 100 μl of 100× dilution virus was used to infect adult specific-pathogen-free (SPF) Litopenaeus vannamei (mean weight 35 g). Collected hemolymph from moribund shrimp was centrifuged at 1,000×g for 10 minutes at 4° C., and the supernatant was diluted 5× with PBS. This suspension was stored at −80° C. and used as a viral stock.
Healthy Procambarus clarkii crayfish were used for this procedure. The WSSV-TW (GeneBank accession no. AF440570) viral stock was diluted with PBS (1:2.5 dilution in PBS; 5 μl/g of body weight) and injected intramuscularly into healthy crayfish. After 4 to 6 days, hemolymph was extracted from moribund crayfish, and virions were purified as described previously. (see Genomic and proteomic analysis of thirty-nine structural proteins of shrimp white spot syndrome virus. Journal of Virology 78:11360-11370, which was incorporated herein by reference.)
Shrimp stomachs from healthy (0 hour post-injection; hpi) and WSSV-infected shrimp (at 12, 24, 36, 48, 60, and 72 hpi) were ground in liquid nitrogen, and lysed with ⅓× ice-cold PBS with complete protease inhibitor cocktail (Roche). Supernatants were obtained after 12000×g centrifugation at 4° C. for 15 min, and concentrations of total protein were quantified using the Bio-Rad Bradford Protein Assay. Total protein (18 μg) of each extract was separated on 15% SDS-PAGE, and the gel was transferred onto polyvinyl difluoride (PVDF) membrane (PerkinElmer) using the wet transfer method (Hoefer apparatus).
For the time course expression experiments, the blots were blocked with 5% non-fat milk in TBS buffer for 16 h overnight at 4° C. Next, the blots were probed with primary antiserum against ICP35, VP28, VP51C or β-tubulin diluted 1:10,000 in 5% non-fat milk in TBST (0.05% of Tween 20 in TBS buffer) for 1 h at room temperature (RT), and this was followed by washing three times for 10 min at RT with TB ST. The blots were then probed with anti-Rabbit IgG secondary antibody (Santa Cruz Biotechnology) diluted 1:10,000 in 5% non-fat milk in TBST for 1 h at RT, and washed three times for 10 min at RT with TBST. The blots were incubated with chemiluminescent substrate using Western Lightning® Plus-ECL reagents (PerkinElmer) and exposed to film for signal detection.
The genes for wssv icp35, EGFP, Spodoptera frugiperda RPS10, and RPS19 were amplified by the primer sets icp35-F1/icp35-R687, EGFP-F/EGFP-R, Sf-RPS10-F/Sf-RPS10-R, and Sf-RPS19-F/Sf-RPS19-R respectively (Table 1) with the following profile: 94° C. for 3 minutes; 30 cycles at 94° C. for 30 seconds, 55° C. for 30 seconds and 72° C. for 30 seconds (30 cycles), and then a final extension at 72° C. for 20 minutes. A T7 promoter was attached to the 5′-end of these purified PCR products by amplification with, the primer sets icp35-dsRNA-T7-F1/icp35-R687, EGFP-T7-F/EGFP-R, Sf-RPS10-dsRNA-T7-F/Sf-RPS10-121 R and Sf-RPS19-dsRNA-T7-F/Sf-RPS19-R respectively (Table 1). The T7 promoter was likewise attached to the 3′-end with the respective primer sets icp35-F1/icp35-dsRNA-T7-R687, EGFP-F/EGFP-T7-R, Sf-RPS10-F/Sf-RPS10-dsRNA-T7-R, and Sf-RPS19-F/Sf-RPS19-dsRNA-T7-R (Table 1).
1 μg of each PCR product having 5′-end and 3′-end T7 promoter was used as DNA template, and then used to perform in vitro transcription by RiboMAX™ Large Scale RNA Production System-T7 (Promega) according to the manufacturer's instructions. The resulting sense and antisense ssRNAs generated from the two kinds of DNA template were mixed together equally, heated at 70° C. for 10 minutes, and incubated at RT for at least 20 minutes to anneal the two complementary ssRNAs into dsRNA. The DNA templates were removed by adding DNase I (Invitrogen), and phenol/chloroform extraction and ethanol precipitation were used to obtain the dsRNA products. The concentration of the dsRNA products was estimated using a NanoDrop® (ND-1000) spectrophotometer.
For the dsRNAi experiment, shrimps were randomly divided into two sets, and then each set was further divided into three experimental groups. Two experimental groups in one set of 10-12 shrimp were injected with icp35 dsRNA or EGFP dsRNA at a concentration of 4 μg (1 μg/g body weight) in 50 μl of PBS, and another group with 50 μl of PBS only as control. Two days later, these groups were injected with 50 μl of WSSV inoculum (50× dilution of the virus stock), while three experimental groups in another set was injected with 50 μl of PBS as a negative control. At 0, 24, 48, 96 hpi, pleopod samples were excised from 3 randomly selected shrimp in each group. One pleopod from each shrimp was subjected to RT-PCR to evaluate the gene expression level after dsRNAi-mediated gene knockdown, and a second pleopod was subjected to real-time PCR to quantify the WSSV viral load. Both of these procedures are described below. In addition, a second replicate was made of this entire experiment. In this replication, instead of taking samples from the shrimp, the mortality was observed and recorded in each group every 12 h.
Total RNA (1 μg) isolated from the pleopod samples by Trizol reagent (Invitrogen) was pretreated with DNase I (Invitrogen) and then reverse transcribed by SuperScript III Reverse Transcriptase (Invitrogen). RT-PCR was performed with primer sets for icp35, ie1, and EF-1α (Table 1) using the following profile: 94° C. for 3 minutes; 30 cycles at 94° C. for 30 seconds, 55° C. for 30 seconds, and 72° C. for 30 seconds (35 cycle); a final extension at 72° C. for 20 minutes.
The IQ REAL WSSV quantitative system (GeneReach Biotechnology Corp.) was used to absolutely quantify the WSSV loads in the dsRNA-mediated gene silencing experiments. From the pleopod samples taken at 48 and 96 hpi, shrimp genomic DNA was isolated using the silica-based resin supplied with the commercial kit and quantified according to the manufacturer's instructions using a TaqMan assay strategy. Reactions were performed on an ABI PRISM 7300. The WSSV load was calculated by the relative ratio of copy number of WSSV genomic DNA to shrimp genomic DNA. WSSV load data were presented as mean±SD (standard deviation) for 3 shrimp from each group, and one-way analysis of variance (ANOVA) tests were used to check for significant difference with the P value<0.005.
Schematics for all of the plasmids used in all Examples can be found in the Results section. The backbone plasmid used for the dual luciferase assays was constructed as described previously (see Kang, S. T., J. H. Leu, H. C. Wang, L. L. Chen, G. H. Kou, and C. F. Lo. 2009. Polycistronic mRNAs and internal ribosome entry site elements (IRES) are widely used by white spot syndrome virus (WSSV) structural protein genes. Virology 387:353-363, which was incorporated herein by reference). Basically, the firefly luciferase from pGL3 plasmid (Promega) was inserted into the pRL-null plasmid (Promega) to give the dual luciferase plasmid T7/pRL-FL (see Bieleski, L., and S. J. Talbot. 2001. Kaposi's sarcoma-associated herpesvirus vCyclin open reading frame contains an internal ribosome entry site. J Virol 75:1864-1869, which was incorporated herein by reference), but before transient DNA transfection in Spodoptera frugiperda Sf9 cells, the T7 promoter was replaced by the WSSV ie1 promoter (−94/+52). This substitution was achieved by PCR amplification with the primers ie1-promoter-SacI-F and ie1-promoter-NheI-R (Table 1) to clone the ie1 promoter into the SacI-NheI sites of T7/pRL-FL to produce the construct ie1/pRL-FL. The putative IRES elements in the 5′ UTR of icp35 and its antisense sequence (as a negative control) were PCR amplified using KOD+Taq polymerase (TOYOBO) with the primer sets listed in Table 1 to clone each respective element into the reporter construct ie1/pRL-FL to generate the corresponding plasmids. The two previously published WSSV IRES elements, IRES123-919 (i.e. vp39b-IRES) and vp28-IRES, were also cloned into the reporter plasmid and used as controls for comparison. Primer sets were listed in Table 1. The empty vector ie1/pRL-FL was used as a negative control.
For the promoterless assays, the ie1 promoters of ie1/pRL-icp35(-291/-1)-FL and ie1/pRL-icp35(-198/-1)-FL were removed by digestion with SacI and NheI followed by Klenow treatment and religation to generate pRL-icp35(-291/-1)-FL(−P) and pRL-icp35(-198/-1)-FL(−P), respectively.
To rule out the possibility of ribosomal read-through, a 28-bp stable stem-loop structure with a free energy of −62 kcal/mol (5′-GCTAGCGGTACGGCAGTGCCGTACGACGAATTCGT CGTACGGCACTGCCGTACCGCTAGC-3′, SEQ ID NO: 4) was introduced upstream of the Renilla luciferase ORF using the NheI site as described previously (see Bieleski, L., and S. J. Talbot. 2001. Kaposi's sarcoma-associated herpesvirus vCyclin open reading frame contains an internal ribosome entry site. J Virol 75:1864-1869, which was incorporated herein by reference.) to generate the plasmid ie1/SL-pRL-icp35(-198/-1)-FL.
The RNA secondary structure of the putative IRES element of icp35(-468/-1) was predicted by the RNA Folding Form software on the mfold Web Server (http://mfold.rna.albany.edu/?q=mfold/RNA-Folding-Form2.3). The folding temperature was set to 27 since the optimal temperature for WSSV infection was 27.
For the IRES activity assays, Sf9 cells were seeded in 24-well plates (1.2×105 cells/well) and grown in Sf-900 II medium (Invitrogen) supplemented with 10% Fetal Bovine Serum (FBS; Gibco®) overnight at 27° C. Plasmid DNAs (0.4 μg of plasmid DNA per well) were transfected into the cells using Effectene transfection reagent (Qiagen) according to the manufacturer's recommendations. Cells were harvested 48 h after transfection and analyzed for dual luciferase activities using the Dual-Luciferase® Reporter Assay System (Promega). Briefly, transfected cells were washed twice with 1×PBS, lysed with 100 ul of passive lysis buffer, and then incubated for 15 min at RT on an orbital shaker with gentle shaking. Luciferase activities in the cell lysates (10 μl) were measured with a Labsystems benchtop luminometer. The ratio of firefly luciferase activity to Renilla luciferase activity was used as an index of IRES activity. Transfection assays were performed in triplicate with three independent experiments. Data are presented as mean±SD (standard derivation) from the three independent triplicate experiments.
1. Abnormal Splicing Test
To check whether abnormal splicing occurred during transfection, RT-PCR was performed with the primer set P1/P2 (Table 1) using the following profile: 94° C. for 3 minutes; 30 cycles at 94° C. for 30 seconds, 60° C. for 30 seconds, and 72° C. for 2 minutes (32 cycle or 40 cycle); a final extension at 72° C. for 20 minutes. The PCR products were then cloned into the vector pGEM-T (Promega) and sequenced. In addition, a quantitative real-time PCR was performed using the KAPA SYBR® FAST Universal Kit (Kapa Biosystems) on an ABI Prism 7300 sequence detection system (Applied Biosystems) according to the manufacturer's instructions. The mRNA expression levels of the Renilla luciferase and firefly luciferase genes were detected with the primer sets
Rluc-qPCR-F/Rluc-qPCR-R (Table 1) and Fluc-qPCR-F/Fluc-qPCR-R (Table 1), respectively. The mRNA expression level of Sf9EF-1α was used as an internal control (Table 1).
2. IRES Activity Experiment by the Inducement of Endoplasmic Reticulum (ER) Stress with Tunicamycin
For the tunicamycin experiments, Sf9 cells were transfected with the bicistronic plasmid ie1/pRL-icp35(-198/-1)-FL (1.0 μg of plasmid DNA per well of a 24-well plate) using the SuperFect transfection
reagent (Qiagen) according to the manufacturer's recommendations, and 2.5 μg/ml final concentration of tunicamycin (Sigma) or DMSO control (100%) was added at 24 h post-transfection.
After 15 h of treatment, cells were harvested. One replicate of the harvested cells was subjected to a dual luciferase assay as described above. The other replicate was subjected to Western blotting to monitor the level of the phosphorylated proteins. For this assay, 2× sample buffer was added to the cells and the mixture was boiled for 10 minutes. An aliquot (15 μl) was separated on 15% SDS-PAGE and transferred onto the PVDF membrane as described above. The blots were then blocked with 5% BSA in TBST (0.01% of Tween 20 in TBS buffer) for 1 h at RT, and probed with the commercial primary antibodies Phospho-eIF2α (Ser51) (Cell Signaling) or EIF2S1 (Abeam). The antibodies were diluted 1:1,000 in 2.5% non-fat milk in TBST and applied for 16 h at 4° C. This step was followed by washing three times for 10 min at RT with TBST. The blots were then probed with anti-Rabbit or anti-Mouse IgG secondary antibody (Santa Cruz Biotechnology) diluted 1:10,000 in 2.5% BSA in TBST for 1 hr at RT, and washed three times for 10 min at RT with TBST. Western Lightning® Plus-ECL reagents were used for visualization as described above.
3. Quinacrine (QC) Effect on Transcription Regulated by Icp35 IRES
For the quinacrine (QC) experiment, Sf9 cells were first transfected with the bicistronic plasmid ie1/pRL-icp35(-198/-1)-FL (0.4 μg of plasmid DNA per well of a 24-well plate) using the Effectene transfection reagent (Qiagen) according to the manufacturer's recommendations, and then quinacrine dihydrochioride (Sigma-Aldrich; dissolved to a 10 mM stock solution in ddH2O, and filtered through a 0.22 μm filter) was added directly to the well at a final concentration of 25 μM or 30 μM. Renilla and firefly luciferase activities were measured after 48 h of treatment.
Transfection assays were performed in triplicate with three independent experiments. Data are presented as mean±SD (standard derivation) from the three independent triplicate experiments.
4. The Effect of RPS10 and RPS19 Genes on IRES Activity by Double-Stranded RNA Interference (dsRNAi)
For the dsRNAi experiment, Sf9 cells were co-transfected with the bicistronic plasmid ie1/pRL-icp35(-198/-1)-FL (0.3 μg of plasmid DNA per well of a 24-well plate) and RPS10 or RPS19 dsRNA (0.1 μg) using the Effectene transfection reagent (Qiagen) according to the manufacturer's recommendations. At 72 h post-transfection, cells were harvested and the dual luciferase activities were measured as described above.
To generate an icp35 IRES-based bicistronic baculovirus transfer vector, the icp35(-198/-1) fragment was amplified with the primer set icp35(-198)-BamHI/icp35(-1)-XhoI (Table 1) and cloned into the pBacPAK9 transfer vector (Clonetech) using the BamHI and XhoI sites. Sf9 cells were co-transfected with the IRES-based transfer vector and a Bsu36 I-digested BacPAK6 viral DNA to produce an AcMNPV-RL/icp35 IRES/FL recombinant virus according to the manufacturer's instructions (Clonetech).
Sf9 cells were infected by AcMNPV-RL/icp35 IRES/FL recombinant viruses for 4-5 days. The cells were then harvested and Trizol reagent (Invitrogen) was used to extract total RNA. The total RNA was electrophoresed on a 1% formaldehyde gel, transferred to a positively charged membrane (Roche Applied Science), and detected by digoxigenin (DIG)-labeled RNA probes for firefly luciferase as described below. For a negative control, the same protocols were applied to uninfected Sf9 cells.
The DIG-labeled RNA probe was created as described previously (see Kang, S. T., J. H. Leu, H. C. Wang, L. L. Chen, G. H. Kou, and C. F. Lo. 2009. Polycistronic mRNAs and internal ribosome entry site elements (IRES) are widely used by white spot syndrome virus (WSSV) structural protein genes. Virology 387:353-363, which was incorporated herein by reference). Briefly, a partial fragment of firefly luciferase was amplified with the primer set FL-F/FL-R (Table 1), ligated with T7 adaptor (Table 1) using T4 ligase (Promega), and then subjected to PCR amplification with a primer set FL-F/T7 adaptor primer 1. The resulting DNA template (200 ng) was then reacted in vitro at 37° C. for 2 h with 5× DIG-RNA labeling mix (Roche Applied Science) and T7 RNA polymerase (Promega). The reaction mixture was then treated with DNase I (RNase-free; Invitrogen) to remove the DNA template and leave only the DIG-labeled RNA probe. For hybridization with the target RNA, and the DIG-labeled RNA probes were denatured by heating at 95° C. for 5 min and cooling on ice for 1 min, and then mixed with preheated (68° C.) DIG Easy Hyb (Roche Applied Science). Hybridization then proceeded for 16 h at 68° C. (DIG System Users Guide from Roche Applied Science).
After hybridization, the membrane was incubated with Anti-Digoxigenin-AP Fab fragment (1:20,000 dilution) (Roche Applied Science), detected with CDP-Star (ready-to-use; Roche Applied Science), and overexposed to X-ray film at RT overnight.
To determine the optimal QC dosage for the challenge experiment, four groups of shrimp (20 shrimp per group) were injected with different doses (0.5 μ/g, 2.5 μg/g, or 5 μg/g body weight) of QC (10 mg/mL stock solution in PBS) or with PBS (negative control). The mortality of each group was observed and recorded every 12 h.
All but one of the shrimp (in the 0.5 μg/g group) survived for 2 weeks, and the highest dosage of 5 μg/g was therefore used in the following WSSV challenge experiment. For the WSSV challenge experiment, groups of shrimp (14 shrimp per group) were injected either with WSSV (50 μl of 100× dilution of the virus stock) or with PBS as a negative control. These first injections were then immediately followed by a second injection of 50 μl of either QC (5 μg/g) or PBS. Pleopods were excised from 3 randomly selected shrimp from each group at 0, 1, 2, 6 and 9 days post-injection (dpi) and tested for WSSV infection as described below. A second replicate of this entire experiment was performed at the same time. No pleopods were collected from the shrimp in this second replicate. Instead, mortality was observed and recorded every 12 h.
A commercial WSSV diagnostic kit (IQ2000 WSSV Detection and Prevention System, GeneReach Biotechnology Corp.) was used to determine WSSV infection levels. Briefly, genomic DNA extracted from the shrimp pleopod samples was isolated using the DTAB/CTAB extraction procedure according to the manufacturer's instructions. Next, competitive nested PCR analysis was performed using the extracted DNA samples. The PCR products were analyzed by electrophoresis with a 2% agarose gel. WSSV infection levels were determined according to the pattern of bands indicated in the kit's instructions.
Results
1. WSSV ICP35 was a Highly Expressed Non-Structural Protein
wssv019 was originally thought to encode a structural protein (see Chen, L. L., J. H. Leu, C. J. Huang, C. M. Chou, S. M. Chen, C. H. Wang, C. F. Lo, and G. H. Kou. 2002. Identification of a nucleocapsid protein (VP35) gene of shrimp white spot syndrome virus and characterization of the motif important for targeting VP35 to the nuclei of transfected insect cells. Virology, which was incorporated herein by reference), and to confirm this, Western blot analysis of purified WSSV virions was performed using the anti-WSSV019 antibody. Anti-VP28 (envelope protein)
and anti-VP5 1C (nucleocapsid protein) antibodies were used as positive controls. However, since no signal was detected from purified WSSV virions by the anti-WSSV019 antibody (
A time course study of ICP35 protein levels in shrimp pleopod was performed using Western blot analysis.
2. dsRNA Silencing of Icp35 Reduced WSSV Replication in Shrimp
To investigate the importance of ICP35 to WSSV pathogenesis, dsRNA gene silencing was used to knock down the expression of icp35 during WSSV infection. First, RT-PCR analysis was used to confirm the knockdown efficiency of icp35 dsRNA at 24, 48 and 96 hpi in WSSV-challenged shrimp (
The expression of the housekeeping gene EF-1α was not affected by icp35 or EGFP dsRNA treatment at 24, 48 or 96 hpi (
Real-time PCR analysis by the IQ REAL WSSV quantitative system (GeneReach Biotechnology Corp.) showed that the WSSV loads of the icp35 and EGFP dsRNA-treated groups were significantly lower than the PBS group (P<0.005) at 48 hpi (
3. The 5′ UTR of Icp35 mRNA Mediated Internal Initiation of Translation
To determine if an IRES element was located upstream of icp35, a 468 by region (-468/-1) between multiple repeats TTTTTCTCC and the icp35 translational start codon (ATG, which A is in +1 position) was cloned into the bicistronic vector ie1/pRL-FL with the WSSV ie1(-94/+52) promoter (
In this example, the first cistron (i.e. Renilla) was translated by a cap-dependent translation mechanism, whereas translation of the second cistron (i.e. firefly) would suggest that the intercistronic region contains an IRES element. The previously reported IRES elements associated with VP39B and VP28 were used for comparison, while the empty ie1/pRL-FL bicistronic vector was used as a negative control. These results suggested that the 5′ UTR of icp35 contained an active IRES element (
Next step in Examples was to more precisely locate the active IRES element within the icp35 5′ UTR. Based on the predicted RNA secondary structure of the putative IRES region of the 5′ UTR of icp35(-468/-1) (
Using the same strategy, further refinement of the icp35(-198/-1) sequence showed that the icp35(-171/-38) fragment (
4. The icp35 IRES Element Did not Contain a Cryptic Promoter or Splice Sites
To exclude the possibility that stem-loops VII and VIII were harboring a cryptic promoter that was driving the expression of FL, a promoterless assay was done using a bicistronic plasmid with the wssv ie1 promoter removed (
An alternative explanation of the observed
FL activity of ie1/pRL-icp35(-198/-1)-FL(+P) (
As shown in the oversaturated right-hand panel of
These conclusions were further supported by using quantitative real-time PCR to accurately determine the gene expression levels of RL and FL. As shown in
produced by the ie1/pRL-icp35(-198/-1)-FL plasmid was only 80% of that produced by ie1/pRL-FL. A Student's t-test found no significant difference between these two ratios, and this result therefore confirmed that during transfection by the ie1/pRL-icp35(-198/-1)-FL plasmid, no cryptic promoter was acting to increase the mRNA level of FL and no abnormal splicing event was occurring to reduce the mRNA level of RL.
The integrity of the bicistronic mRNA was also confirmed by Northern blot analysis with a DIG-labeled RNA probes for firefly luciferase (
5. Ribosomal Read-Through Did not Responsible for Icp35 IRES Activity
To rule out the possibility that the FL activity was caused by read-through of the RL termination codon, a stable stem-loop was inserted upstream of the RL ORF (
RL activity of the
ie1/SL-pRL-icp35(-198/-1)-FL plasmid, while the FL activity was not affected (
6. Initiation of Translation on the Icp35 IRES was eIF2-Dependent
To determine whether eIF2 is required for icp35 IRES-mediated translation, the translational machinery was shut down by inducing phosphorylation of eIF2a with the ER stress chemical reagent tunicamycin. It was found that the ER stress induced by tunicamycin inhibited both cap-dependent and icp35 IRES-dependent translation (
7. Quinacrine (QC) Reduced Icp35 IRES Activity and has an Anti-WSSV Effect
QC is an intercalating drug that inhibits DNA replication and RNA transcription. Nucleic acid intercalating drugs were shown to inhibit IRES-mediated translation more than cap-dependent translation at low concentrations (10 to 20 μM). It was subsequently shown that QC inhibited the IRES activities of Encephalomyocarditis Virus (EMCV), hepatitis C virus (HCV) and poliovirus in a dose-dependent manner by interacting with the highly complex secondary structures of their IRES regions (see Gasparian, A. V., N. Neznanov, S. Jha, O. Galkin, J. J. Moran, A. V. Gudkov, K. V. Gurova, and A. A. Komar. 2010. Inhibition of encephalomyocarditis virus and poliovirus replication by quinacrine: implications for the design and discovery of novel antiviral drugs. J Virol 84:9390-9397, which was incorporated by reference). The same study also found that the cellular p53 IRES, which has a much less complex secondary structure, is not sensitive to QC. Since the IRES region of icp35 was predicted to be complex (
Having shown that QC suppressed icp35 IRES activity in vitro, the effect of this drug on WSSV-challenged shrimp in vivo was next investigated.
Nested competitive PCR analysis detected a high virus load (heavy infection) in all but one of the shrimp that died (
8. RPS10 Knockdown Selectively Inhibited the Icp35 IRES Activity
Knockdown of certain ribosomal proteins could have a differential effect on cap-dependent versus IRES-mediated
translation. Spodoptera frugierda RPS10 and RPS19 genes were selected for this experiment based on applicant's unpublished proteomics data and a literature review for other viral IRES element (see Babaylova, E., D. Graifer, A. Malygin, J. Stahl, I. Shatsky, and G. Karpova. 2009. Positioning of subdomain IIId and apical loop of domain II of the hepatitis C IRES on the human 40S ribosome. Nucleic Acids Res 37:1141-1151; Otto, G. A., P. J. Lukaysky, A. M. Lancaster, P. Sarnow, and J. D. Puglisi. 2002. Ribosomal proteins mediate the hepatitis C virus IRES-HeLa 40S interaction. RNA 8:913-923; Sarnow, P. 2003. Viral internal ribosome entry site elements: novel ribosome-RNA complexes and roles in viral pathogenesis. J Virol 77:2801-2806. All of them were incorporated by reference). After first using RT-PCR analysis to confirm the knockdown efficiency of RPS10 and RPS19 dsRNA at 72 hpi (
B. Preparation and Analysis for the Dual-Fluorescence Reporter Vector (Ie1/pIZV5- DR-Icp 35 IRES-GFP, Including a Sequence of SEQ ID NO: 3)
The pIZ/V5-His PCR fragment without the OpIE2 promoter (base 4-552) was amplified using KOD+ Taq polymerase (TOYOBO) with the primer set pIZ/V5-His-HindIII-F-ΔOpIE2p and pIZ/V5-His-R-ΔOpIE2p (Table 1) from the pIZ/V5-His vector. The WSSV ie1 promoter was amplified using KOD+Taq polymerase (TOYOBO) with the primer set ie1-Pro2-Bgl II and ie1-R-HindIII (Table 1), digested with HindIII and then cloned into the HindIII site of pIZ/V5-HisAOpIE2 (4-552) to generate the construct ie1/pIZ/V5-His. The pBacDIRE plasmid was a gift from Dr. Wu (Chung Yuan Christian University, Taiwan, ROC)(Chen et al., 2005, which was incorporated by reference). The fragment DIRE containing DsRed, IRES, and EGFP gene was amplified using the primer set DsRed-KpnI-F and EGFP-R from the pBacDIRE plasmid (Table 1). The PCR amplified fragment was digested with KpnI, and then cloned into KpnI-XbaI (Klenow filled) sites of ie1/pIZ/V5-His vector to generate the construct ie1/pIZ/V5-DIRE. The IRES was removed by digestion with BamHI from the ie1/pIZ/V5-DIRE, and then religated to produce the construct ie1/pIZ/V5-D-E. The icp35(-198/-1) IRES element was amplified with the primer set icp35(-198)-BamHI-F and icp35(-1)-BamHI-R (Table 1) and then cloned into the BamHI site of the ie1/pIZ/V5-D-E to produce the bicistronic plasmid ie1/pIZ/V5-D-icp35(-198/-1)-E.
Results
Embodiments of the inventive concept of the present invention may be modified in various forms, and the scope and spirit of the present invention should not be construed as being limited by the above-described embodiments. Therefore, the above-disclosed Embodiments are to be considered illustrative, and not restrictive, and the appended claims are intended to cover all such modifications, enhancements, and other embodiments, which fall within the true spirit and scope of the present invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 103104691 | Feb 2014 | TW | national |
