- Email: [email protected]
Extensive characterization of a lentiviral-derived stable cell line expressing rabbit hemorrhagic disease virus VPg protein
Journal of Virological Methods 237 (2016) 86–91
Contents lists available at ScienceDirect
Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Short communication
Extensive characterization of a lentiviral-derived stable cell line expressing rabbit hemorrhagic disease virus VPg protein Jie Zhu, Qiuhong Miao, Yonggui Tan, Huimin Guo, Chuanfeng Li, Zongyan Chen, Guangqing Liu ∗ Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, 200241, PR China
a b s t r a c t Article history: Received 3 May 2016 Received in revised form 1 August 2016 Accepted 4 September 2016 Available online 5 September 2016 Keywords: VPg Rabbit hemorrhagic disease virus Cell line
Rabbit hemorrhagic disease virus (RHDV) is an important member of the caliciviridae family. Currently, no suitable tissue culture system is available for proliferating RHDV, which limits the study of its pathogenesis. To bypass this obstacle, we established a cell line, RK13-VPg, stably expressing the VPg gene with a lentivirus packaging system in this study. In addition, the recently constructed RHDV replicon in our laboratory provided an appropriate model for studying the pathogenesis of RHDV without in vitro RHDV propagation and culture. Using this RHDV replicon and RK13-VPg cell line, we further demonstrated that the presence of VPg protein is essential for efficient translation of an RHDV replicon. Therefore, the RK13-VPg cell line is a powerful tool for studying the replication and translation mechanisms of RHDV. © 2016 Published by Elsevier B.V.
Rabbit hemorrhagic disease (RHD) is a highly contagious, fatal disease in adult rabbits that is often associated with liver necrosis, hemorrhage, and high mortality (Parra and Prieto, 1990), and it is responsible for significant economic losses in the rabbit industry (Asgari et al., 1998; Le Gall-Recule et al., 2003). It was first described in China in 1984 (Liu et al., 1984); a few years later, it spread worldwide (Gregg et al., 1991; Lee and Park, 1987; Morisse et al., 1991; Pu et al., 1985). The etiological agent for RHD is rabbit hemorrhagic disease virus (RHDV), which has a single-stranded, positive sense, polyadenylated RNA genome of 7.5 kb (Meyers et al., 1991a) and belongs to the Lagovirus genus of the Caliciviridae family, which also includes the Norovirus, Nebovirus, Sapovirus, and Vesivirus genera (Meyers et al., 1991b; Moussa et al., 1992; Parra and Prieto, 1990). As shown in Fig. 1, the RHDV genome encodes two open reading frames (ORFs), ORF1 and ORF2. ORF1 encodes a polyprotein that produces several non-structural proteins and the VP60 capsid protein. ORF2 encodes the minor structural protein, VP10. The VPg is a genome-linked protein that is covalently attached to the 5 end of the RNA genome. The 3 end of the positive sense RNA molecule is polyadenylated (Abrantes et al., 2012; Chang et al., 2006; Rasschaert et al., 1995). VPg is a general term for a protein encoded within a viral genome attached by a covalent linkage to the 5 end of genomic and subgenomic RNA. VPg plays a variety of roles in the synthesis
∗ Corresponding author. E-mail address: [email protected] (G. Liu). http://dx.doi.org/10.1016/j.jviromet.2016.09.006 0166-0934/© 2016 Published by Elsevier B.V.
of progeny viruses, including participating in genome replication, protein synthesis, and the packaging of RNA into viral capsids. VPg proteins are attached to the genomes of many plant and animal viruses, as described below. The positive sense RNA genomes of the Luteoviridae, Comoviridae, and Potyviridae plant virus families are covalently linked with VPg (Sadowy et al., 2001). Viruses of the Caliciviridae family are covalently linked at the 5 end of genomic and subgenomic RNAs to VPg (Burroughs and Brown, 1978; Meyers et al., 1991a). Recently, the role for VPg in the translation of feline calicivirus (FCV) and murine norwalk virus (MNV) has been demonstrated (Daughenbaugh et al., 2006; Goodfellow et al., 2005; Hosmillo et al., 2014). However, the specific function of RHDV VPg remains unclear because of the lack of a suitable tissue culture system. Moreover, studies on the mechanism of RHDV translation have been greatly limited. To bypass the lack of a tissue culture system, we tried to establish a stable VPg gene-expressing RK13 cell line with a lentivirus packaging system in this study. First, we constructed a pLOV-VPg plasmid, which encodes RHDV VPg protein, by inserting VPg cDNA into a pLOV-CMV (Neuron Biotech, China) using NheI and NotI. Briefly, the VPg-encoding sequence, which is described in our previous study (Liu et al., 2006), was amplified by PCR with sense VPg-F (5 -CTAGCTAGCATGGGCGTGAAAGGCAAAACA-3 ) and antisense VPg-R (5 -ATTTGCGGCCGCCTCGTAGTCATTGTC-3 ) primers. The package plasmid psPAX2 containing the specific lentivirus genes, Gag, Pol, etc., and the pMD2.G plasmid, which expresses the G protein of the vesicular stomatitis virus (VSV-G), were purchased from Neuron Biotech. In addition, the pRHDV-luc plasmid, wherein
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Fig. 1. A schematic presentation of the RHDV genome. A schematic presentation of the RHDV genome, which shows the nonstructural proteins (NSP1-7), virion coat protein VP60, and a minor structural protein (VP10).
the Fluc gene replaces the VP60 and partial VP10 genes, and VPgdeletion mutant of the pRHDV-luc (pRHDV-luc/VPg) plasmids were generated in our previous study (Binbin et al., 2013). To generate recombinant lentivirus containing the VPg gene, we transfected pMD2.G (12 g), psPAX2 (10 g), and pLOV-VPg (22 g) plasmids into 293T cells, which were seeded (1 × 106 cells) onto 100-mm dishes, using a calcium phosphate transfection reagent (Invitrogen, USA). At 48 h after transfection, we collected culture medium containing recombinant lentivirus. In addition, the control group was co-transfected the pMD2.G (12 g), psPAX2 (10 g), and pLOV-CMV-eGFP (22 g) plasmids. Then, we treated a RK13 cell line with supernatant containing recombinant lentivirus, which was diluted 1:1 in complete culture medium. The cells were at approximately 70% confluence and seeded in 100-mm dishes (1 × 106 cells in 10 ml of medium). After 48 h post infection, we selected the positive cells with 3 g/ml puromycin (Sigma, USA) and named the resulting cell population RK13-VPg cells. After 10 passages, RT-PCR, Western blot, and IFA were used to analyze the VPg protein expression levels. To verify that the VPg gene integrated into the RK13-VPg genome, we performed genetic analysis for the RK13-VPg cells by PCR with cellular DNA extracted from the 10th passage RK13VPg cells. Total cellular DNA was extracted using the E.Z.N.A. Tissue DNA kit (OMEGA, USA) according to the manufacturer’s protocol. The VPg gene of the RK13-VPg cells was detected by PCR with the forward primer VPg-F and antisense primer VPg-R. The -actin gene was used as an internal control with
-actin-F (5 -ATCGAGCACGGCATCGTCACC-3 ) and -actin-R (5 CACGTCACACTTCATGATGGA-3 ) primers. Moreover, the negative control for PCR was the total DNA from RK13 cells. PCR was performed in 30 cycles using Ex Taq polymerase (Takara, Japan). The PCR products were analyzed on a 1.0% agarose gel. As shown in Fig. 2A, the VPg gene have successfully inserted into the RK13-VPg cell genome. Then, to explore the transcription efficiency of the VPg gene, we performed RT-PCR analysis using cellular RNA extracted from the 10th passage of RK13-VPg cells. Total cellular RNA was extracted with Trizol reagent (Invitrogen, USA) according to the manufacturer’s protocol, and the RNA samples were stored at −70 ◦ C. DNA was removed from the isolated RNA using DNaseI (Takara, Japan). Then, cDNA was produced using M-MLV reverse transcriptase (Promega, USA) and random hexamers. The VPg gene of RK13-VPg cells was detected at the mRNA level by RT-PCR with the forward and antisense primers, VPg-F and VPg-R, respectively. Then, the -actin gene was used as the internal control with the -actin-F and -actin-R primers. In addition, PCR was performed which the extracted RNA treated with DNase I as temples. As shown in Fig. 2B, the VPg gene was efficiently expressed at the mRNA level in the 10th generation RK13-VPg cells. -actin was used as an internal control in both the DNA PCR and RT-PCR analyses. The results suggested that the RHDV VPg gene have been successfully integrated into the RK13-VPg cell genome and has high transcription level in the stable cell line.
Fig. 2. PCR and RT-PCR for VPg gene analysis in RK13-VPg cells. (A) PCR confirmation of the VPg gene in the RK13-VPg cell line genome. The -actin gene was used as an internal control in the RK13 and RK13-VPg cell lines. M: 2-kb plus DNA ladder, Lane 1: RK13-VPg cell genome, Lane 2: RK13 cell genome, and C: Blank control. (B) RT-PCR analysis of the VPg gene at the mRNA expression level in the RK13-VPg cell line. The -actin gene (651 bp) was used as an internal control. M: 2-kb plus DNA ladder, Lane 1: RK13-VPg cell cDNA, Lane 2: Negative control (RK13 cells cDNA), and C: Blank control.
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Fig. 3. The expression level of the VPg protein in the RK13-VPg cell line. (A) Western blot analysis of the VPg protein in the RK13-VPg cell line at different passage intervals (10 passages, 20 passages, and 30 passages). -actin was used as an internal control. (B) IFA analysis of VPg protein in the RK13-VPg cell line at 10, 20, and 30 passages, respectively. Normal RK13 cells acted as the negative control. RK13-eGFP cells acted as the positive control. The VPg rabbit polyclonal antibodies were used as the primary antibody.
Moreover, to explore the protein expression efficiency of the VPg gene, we performed Western blot and IFA analyses for RK13VPg cells. First, the 10th, 20th, and 30th passages of RK13-VPg cells were lysed with cell lysis buffer (0.15 M NaCl, 0.025 M Tris–HCl, 0.001 M EDTA, 1% NP-40, 5% glycerol; pH 7.4). Based on the protein concentration, equal levels of total protein samples were loaded in each well of a 12% SDS-PAGE gel and transferred by electrophoresis to a nitrocellulose membrane according to the manufacturer’s protocol (Bio-Rad, USA). After blocking with a 5% skimmed milk solution to reduce non-specific binding, the blots were incubated with VPg rabbit polyclonal antibodies obtained in our previous work (Binbin et al., 2013), which was followed by incubation with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (ZSGB-BIO, Beijing, China). The membranes were developed using a chemiluminescence (ECL) detection kit (Pierce Thermo, USA) and imaged using X-ray film (Eastman Kodak, NY, USA). Equal loading of the protein samples was confirmed by stripping the blots and restaining them with -actin monoclonal antibodies (ZSGB-BIO, Beijing, China). The Western blot results shown in Fig. 3A demonstrated that the VPg protein expression levels were high in the 10th, 20th, and 30th generation RK13-VPg cells, respectively. For the indirect immunofluorescence assay (IFA), the 10th, 20th, and 30th passages of RK13-VPg cells were fixed in 3.7% paraformalde-
hyde in PBS (pH 7.5). Then, the fixed cells were incubated with VPg rabbit polyclonal antibodies that were obtained from our previous work (Binbin et al., 2013). After incubation with the primary antibody, the samples were incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (ZSGB-BIO, Beijing, China). Finally, the samples were observed under a fluorescence microscope that was equipped with a video documentation system. The IFA revealed that the VPg protein was expressed in the nucleus and cytoplasm of the RK13-VPg cells (Fig. 3B). Meanwhile, there was no specific fluorescence observed in mock cells (RK13 cells). The results suggested that the VPg protein was stably expressed in the RK13-VPg cells. In addition, we analyzed the growth kinetics of RK13-VPg cells under standard culture conditions. First, RK13-VPg cells were seeded into 6-well plates at a density of 5 × 104 cells/well in 2.0 ml of MEM containing 10% FBS and incubated in 5% CO2 at 37 ◦ C. The cells were not passaged during the 7-day experimental periods. Moreover, cell numbers were normalized by flow cytometry. In addition, the cell growth curve was drawn with culture time as the abscissa and cell numbers as the ordinate. RK13 cells served as the control group. Each cell count was conducted in triplicate. As shown in Fig. 4A, RK13-VPg cells proliferated from 5 × 104 to 2.2 × 105 cells/well over 7 days, and they were similar to RK13 cells through-
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Fig. 4. Cell morphology and growth curve of RK13-VPg cells. (A) Cell growth curve shows that RK13-VPg cells were similar to RK13 cells. (B) Morphologic characteristics of RK13-VPg cells.
out all observation periods. In addition, RK13-VPg cells have the same shape as RK13 cells (Fig. 4B). To explore the relationship between VPg and RHDV translation, an RHDV replicon system, pRHDV-luc, was included in the present study. The luciferase activity of the RK13-VPg cells transfected with pRHDV-luc, pRHDV-luc/VPg, and pGL4.75 (Rluc) was analyzed and compared. We also analyzed the luciferase activity of RK13 cells that were transfected with pRHDV-luc, pRHDV-luc/VPg, and pGL4.75 (Rluc). The X-treme GENE HP DNA transfection reagent (Roche) was used to transfect cells using the relative plasmids mentioned above according to the manufacturer’s instructions. In addition, the pGL4.75 Vector encoding the luciferase reporter gene Renilla reniformis (Rluc) and a CMV promoter (pGL4.75; Promega, USA) were used as the internal controls for normalizing the luciferase values obtained from the cells transfected with the firefly luciferase replicon. The stability of Fluc mRNA in the RK13-VPg cells or normal RK 13 cells was evaluated using qRT-PCR. qRT-PCR was conducted in SYBR green PCR mix (Takara, Japan) with qFluc-F (5 -TTCGGTTGGCAGAAGCTATG-3 ) and qFluc-R (5 -GGTAGGCTGC GAAATGCCCA-3 ) primers. The GAPDH gene was used as an internal control with qGAPDH-F (5 -AGGGCTGCTTTTAACTCTGGTAAA3 ) and qGAPDH-R (5 -CATATTGGAACATGTAAACCATGTAGTTG-3 ) primers. Each qRT-PCR was performed in three biological replicates. Amplification was conducted in an Mastercycler ep realplex real-time PCR system (Eppendorf, Germany) using the following program: 95 ◦ C for 10 min followed by 40 cycles of 95 ◦ C for 15 s, 55 ◦ C for 45 s, and 72 ◦ C for 45 s. RNase-free water and cDNA of un-transfected RK13 cells were used as blank and negative controls, respectively. False-positive qRT-PCR results caused by primer dimers or nonspecific amplicons were eliminated from the final analysis based on a melting curve analysis of 95 ◦ C for 15 s, 60 ◦ C for 30 s, and 95 ◦ C for 15 s. The relative expression level of the Fluc gene compared to the control (pRHDV-luc) was analyzed on the basis of a comparative threshold cycle (CT ). Briefly, the Fluc CT value was normalized according to C = CT(Fluc) − CT(GAPDH) , and CT = CT(sample) − CT(control) was used to determine the relative expression levels of genes. The relative expression of Fluc was determined according to the 2– ct formula. In addition, luciferase activities were analyzed using a FB12 Luminometer (Berthold). Twenty-four hours after transfection, the cells were washed with PBS and lysed in 200 l of passive lysis buffer (Promega). After gentle shaking for 15 min at room temperature,
the cell lysate was transferred to a tube and centrifuged for 2 min at 12,000 × g and 4 ◦ C. The supernatant (20 l) was added to 100 l of luciferase assay substrate to evaluate firefly luciferase (Fluc) and Renilla luciferase (Rluc) activities using a dual-luciferase reporter assay system (Promega) based on relative light units (RLUs). To normalize the luciferase values obtained from cells transfected with the firefly luciferase replicon, the Rluc activity was used as an internal control. The levels of Fluc mRNA of pRHDV-luc and pRHDV-luc/VPg transfected in RK13-VPg cells were approximately 140% and 80% of that for pRHDV-luc transfected in normal RK 13 cells, respectively (Fig. 5A). As shown in Fig. 5B, VPg deletion significantly reduced the Fluc gene expression, but the expression was restored in RK13-VPg cells. In addition, the Fluc gene of pRHDV-luc was over-expressed in RK13-VPg cells. These results are similar to the previous results (Zhu et al., 2015). Therefore, we further demonstrated that VPg is essential for viral translation. There are many methods of establishing stable cell lines. For example, after directly transfecting cells with lipofectin reagents, some target genes can be integrated into the cell chromosome with the continuous pressure of antibiotics. However, the efficiency of these methods is usually poor because of the low transfection efficiency. Currently, vectors derived from human immunodeficiency virus type 1 are widely used for gene delivery, mainly because they can transduce both dividing and non-dividing cells, leading to stable and long-term gene expression (Naldini, 1998). In addition, these vector types are safe and have a low toxicity and high stability and cell type specificity (Follenzi and Naldini, 2002; Naldini and Verma, 2000). Therefore, this work aimed to screen an RK13VPg cell line for stable expression of the RHDV VPg protein using a lentivirus-based plasmid system. The RK13-VPg cells have similar cell morphology and growth kinetics as RK13 cells. Therefore, we can cultivate a large number of the cells under standard culture conditions. In addition, the RK13-VPg cell line may be useful for better exploring the biological functions of RHDV VPg in RHDV translation or replication. Like most Caliciviruses, RHDV cannot proliferate in vitro. Therefore, many molecular biology mechanisms and characteristics of RHDV remain unclear, including the translation and replication mechanisms of RHDV, which are fundamental to understanding RNA viruses. These culturing problems have limited our understanding of this virus. The role of the calicivirus VPg protein in viral
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Fig. 5. VPg plays crucial roles in RHDV translation. (A) Thirty-two hours after transfection, the luciferase mRNA levels in RK13-VPg or RK13 cells transfected with pRHDV-luc and pRHDV-luc/VPg were evaluated by qRT-PCR. The Student’s t test and ANOVA were applied for the statistical analyses; P <0.05 was considered statistically significant (*), and P <0.01 was considered extremely statistically significant (**). (B) Relative luciferase activity in RK13-VPg or RK13 cells carrying pRHDV-luc/VPg and the parental pRHDV-luc genotype at 12, 24, 48, and 72 h post-transfection. The luciferase activity in the cells was evaluated by measuring the firefly luciferase activity at different time points after transfection. Renilla luciferase activity, measured at the same time points, was used to normalize the transfection efficiency. The differences in the luciferase activities associated with RK13-VPg and RK13 cells were compared using SAS 9.1 software. The Student’s t test and ANOVA were used for the statistical analyses. P <0.05 was considered statistically significant (*), and P <0.01 was considered extremely statistically significant (**). The experiments were conducted in triplicate, and similar results were obtained from three independent experiments.
translation was first reported in feline calicivirus (FCV) (Herbert et al., 1997; Mitra et al., 2004) and indicated that the VPg protein may play a role in replication and function as a protein primer of FCV RNA polymerase (Han et al., 2010). Because RHDV and FCV belong to the same Caliciviradae family, we speculated that VPg might also play an important role in the translation and/or the replication mechanisms of RHDV. To bypass the barriers of RHDV proliferation in vitro and provide a platform for exploring the biological function of RHDV VPg, we successfully established a cell line that stably expresses RHDV VPg. With the help of this cell line, we found that VPg deletion significantly reduced the reporter (Fluc) expression level. However, the Fluc expression level was restored through a trans-supplemental VPg protein in the RK13-VPg cell line. These results further imply that the VPg protein plays a critical role in the RHDV life cycle, and the details of its potential role in RHDV replication and translation need to be further studied. Acknowledgments This work was supported by the Special Fund for Agro-scientific Research in the Public Interest (201303046), Chinese Natural Sci-
ences Foundation (31101848, 31270194, 31101848), Innovation Project of Science and Technology Commission Foundation of Shanghai (No. 13391901602), and National Advanced Technology Research and Development Program of China (863 Program) (No. 2011AA10A200).
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Contents lists available at ScienceDirect
Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Short communication
Extensive characterization of a lentiviral-derived stable cell line expressing rabbit hemorrhagic disease virus VPg protein Jie Zhu, Qiuhong Miao, Yonggui Tan, Huimin Guo, Chuanfeng Li, Zongyan Chen, Guangqing Liu ∗ Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, 200241, PR China
a b s t r a c t Article history: Received 3 May 2016 Received in revised form 1 August 2016 Accepted 4 September 2016 Available online 5 September 2016 Keywords: VPg Rabbit hemorrhagic disease virus Cell line
Rabbit hemorrhagic disease virus (RHDV) is an important member of the caliciviridae family. Currently, no suitable tissue culture system is available for proliferating RHDV, which limits the study of its pathogenesis. To bypass this obstacle, we established a cell line, RK13-VPg, stably expressing the VPg gene with a lentivirus packaging system in this study. In addition, the recently constructed RHDV replicon in our laboratory provided an appropriate model for studying the pathogenesis of RHDV without in vitro RHDV propagation and culture. Using this RHDV replicon and RK13-VPg cell line, we further demonstrated that the presence of VPg protein is essential for efficient translation of an RHDV replicon. Therefore, the RK13-VPg cell line is a powerful tool for studying the replication and translation mechanisms of RHDV. © 2016 Published by Elsevier B.V.
Rabbit hemorrhagic disease (RHD) is a highly contagious, fatal disease in adult rabbits that is often associated with liver necrosis, hemorrhage, and high mortality (Parra and Prieto, 1990), and it is responsible for significant economic losses in the rabbit industry (Asgari et al., 1998; Le Gall-Recule et al., 2003). It was first described in China in 1984 (Liu et al., 1984); a few years later, it spread worldwide (Gregg et al., 1991; Lee and Park, 1987; Morisse et al., 1991; Pu et al., 1985). The etiological agent for RHD is rabbit hemorrhagic disease virus (RHDV), which has a single-stranded, positive sense, polyadenylated RNA genome of 7.5 kb (Meyers et al., 1991a) and belongs to the Lagovirus genus of the Caliciviridae family, which also includes the Norovirus, Nebovirus, Sapovirus, and Vesivirus genera (Meyers et al., 1991b; Moussa et al., 1992; Parra and Prieto, 1990). As shown in Fig. 1, the RHDV genome encodes two open reading frames (ORFs), ORF1 and ORF2. ORF1 encodes a polyprotein that produces several non-structural proteins and the VP60 capsid protein. ORF2 encodes the minor structural protein, VP10. The VPg is a genome-linked protein that is covalently attached to the 5 end of the RNA genome. The 3 end of the positive sense RNA molecule is polyadenylated (Abrantes et al., 2012; Chang et al., 2006; Rasschaert et al., 1995). VPg is a general term for a protein encoded within a viral genome attached by a covalent linkage to the 5 end of genomic and subgenomic RNA. VPg plays a variety of roles in the synthesis
∗ Corresponding author. E-mail address: [email protected] (G. Liu). http://dx.doi.org/10.1016/j.jviromet.2016.09.006 0166-0934/© 2016 Published by Elsevier B.V.
of progeny viruses, including participating in genome replication, protein synthesis, and the packaging of RNA into viral capsids. VPg proteins are attached to the genomes of many plant and animal viruses, as described below. The positive sense RNA genomes of the Luteoviridae, Comoviridae, and Potyviridae plant virus families are covalently linked with VPg (Sadowy et al., 2001). Viruses of the Caliciviridae family are covalently linked at the 5 end of genomic and subgenomic RNAs to VPg (Burroughs and Brown, 1978; Meyers et al., 1991a). Recently, the role for VPg in the translation of feline calicivirus (FCV) and murine norwalk virus (MNV) has been demonstrated (Daughenbaugh et al., 2006; Goodfellow et al., 2005; Hosmillo et al., 2014). However, the specific function of RHDV VPg remains unclear because of the lack of a suitable tissue culture system. Moreover, studies on the mechanism of RHDV translation have been greatly limited. To bypass the lack of a tissue culture system, we tried to establish a stable VPg gene-expressing RK13 cell line with a lentivirus packaging system in this study. First, we constructed a pLOV-VPg plasmid, which encodes RHDV VPg protein, by inserting VPg cDNA into a pLOV-CMV (Neuron Biotech, China) using NheI and NotI. Briefly, the VPg-encoding sequence, which is described in our previous study (Liu et al., 2006), was amplified by PCR with sense VPg-F (5 -CTAGCTAGCATGGGCGTGAAAGGCAAAACA-3 ) and antisense VPg-R (5 -ATTTGCGGCCGCCTCGTAGTCATTGTC-3 ) primers. The package plasmid psPAX2 containing the specific lentivirus genes, Gag, Pol, etc., and the pMD2.G plasmid, which expresses the G protein of the vesicular stomatitis virus (VSV-G), were purchased from Neuron Biotech. In addition, the pRHDV-luc plasmid, wherein
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Fig. 1. A schematic presentation of the RHDV genome. A schematic presentation of the RHDV genome, which shows the nonstructural proteins (NSP1-7), virion coat protein VP60, and a minor structural protein (VP10).
the Fluc gene replaces the VP60 and partial VP10 genes, and VPgdeletion mutant of the pRHDV-luc (pRHDV-luc/VPg) plasmids were generated in our previous study (Binbin et al., 2013). To generate recombinant lentivirus containing the VPg gene, we transfected pMD2.G (12 g), psPAX2 (10 g), and pLOV-VPg (22 g) plasmids into 293T cells, which were seeded (1 × 106 cells) onto 100-mm dishes, using a calcium phosphate transfection reagent (Invitrogen, USA). At 48 h after transfection, we collected culture medium containing recombinant lentivirus. In addition, the control group was co-transfected the pMD2.G (12 g), psPAX2 (10 g), and pLOV-CMV-eGFP (22 g) plasmids. Then, we treated a RK13 cell line with supernatant containing recombinant lentivirus, which was diluted 1:1 in complete culture medium. The cells were at approximately 70% confluence and seeded in 100-mm dishes (1 × 106 cells in 10 ml of medium). After 48 h post infection, we selected the positive cells with 3 g/ml puromycin (Sigma, USA) and named the resulting cell population RK13-VPg cells. After 10 passages, RT-PCR, Western blot, and IFA were used to analyze the VPg protein expression levels. To verify that the VPg gene integrated into the RK13-VPg genome, we performed genetic analysis for the RK13-VPg cells by PCR with cellular DNA extracted from the 10th passage RK13VPg cells. Total cellular DNA was extracted using the E.Z.N.A. Tissue DNA kit (OMEGA, USA) according to the manufacturer’s protocol. The VPg gene of the RK13-VPg cells was detected by PCR with the forward primer VPg-F and antisense primer VPg-R. The -actin gene was used as an internal control with
-actin-F (5 -ATCGAGCACGGCATCGTCACC-3 ) and -actin-R (5 CACGTCACACTTCATGATGGA-3 ) primers. Moreover, the negative control for PCR was the total DNA from RK13 cells. PCR was performed in 30 cycles using Ex Taq polymerase (Takara, Japan). The PCR products were analyzed on a 1.0% agarose gel. As shown in Fig. 2A, the VPg gene have successfully inserted into the RK13-VPg cell genome. Then, to explore the transcription efficiency of the VPg gene, we performed RT-PCR analysis using cellular RNA extracted from the 10th passage of RK13-VPg cells. Total cellular RNA was extracted with Trizol reagent (Invitrogen, USA) according to the manufacturer’s protocol, and the RNA samples were stored at −70 ◦ C. DNA was removed from the isolated RNA using DNaseI (Takara, Japan). Then, cDNA was produced using M-MLV reverse transcriptase (Promega, USA) and random hexamers. The VPg gene of RK13-VPg cells was detected at the mRNA level by RT-PCR with the forward and antisense primers, VPg-F and VPg-R, respectively. Then, the -actin gene was used as the internal control with the -actin-F and -actin-R primers. In addition, PCR was performed which the extracted RNA treated with DNase I as temples. As shown in Fig. 2B, the VPg gene was efficiently expressed at the mRNA level in the 10th generation RK13-VPg cells. -actin was used as an internal control in both the DNA PCR and RT-PCR analyses. The results suggested that the RHDV VPg gene have been successfully integrated into the RK13-VPg cell genome and has high transcription level in the stable cell line.
Fig. 2. PCR and RT-PCR for VPg gene analysis in RK13-VPg cells. (A) PCR confirmation of the VPg gene in the RK13-VPg cell line genome. The -actin gene was used as an internal control in the RK13 and RK13-VPg cell lines. M: 2-kb plus DNA ladder, Lane 1: RK13-VPg cell genome, Lane 2: RK13 cell genome, and C: Blank control. (B) RT-PCR analysis of the VPg gene at the mRNA expression level in the RK13-VPg cell line. The -actin gene (651 bp) was used as an internal control. M: 2-kb plus DNA ladder, Lane 1: RK13-VPg cell cDNA, Lane 2: Negative control (RK13 cells cDNA), and C: Blank control.
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Fig. 3. The expression level of the VPg protein in the RK13-VPg cell line. (A) Western blot analysis of the VPg protein in the RK13-VPg cell line at different passage intervals (10 passages, 20 passages, and 30 passages). -actin was used as an internal control. (B) IFA analysis of VPg protein in the RK13-VPg cell line at 10, 20, and 30 passages, respectively. Normal RK13 cells acted as the negative control. RK13-eGFP cells acted as the positive control. The VPg rabbit polyclonal antibodies were used as the primary antibody.
Moreover, to explore the protein expression efficiency of the VPg gene, we performed Western blot and IFA analyses for RK13VPg cells. First, the 10th, 20th, and 30th passages of RK13-VPg cells were lysed with cell lysis buffer (0.15 M NaCl, 0.025 M Tris–HCl, 0.001 M EDTA, 1% NP-40, 5% glycerol; pH 7.4). Based on the protein concentration, equal levels of total protein samples were loaded in each well of a 12% SDS-PAGE gel and transferred by electrophoresis to a nitrocellulose membrane according to the manufacturer’s protocol (Bio-Rad, USA). After blocking with a 5% skimmed milk solution to reduce non-specific binding, the blots were incubated with VPg rabbit polyclonal antibodies obtained in our previous work (Binbin et al., 2013), which was followed by incubation with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (ZSGB-BIO, Beijing, China). The membranes were developed using a chemiluminescence (ECL) detection kit (Pierce Thermo, USA) and imaged using X-ray film (Eastman Kodak, NY, USA). Equal loading of the protein samples was confirmed by stripping the blots and restaining them with -actin monoclonal antibodies (ZSGB-BIO, Beijing, China). The Western blot results shown in Fig. 3A demonstrated that the VPg protein expression levels were high in the 10th, 20th, and 30th generation RK13-VPg cells, respectively. For the indirect immunofluorescence assay (IFA), the 10th, 20th, and 30th passages of RK13-VPg cells were fixed in 3.7% paraformalde-
hyde in PBS (pH 7.5). Then, the fixed cells were incubated with VPg rabbit polyclonal antibodies that were obtained from our previous work (Binbin et al., 2013). After incubation with the primary antibody, the samples were incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (ZSGB-BIO, Beijing, China). Finally, the samples were observed under a fluorescence microscope that was equipped with a video documentation system. The IFA revealed that the VPg protein was expressed in the nucleus and cytoplasm of the RK13-VPg cells (Fig. 3B). Meanwhile, there was no specific fluorescence observed in mock cells (RK13 cells). The results suggested that the VPg protein was stably expressed in the RK13-VPg cells. In addition, we analyzed the growth kinetics of RK13-VPg cells under standard culture conditions. First, RK13-VPg cells were seeded into 6-well plates at a density of 5 × 104 cells/well in 2.0 ml of MEM containing 10% FBS and incubated in 5% CO2 at 37 ◦ C. The cells were not passaged during the 7-day experimental periods. Moreover, cell numbers were normalized by flow cytometry. In addition, the cell growth curve was drawn with culture time as the abscissa and cell numbers as the ordinate. RK13 cells served as the control group. Each cell count was conducted in triplicate. As shown in Fig. 4A, RK13-VPg cells proliferated from 5 × 104 to 2.2 × 105 cells/well over 7 days, and they were similar to RK13 cells through-
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Fig. 4. Cell morphology and growth curve of RK13-VPg cells. (A) Cell growth curve shows that RK13-VPg cells were similar to RK13 cells. (B) Morphologic characteristics of RK13-VPg cells.
out all observation periods. In addition, RK13-VPg cells have the same shape as RK13 cells (Fig. 4B). To explore the relationship between VPg and RHDV translation, an RHDV replicon system, pRHDV-luc, was included in the present study. The luciferase activity of the RK13-VPg cells transfected with pRHDV-luc, pRHDV-luc/VPg, and pGL4.75 (Rluc) was analyzed and compared. We also analyzed the luciferase activity of RK13 cells that were transfected with pRHDV-luc, pRHDV-luc/VPg, and pGL4.75 (Rluc). The X-treme GENE HP DNA transfection reagent (Roche) was used to transfect cells using the relative plasmids mentioned above according to the manufacturer’s instructions. In addition, the pGL4.75 Vector encoding the luciferase reporter gene Renilla reniformis (Rluc) and a CMV promoter (pGL4.75; Promega, USA) were used as the internal controls for normalizing the luciferase values obtained from the cells transfected with the firefly luciferase replicon. The stability of Fluc mRNA in the RK13-VPg cells or normal RK 13 cells was evaluated using qRT-PCR. qRT-PCR was conducted in SYBR green PCR mix (Takara, Japan) with qFluc-F (5 -TTCGGTTGGCAGAAGCTATG-3 ) and qFluc-R (5 -GGTAGGCTGC GAAATGCCCA-3 ) primers. The GAPDH gene was used as an internal control with qGAPDH-F (5 -AGGGCTGCTTTTAACTCTGGTAAA3 ) and qGAPDH-R (5 -CATATTGGAACATGTAAACCATGTAGTTG-3 ) primers. Each qRT-PCR was performed in three biological replicates. Amplification was conducted in an Mastercycler ep realplex real-time PCR system (Eppendorf, Germany) using the following program: 95 ◦ C for 10 min followed by 40 cycles of 95 ◦ C for 15 s, 55 ◦ C for 45 s, and 72 ◦ C for 45 s. RNase-free water and cDNA of un-transfected RK13 cells were used as blank and negative controls, respectively. False-positive qRT-PCR results caused by primer dimers or nonspecific amplicons were eliminated from the final analysis based on a melting curve analysis of 95 ◦ C for 15 s, 60 ◦ C for 30 s, and 95 ◦ C for 15 s. The relative expression level of the Fluc gene compared to the control (pRHDV-luc) was analyzed on the basis of a comparative threshold cycle (CT ). Briefly, the Fluc CT value was normalized according to C = CT(Fluc) − CT(GAPDH) , and CT = CT(sample) − CT(control) was used to determine the relative expression levels of genes. The relative expression of Fluc was determined according to the 2– ct formula. In addition, luciferase activities were analyzed using a FB12 Luminometer (Berthold). Twenty-four hours after transfection, the cells were washed with PBS and lysed in 200 l of passive lysis buffer (Promega). After gentle shaking for 15 min at room temperature,
the cell lysate was transferred to a tube and centrifuged for 2 min at 12,000 × g and 4 ◦ C. The supernatant (20 l) was added to 100 l of luciferase assay substrate to evaluate firefly luciferase (Fluc) and Renilla luciferase (Rluc) activities using a dual-luciferase reporter assay system (Promega) based on relative light units (RLUs). To normalize the luciferase values obtained from cells transfected with the firefly luciferase replicon, the Rluc activity was used as an internal control. The levels of Fluc mRNA of pRHDV-luc and pRHDV-luc/VPg transfected in RK13-VPg cells were approximately 140% and 80% of that for pRHDV-luc transfected in normal RK 13 cells, respectively (Fig. 5A). As shown in Fig. 5B, VPg deletion significantly reduced the Fluc gene expression, but the expression was restored in RK13-VPg cells. In addition, the Fluc gene of pRHDV-luc was over-expressed in RK13-VPg cells. These results are similar to the previous results (Zhu et al., 2015). Therefore, we further demonstrated that VPg is essential for viral translation. There are many methods of establishing stable cell lines. For example, after directly transfecting cells with lipofectin reagents, some target genes can be integrated into the cell chromosome with the continuous pressure of antibiotics. However, the efficiency of these methods is usually poor because of the low transfection efficiency. Currently, vectors derived from human immunodeficiency virus type 1 are widely used for gene delivery, mainly because they can transduce both dividing and non-dividing cells, leading to stable and long-term gene expression (Naldini, 1998). In addition, these vector types are safe and have a low toxicity and high stability and cell type specificity (Follenzi and Naldini, 2002; Naldini and Verma, 2000). Therefore, this work aimed to screen an RK13VPg cell line for stable expression of the RHDV VPg protein using a lentivirus-based plasmid system. The RK13-VPg cells have similar cell morphology and growth kinetics as RK13 cells. Therefore, we can cultivate a large number of the cells under standard culture conditions. In addition, the RK13-VPg cell line may be useful for better exploring the biological functions of RHDV VPg in RHDV translation or replication. Like most Caliciviruses, RHDV cannot proliferate in vitro. Therefore, many molecular biology mechanisms and characteristics of RHDV remain unclear, including the translation and replication mechanisms of RHDV, which are fundamental to understanding RNA viruses. These culturing problems have limited our understanding of this virus. The role of the calicivirus VPg protein in viral
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Fig. 5. VPg plays crucial roles in RHDV translation. (A) Thirty-two hours after transfection, the luciferase mRNA levels in RK13-VPg or RK13 cells transfected with pRHDV-luc and pRHDV-luc/VPg were evaluated by qRT-PCR. The Student’s t test and ANOVA were applied for the statistical analyses; P <0.05 was considered statistically significant (*), and P <0.01 was considered extremely statistically significant (**). (B) Relative luciferase activity in RK13-VPg or RK13 cells carrying pRHDV-luc/VPg and the parental pRHDV-luc genotype at 12, 24, 48, and 72 h post-transfection. The luciferase activity in the cells was evaluated by measuring the firefly luciferase activity at different time points after transfection. Renilla luciferase activity, measured at the same time points, was used to normalize the transfection efficiency. The differences in the luciferase activities associated with RK13-VPg and RK13 cells were compared using SAS 9.1 software. The Student’s t test and ANOVA were used for the statistical analyses. P <0.05 was considered statistically significant (*), and P <0.01 was considered extremely statistically significant (**). The experiments were conducted in triplicate, and similar results were obtained from three independent experiments.
translation was first reported in feline calicivirus (FCV) (Herbert et al., 1997; Mitra et al., 2004) and indicated that the VPg protein may play a role in replication and function as a protein primer of FCV RNA polymerase (Han et al., 2010). Because RHDV and FCV belong to the same Caliciviradae family, we speculated that VPg might also play an important role in the translation and/or the replication mechanisms of RHDV. To bypass the barriers of RHDV proliferation in vitro and provide a platform for exploring the biological function of RHDV VPg, we successfully established a cell line that stably expresses RHDV VPg. With the help of this cell line, we found that VPg deletion significantly reduced the reporter (Fluc) expression level. However, the Fluc expression level was restored through a trans-supplemental VPg protein in the RK13-VPg cell line. These results further imply that the VPg protein plays a critical role in the RHDV life cycle, and the details of its potential role in RHDV replication and translation need to be further studied. Acknowledgments This work was supported by the Special Fund for Agro-scientific Research in the Public Interest (201303046), Chinese Natural Sci-
ences Foundation (31101848, 31270194, 31101848), Innovation Project of Science and Technology Commission Foundation of Shanghai (No. 13391901602), and National Advanced Technology Research and Development Program of China (863 Program) (No. 2011AA10A200).
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