English

• 1.   Introduction

  Human respiratory syncytial virus (RSV) is the most important cause of lower respiratory tract diseases during infants and children under two years of age worldwide [1, 2], which has caused serious socioeconomic burden [3]. Currently, Palivizumab and Ribavirin are the few options for RSV specific-treatment and prophylaxis. Palivizumab is restricted to infants who are at high risk to develop severe RSV infection and costs high, while the application of ribavirin is impeded by questionable efficacy and safety reasons [4]. Therefore, the development of novel anti-RSV drugs becomes very urgent.

  RSV contains single-stranded, negative-sense, non-segmented RNA genomes. The gene order is as follows: 30-Leader-NS1-NS2-NP-M-SH-G-F-M2-1/M2-2-L-trailer-50 [2]. Four of the viral proteins, N, P, M2-1, and L, are associated with the viral genomic RNA to form the ribonucleoprotein (RNP) complex [3]. The untranslated leader region and trailer region at the 30 and 50 terminals of genome, and gene end sequence (GS) as well as gene start sequence (GE) flanking the subgenomes have been shown to be necessary and sufficient cis-acting elements for viral RNA replication and transcription [4]. Consequently, RSV minigenome containing a single reporter gene as an alternative of the RSV coding genes can simulates the processes of transcription and replication of RSV genome. T7 polymerase-driven RSV minigenome had been used as a tool in quantitating RSV replication and screening anti-RSV drugs. However, T7 polymerase-driven RSV minigenome needs an exogenously introduced T7 polymerase, which potentially limits the system?ˉs efficiency because of technical difficulties in the expression of T7 in all cells. Another substantial limitation of T7 polymerase-driven RSV minigenome involves the incorporation of an ribozyme sequence of hepatitis D virus (HDV), neighboring the minigenome and at the 30 end of the transcription cassette [5]. While the cleavage by the HDV ribozyme can produce an authentic 30 or 50 terminus of RSV genome, the cleavage efficiency of the transcript by HDV ribozyme is very low and therefore additional potential to limit the production of functional minigenome transcripts would occur within this system [6]. In contrast, the RNA polymerase I (Pol I) is a eukaryotic host cell polymerase which is normally localized to the nucleoli and can provide a substantial advantage in terms of the development of minigenome systems, since it alleviates the need to supply the polymerase in trans [6]. Pol I-driven paramyxovirus minigenome detection model has been successfully established and applied, and the Pol I-driven minigenome system has the same or even better signal strength compared with T7 [6, 7]. Taken together, these results suggest that the Pol I-driven minigenome expression system is another effective means to analyze the replication of paramyxovirus. In this article, an RNA polymerase I-driven RSV minigenome applied for quantitating RSV replication and screening anti-RSV drugs is investigated. Pol I was used to transcribe RSV minigenome from the constructed plasmid, designated pHM-RSV-Gluc, of minigenome cDNA which comprised trailer region, GS, reverse complement copy of Gaussia luciferase (Gluc) gene, an alternative of viral genes, and GE as well as leader region in the direction of 50¨C30 end and was flanked by promoter and terminator of Pol I. After pHMRSV-Gluc being transfected into RSV infected HEp-2 cells, (-) vRNA is generated by Pol I in cell nucleus. Then (+) mRNA is generated by RNA dependent RNA polymerase of RSV, and Gluc is expressed, which is an indicator of the replication level of RSV.

  In summary, the constructed RSV minigenome under the control of Pol I can be used as a tool for quantitating RSV replication and screening anti-RSV drugs.

  2.   Experimental materials and methods

  2.1   Cell and virus

  HEp-2 (Human laryngeal carcinoma, ATCC, Rockefeller, MD, USA) cells were grown in DMEM (Gibco BRL, Gaithersburg, USA) containing 2 mmol/L L-glutamine (Amresco, Solon, USA) and 10% fetal bovine serum (FBS, Hyclone, Logan, USA). Subgroup A RSV Long strain was kindly provided by Prof. Y. Qian, Capital Institute of Pediatrics, Beijing, China. All experiments with infectious virus were performed in the BSL-2 laboratory at the Beijing Jiaotong University, Beijing, China.

  2.2   Purification and titration of RSV

  Subgroup A RSV Long strain was propagated in HEp-2 cells in DMEM supplemented with 2% FBS (Invitrogen), L-glutamine (2 mmol/L), penicillin G (40 U/mL), streptomycin (100 mg/mL) and 0.2% sodium bicarbonate. RSV was purified by ultracentrifugation and titrated for infectivity by immunoenzyme assay. RSV titers were expressed as plaque-forming units (pfu).

  2.3   Construction of pHM-RSV-Gluc

  To construct pHM-RSV-Gluc, the sequences of RSV-Gluc (shown as Fig. 1) were synthesized by BGI (Shenzhen, China). The RSV-Gluc expressing cassette with Pol I enhancer/promoter and terminator was cloned into the pHM vector to produce pHM-RSV-Gluc.

  图 1

  

  图 1  Schematic representation of reporter gene expression from vRNA-oriented polymerase I-driven minigenome. vRNA-oriented polymerase I-driven minigenome contained a trailer region, GS (gene start sequence), NC1 (non-coding sequence of NS1), a reverse complement copy of Gaussia luciferase (Gluc) gene in place of viral genes, NC2 (non-coding sequence of L), GE (gene end sequence), and a leader region in the direction of 50¨C30 end and was flanked by a promoter and a terminator of RNP I.

  Figure 1.  Schematic representation of reporter gene expression from vRNA-oriented polymerase I-driven minigenome. vRNA-oriented polymerase I-driven minigenome contained a trailer region, GS (gene start sequence), NC1 (non-coding sequence of NS1), a reverse complement copy of Gaussia luciferase (Gluc) gene in place of viral genes, NC2 (non-coding sequence of L), GE (gene end sequence), and a leader region in the direction of 50¨C30 end and was flanked by a promoter and a terminator of RNP I.

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  2.4   Gaussia luciferase assays

  The supernatant from the transfected and infected HEp-2 cells was transferred into 96 well plate, Gluc activity was assayed by use of LUMIstar (BMG LABTECH, Ortenberg, Germany) according to the manufacturer?ˉs instructions after adding the chromogenic substrate of Gaussia Luciferase Flex Assay Kit (NEB, Ipswich, USA). The results were documented by photography and/or evaluated with OPTIMA software (BMG LABTECH, Ortenberg, Germany). The resultant signal strength was expressed as relative light unit (RLU).

  2.5   Cell viability assays

  Cell viability was detected by the MTS method. Assays are performed by adding MTS (Promega, Madison, USA) mixture directly to culture wells. After incubating for 4 h, the absorbance was recorded at 490 nm with an ELISA plate reader (Tecan, Ma?§nnedorf, Switzerland).

  2.6   Statistical analyses

  Statistical analyses were performed using the SPSS 11.5 software (SPSS, Chicago, USA). Comparison of differences was conducted using the Tukey test. P < 0.05 was considered significant.

  3.   Results and discussion

  3.1   The construction of pHM-RSV-Gluc and the purification of RSV

  In order to ensure the stability of the experiment, the purified RSV with titers above 10⁸ pfu/mL was obtained and used through this experiment. The feature of RSV, typical fluff edge, was observed under electron microscope (Fig. 2).

  图 2

  

  图 2  RSV electron micrograph after purification.

  Figure 2.  RSV electron micrograph after purification.

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  Then the pHM-RSV-Gluc containing RSV-Gluc sequences was constructed and confirmed by gene sequencing and restriction enzyme assay. As shown in Fig. 3a and b, the sequences of pHM-RSV-Gluc were consistent with the anticipated.

  图 3

  

  图 3  Construction of pHM-RSV-Gluc. (a) The sequence analysis. (b) Nucleic acid electrophoresis of restriction analysis.

  Figure 3.  Construction of pHM-RSV-Gluc. (a) The sequence analysis. (b) Nucleic acid electrophoresis of restriction analysis.

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  3.2   Identification of the Gluc expression from pHM-RSV-Gluc transfected HEp-2 cells

  At 24 h and 48 h post RSV infection, the pHM-RSV-Gluc was transfected into HEp-2 cells and the expression of Gluc was detected using Gaussia Luciferase Flex Assay Kit, respectively, in which the expression level of RLU was much high compared to the negative control groups treated without RSV or pHM-RSV-Gluc, or both (P < 0.05). It showed RSV infection was indispensable for the expression of Gluc from pHM-RSV-Gluc transfected HEp-2 cells (Fig. 4).

  图 4

  

  图 4  The expression of Gluc. After HEp-2 cells were infected with RSV for 24 h, the pHM-RSV-Gluc was transfected into the HEp-2 cells. After 48 h, the expression of Gluc was detected. The group transfected with plasmid without RSV infection, the group infected RSV without plasmid transfection, and the group without RSV infection or plasmid transfection served as negative control.

  Figure 4.  The expression of Gluc. After HEp-2 cells were infected with RSV for 24 h, the pHM-RSV-Gluc was transfected into the HEp-2 cells. After 48 h, the expression of Gluc was detected. The group transfected with plasmid without RSV infection, the group infected RSV without plasmid transfection, and the group without RSV infection or plasmid transfection served as negative control.

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  3.3   The optimized parameters important for quantitative assay of RSV replication

  The optimization of parameters involved in the quantitative assay of RSV replication is of great importance to establish a feasible detection system for screening anti-RSV drugs. These parameters include the detection time, cell density, amounts of plasmid and RSV infection titers.

  For the detection time, HEp-2 cells were infected with RSV at 0.1, 0.2, or 0.4 MOI for 24 h before transfection of the pHM-RSVGluc. Samples were harvested, and Gluc activity was analyzed at five different time points post-transfection. As shown in Fig. 5a, the strongest signal was observed at 48 h post-transfection regardless of RSV titers. So the optimized detection time was 48 h posttransfection (P < 0.05). In order to evaluate the optimized cell density, different amounts of HEp-2 cells were seeded in 96 well plates. As shown in Fig. 5b, the strongest signal was observed when cell density was 20, 000 cells/well at 48 h post-transfection (P < 0.05). Therefore we will employ 20, 000 cells/well in the subsequent experiments. Then we did the experiment to optimize the transfected plasmids. Different amounts of pHM-RSV-Gluc were transfected into HEp-2 cells. At 48 h post-transfection, samples were harvested, and Gluc activity was analyzed. As shown in Fig. 5c, the strongest signal was detected when the plasmid dosage was 0.1 mg/well (P < 0.05), which was consistent with the amount suggested by the manufacturer for the transfection reagent. So the plasmid dosage should be maintained at 0.1 mg/well. Finally, in order to study the relationship between RSV titer and the RLU, HEp-2 cells were infected with different RSV titers, but all the other parameters from the result introduced above. The relationship was dose-dependent between Gluc expression level and the RSV titers ranged from 0.001 MOI to 0.2 MOI (Fig. 5d). Furthermore, we tested the cell viability, impacted by infecting RSV, with MTS assay. With the increasing RSV titers, the cell viability decreased gradually. However, up to 1 MOI of RSV, the viability of the infected cells was still above 80% (Fig. 5e). Taken together, we thought that RSV titers at 0.1 MOI were suitable for the subsequent experiments.

  图 5

  

  图 5  Optimization of conditions. (a) Optimization of the detection time. (b) Optimization of cell density. (c) Optimization of the transfection dose of plasmid. (d) The relationship between RSV titers and RLU of Gluc. (e) The relationship between RSV titers and cell viability. The cell viability was detected using the MTS. Data were shown as average?à standard deviation, and P values were calculated.

  Figure 5.  Optimization of conditions. (a) Optimization of the detection time. (b) Optimization of cell density. (c) Optimization of the transfection dose of plasmid. (d) The relationship between RSV titers and RLU of Gluc. (e) The relationship between RSV titers and cell viability. The cell viability was detected using the MTS. Data were shown as average?à standard deviation, and P values were calculated.

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  3.4   Verification of the screening system using known antiviral compound and drug

  In order to verify the feasibility of using Pol I-driven RSV minigenome in screening antiviral drugs, we used compound P13, a small molecule compound with inhibitory activity on RSV replication [8, 9], and ribavirin, a commercially available drug against RSV infection clinically, as positive control.

  After adding 10-fold serial dilutions of P13 to the system, the Gluc activity displayed significant decline to completely suppressed when the concentration of compound P13 was increased from 0.1 mmol/L to 1 mmol/L, compared to the DMSO control group (P < 0.05). The IC₅₀ value of P13 is 0.32 mmol/L, which is similar with 0.11 mmol/L from the published data [8], which was a suggestive of this assay is qualified to screen RSV inhibitors. Additionally, the cell viability was above 80% following P13 treatment (Fig. 6b). As P13 was dissolved by DMSO, we used the equivalent concentration of DMSO as a control. The results proved that DMSO did have some impacts, but it was not the major factor (Fig. 6a). We used ribavirin to conduct the same experiment, which indicated that ribavirin also had inhibitory effect on RSV. Compared with the negative control group, the Gluc activity showed significant reduction when the concentration of ribavirin was 0.1 mmol/L (P < 0.05) (Fig. 6c). The IC₅₀ value of ribavirin is 36.48 mmol/L, which is slightly higher than 17.59 mmol/L from the published data [12]. Moreover, the cell viability was above 80% following ribavirin treatment (Fig. 6d).

  图 6

  

  图 6  Verification of antiviral drugs. (a) The inhibitory effect of P13. (b) The relationship between P13 and cell viability. (c) The inhibitory effect of ribavirin. (d) The relationship between ribavirin and cell viability. Data were shown as average?à standard deviation, and P values were calculated.

  Figure 6.  Verification of antiviral drugs. (a) The inhibitory effect of P13. (b) The relationship between P13 and cell viability. (c) The inhibitory effect of ribavirin. (d) The relationship between ribavirin and cell viability. Data were shown as average?à standard deviation, and P values were calculated.

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  The system employed Gluc, the smallest known luciferase (185 amino acids), as reporter gene, which is a naturally secreted protein capable of emitting strong fluorescence and advantageous to sensitive detection [10, 11]. A lot of luciferase has characteristics of?°instant glow?±, meaning that the fluorescence intensity will change significantly from the first time, which is not suitable for large scale screening. However Gluc overcomes this issue [12], it is stable in the cell culture medium and insensitive to the changes of pH and temperature. So this method is easy to operate, short-time required and more quickly to reflect the status of the virus. The system detection operates in 96-well plates, which can simultaneously achieve screening on multiple samples. All these features have showed RNA polymerase I-driven RSV minigenome can be used as a tool for quantifying virus titers and screening antiRSV drug.

  However, this method also has some limitations. Due to the highly sensitive nature of Gluc, it can result instability sometimes. The way to solve this problem is to establish the cell lines which can stably express an RNA polymerase I-driven RSV minigenome. In addition, this method can also be extended to other marker proteins such as green fluorescent protein (EGFP). Such visible marker proteins take the advantages of facilitating the observation and identification.

  4.   Conclusion

  The results show the plasmid encoding RSV minigenome under the control of Pol I has been constructed successfully and can be used as a tool for quantitating RSV replication and screening antiRSV drugs.

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  Figure 1  Schematic representation of reporter gene expression from vRNA-oriented polymerase I-driven minigenome. vRNA-oriented polymerase I-driven minigenome contained a trailer region, GS (gene start sequence), NC1 (non-coding sequence of NS1), a reverse complement copy of Gaussia luciferase (Gluc) gene in place of viral genes, NC2 (non-coding sequence of L), GE (gene end sequence), and a leader region in the direction of 50¨C30 end and was flanked by a promoter and a terminator of RNP I.

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  Figure 2  RSV electron micrograph after purification.

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  Figure 3  Construction of pHM-RSV-Gluc. (a) The sequence analysis. (b) Nucleic acid electrophoresis of restriction analysis.

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  Figure 4  The expression of Gluc. After HEp-2 cells were infected with RSV for 24 h, the pHM-RSV-Gluc was transfected into the HEp-2 cells. After 48 h, the expression of Gluc was detected. The group transfected with plasmid without RSV infection, the group infected RSV without plasmid transfection, and the group without RSV infection or plasmid transfection served as negative control.

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  Figure 5  Optimization of conditions. (a) Optimization of the detection time. (b) Optimization of cell density. (c) Optimization of the transfection dose of plasmid. (d) The relationship between RSV titers and RLU of Gluc. (e) The relationship between RSV titers and cell viability. The cell viability was detected using the MTS. Data were shown as average?à standard deviation, and P values were calculated.

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  Figure 6  Verification of antiviral drugs. (a) The inhibitory effect of P13. (b) The relationship between P13 and cell viability. (c) The inhibitory effect of ribavirin. (d) The relationship between ribavirin and cell viability. Data were shown as average?à standard deviation, and P values were calculated.

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