Arch Virol DOI 10.1007/s00705-013-1899-9
BRIEF REPORT
Screening effective short interfering RNA/short hairpin RNA for inhibition of human astrovirus ORF2 gene expression in cultured cells Wei Zhao • Ke Niu • Wen-Hui Liu • Jian Zhao • Yi-Ming Jin • Ting-Ting Sui
Received: 31 July 2013 / Accepted: 15 October 2013 Ó Springer-Verlag Wien 2013
Abstract In this study, we have evaluated four different 21-nt duplexes of small interfering RNA (siRNA-469, siRNA-852, siRNA-1802 and siRNA-1806) that specifically target the ORF2 gene of human astrovirus (HAstV) in inhibiting HAstV capsid protein expression in transfected BHK-21 cells. Furthermore, fluorescence analysis, realtime quantitative PCR (RT-qPCR) and western blot assays showed that pGPU6/GFP/Neo-shRNA inhibits ORF2 gene expression in Caco2 cells. The results indicate that siRNA/ shRNA-469 and siRNA/shRNA-1802 can interfere with capsid protein expression in cell culture, and this provides a powerful tool for the study of HAstV gene functions and the biological properties of the capsid protein. Keywords Human astrovirus siRNA Capsid protein Gene expression shRNA
Astroviruses are recognized as important viral etiological agents, and infections are a major public health concern. They are known to cause sporadic diarrhea or large outbreaks of gastroenteritis in human and animals [1]. Human astrovirus (HAstV) is the main causative agent of diarrhea in young children [2]. In recent reports, it has also been associated with encephalitis and viremia in
W. Zhao (&) School of Basic Medical Sciences, Liaoning Medical University, No. 40, The Third Section of SongPo Rd, Jinzhou 121200, Liaoning, People’s Republic of China e-mail: [email protected] K. Niu W.-H. Liu J. Zhao Y.-M. Jin T.-T. Sui Department of Laboratory Animals of Basic Medical Sciences, Liaoning Medical University, Jinzhou 121200, Liaoning, People’s Republic of China
immunocompromised patients [3, 4]. HAstV is a small, single-stranded, positive-sense RNA virus. The 5’-terminal portion of the genome encodes two major nonstructural proteins, ORF1a and ORF1b. The 3’-terminal one-third of the genome (ORF2) encodes the structural protein (capsid protein) [5]. The astrovirus capsid protein has several unique biological properties. It has been shown to be a unique inhibitor of the complement pathway [6], and its potential action as an enterotoxin increases epithelial barrier permeability by disrupting cellular tight junction complexes [7] and may contribute to diarrhea in vivo. The C-terminal domain of the capsid protein may include a cell-attachment and receptor-binding domain, and it forms the spikes of the virion. This domain is predicted to participate in the early interactions of the virus with the host cell [8]. Small interfering RNAs (siRNAs) are a powerful tool for sequence-specific post-transcriptional gene silencing [9]. It has been utilized successfully in cultured mammalian cells to reduce the expression of specific genes [10]. siRNA or short hairpin RNA (shRNA) has been used recently to interfere with the replication of a number of viruses [11–16]. To investigate whether siRNA can be used to inhibit HAstV capsid protein expression, we designed four siRNAs targeting the ORF2 gene and tested their ability to inhibit human astrovirus ORF2 gene expression in BHK-21 cells. Additionally, an shRNA expression vector was also constructed to detect inhibition of ORF2 expression in Caco2 cells. Four 21-nt siRNAs directed against the sequence of the HAstV ORF2 gene were designed according to QIAGEN’s guidelines (http://www.qiagen.com) and chemically synthesized (Invitrogen, Life Technologies, China). The sequences are shown in Table 1. In order to choose the most effective siRNA, we screened siRNA by fusing the
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W. Zhao et al. Table 1 Four siRNA sequences targeting the ORF2 gene Location
Sense
ORF2-469
5’ CCUCAGAGUUUCACUUAAUTT 3’
5’ AUUAAGUGAAACUCUGAGGTT 3’
ORF2-852
5’ CGCCAUUAAUAAUGAAUGUTT 3’
5’ ACAUUCAUUAUUAAUGGCGTT 3’
ORF2-1802
5’ CUGGCUGGAUCUGGAAUAATT 3’
5’ UUAUUCCAGAUCCAGCCAGTT 3’
ORF2-1862
5’ GCCUGAUUCACCUGAUAAUTT 3’
5’ AUUAUCAGGUGAAUCAGGCTT 3’
targeted sequence and the reporter EGFP gene together to produce a recombinant plasmid that could be used for expression of ORF2-EGFP fusion mRNAs. The positive HAstV I (GenBank ID: KF211475), isolated from human feces from the city of Jinzhou, China, was used as template. The primer 5’-CCGctcgagGGATGGCTAGCAAGT CCAATAAGCAGGTAACT-3’ (sense; lower-case letters indicate an XhoI recognition site) and 5’-CGGggatccC TCGGCGTGGCCGCGGCTTCCAGAAGT-3’ (antisense; lower-case letters indicate a KpnI recognition site) were used to amplify the HAstV ORF2 gene. RT-PCR was performed according to a procedure that was described previously (REFERENCE?). The purified RT-PCR product was then cloned into the XhoI and KpnI sites of pEGFP-N3 (Clontech) to generate the plasmid pEGFP-N3ORF2. To determine whether these ORF2 gene-specific siRNA can suppress the expression of capsid protein, BHK-21 cells were cotransfected with pEGFP-N3-ORF2 and siRNA. The BHK-21 cells were maintained in Dulbecco’s modified Eagle medium (GIBCO/BRL) supplemented with 10 % fetal bovine serum at 37°C in a 5 % CO2 atmosphere. Cells were seeded into a 6-well plate and grown to reach approximately 70 % confluence at the time of transfection. Cells were transfected with 4 lg of pEGFP-N3-ORF2 plasmid together with 20 lM of siRNA, using Lipofectamine 2000 transfected reagent (Invitrogen, Life Technologies, China) according to the manufacturer’s protocol. The cells were harvested 48 h after transfection for EGFP fluorescence analysis and measurement of mRNA and protein expression. One-step SYBR Green RT-qPCR using a SYBR PrimerScriptTM RT-PCR Kit II (TaKaRa, Japan) was carried out using a real-time thermal cycler (Bio-Rad Laboratories, Inc., USA) to measure the effect of siRNA inhibition on the mRNA expression level. The 2-DDCT method was used to evaluate the relative expression level of ORF2 gene. The ORF2-specific primers were 5’TTAATAATGAATGTGCCAGAGGGAAG-3’ (antisense) and 5’-AGCACCTGCCATACTGTGTCTGAG-3’ (antisense). The b-actin F primer was 5’-GCATCCACGAAAC TACATTCAACTC-3’, and the R primer was 5’-CACTG TGTTGGCATAGAGGTCTTTG-3’. BHK-21 cells were harvested 48 h post-transfection. Equal amounts of total protein were separated by 10 % sodium dodecyl sulfate
123
Antisense
polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred to PVDF membranes (BioRad, USA). Protein was detected using Anti-GFP Tag Monoclonal Antibody (Abmart, Shanghai, China). A goatmouse IgG conjugated with HRP was used as secondary antibody (Promega; 1:1000 dilution). The b-actin protein of transfected cells served as a loading control. A gel image scan was performed to find the bands and to measure the grey values of bands using Quantity One software (BioRad, USA). To further study the effects of selected siRNA molecules on ORF2 gene expression in other cell culture models, we constructed a hairpin siRNA expression cassette. The following DNA oligonucleotides were synthesized: ORF21802 (5’-CACCgctggctggatctggaataaTTCAAGAGAttatt ccagatccagccagTTTTTTG-3’), ORF2-469 (5’-CACCgcc tcagagttcacttaatTTCAAGAGAattaagtgaaactctgaggtTTTTT G-3’; lower-case letters indicate the ORF2 sequence, and the stretch of five thymidines at the 3’ end [pol III terminator] are underlined). Primers were annealed and cloned into the BbsI and BamHI sites of the pGPU6/GFP/Neo vector. The corresponding shRNA expression vector pGPU6 contained a human U6 polymerase III (pol-III) promoter and a neomycin resistance gene (NEOr). After cotransfection of BHK-21 cells with pEGFP-N3ORF2 and siRNA, EGFP marker protein was quantified by fluorescence assay. The results show that EGFP protein expression 48 after transfection decreased 50 %, 70 %, 50 % and 60 % when cells were cotransfected with siRNA469, siRNA-852, siRNA-1802 and siRNA-1863, respectively (Fig. 1). To determine whether ORF2 gene siRNA-expressing cells suppressed capsid protein expression, mRNA expression of the HAstV ORF2 gene was measured by RTqPCR 48 hours post-transfection and found to be decreased about 50 %, 59 %, 50 % and 39 % in cells co-transfected with HAstV-specific siRNA-469, siRNA-852, siRNA1802, siRNA-1863, respectively. No significant change was observed, however, in the blank or NC controls (Fig. 2). The HAstV ORF2 gene encodes a protein with an approximate molecular mass of 79 kDa, and the fusion protein ORF2-EGFP has an approximate molecular mass of 110 kDa (79 kDa ? 27 kDa of EGFP protein). To analyze
siRNA/shRNA inhibiting human astrovirus ORF2 gene expression
Fig. 1 Inhibition of EGFP reporter protein expression by siRNA in normal BHK-21 cells (1003). BHK-21 cells were co-transfected with the pEGFP-N3- ORF2 plasmid and various siRNAs. After 48 hours, EGFP marker protein was quantified by fluorescence microscopy. The results show that EGFP protein expression 48 h after transfection decreased 50 %, 70 %, 50 % and 60 % when cells were
Fig. 2 Inhibition of HAstV ORF2 mRNA expression detected by RT-qPCR 48 hours post-transfection. The transcription level of the ORF2 gene 48 hours post-transfection was decreased about 50 %, 59 %, 50 % and 39 % in cells co-transfected with HAstVspecific siRNA-469, siRNA852, siRNA-1802, siRNA-1863, respectively, but no significant change was observed in the blank or NC controls. The 2-DDCT and confidence interval were both derived from the DDCT value. All procedures were performed in triplicate, and data are expressed as means ±S.D.
cotransfected with siRNA-469, siRNA-852, siRNA-1802 and siRNA1863. Normal, BHK-21 cells under white light; Blank, pEGFP-N3ORF2 expression under fluorescence light; Mock, Lipofectamine 2000 transfection reagent control; ORF2-NC, siRNA-NC-FAM control
siRNA-469
siRNA-852
siRNA-1802
ΔCT
2.06 ± 0.27
2.35 ± 0.13
2.07 ± 0.16
ΔΔCT
1.00 ± 0.28
1.29 ± 0.15
0.50
0.41
2-
ΔΔC
T
NC
Blank
1.77 ± 0.23
1.06 ± 0.08
0.96 ± 0.23
1.01 ± 0.18
0.71 ± 0.24
0.00 ± 0.11
-0.10 ± 0.24
0.50
0.61
1.00
1.07
siRNA-1862
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W. Zhao et al. Table 2 shRNA for inhibiting human astrovirus ORF2 gene expression in Caco2 cells
A
B
C ORF2
NC Blank siRNA-469 siRNA-852 siRNA-1802 siRNA-1862
after 48h post-transfection Relative interference expression efficiency 1 1.03273 0.294506 0.758165 0.376226 1.136378
70% 24% 62% 0%
Fig. 3 Inhibition of HAstV ORF2 fusion protein expression detected by western blot analysis 48 hours post-transfection. BHK-21 cell monolayers were co-transfected with pEGFP-N3-ORF2 plasmid expressing HAstV capsid protein and four siRNA molecules. At 48 h post-transfection, lysates were collected, and western blotting was performed to detect ORF2-EGFP fusion protein. b-actin was used as a loading control. The protein expression level is given as a ratio, relative to the amount of GAPDH present in the sample. A, inhibition of protein expression measured by SDS-PAGE; B, histogram of ORF2 expression relative to GAPDH for the ORF2-NC sample; C, semiquantitative measurement of the gray values of bands
the suppression of the ORF2 gene at the protein level, BHK-21 cells were collected for western blot analysis 48 h post-transfection. Western blot analysis showed that ORF2 protein expression was significantly reduced in cells cotransfected with HAstV-specific siRNAs-469 (70 %) and siRNA-1802 (62 %) compared with cells transfected with the blank. siRNA-852 treatment yielded only 24 % inhibition, and treatment with siRNA-1862 did not result in any inhibition (Fig. 3). The data showed that siRNA-469 and siRNA-1802 were more efficient at inhibiting human astrovirus ORF2 gene expression in BHK-21cells. To further study the effects of selected siRNA molecules on ORF2 gene expression in HAstV host cell models, we constructed the pGPU6/GFP/ Neo-shRNA plasmid (shRNA-469 and shRNA-1802). We used the Caco2 cell line, which is stably infected with
123
ORF2
Relative expression of mRNA
NC
1.00
Interference efficiency
Relative expression of protein
Interference efficiency
1
Blank
0.93
shRNA469
0.5
50 %
1.022 0.27
73 %
shRNA1802
0.41
59 %
0.35
65 %
HAstV and is used as a host and in vitro and in vivo model for HAstV infection and replication [17]. The effects of shRNA on ORF2 gene expression was also studied thoroughly by analyzing the levels of mRNA and protein by RT-qPCR analysis and western blot, respectively. The results indicated that treatment with shRNA-469 and shRNA-1802 significantly reduced viral mRNA and protein expression in Caco2 cell culture (Table 2). All the result suggested that a high inhibition efficiency and specificity induced by siRNAs/shRNA had been achieved, especially by siRNA/shRNA-469 and siRNA/shRNA1802. Here, we report the first use of siRNA/shRNA to suppress expression of the HAstV capsid protein in cell culture. We identified four different siRNA target sequences in the HAstV capsid protein. In order to choose the most effective siRNA molecules, we produced recombinant ORF2-EGFP plasmid by fusing the targeted sequence and reporter EGFP gene together, resulting in the expression of ORF2 and EGFP fusion mRNAs. Therefore, we can initially select suitable siRNA duplexes by cotransfection and analysis of siRNA inhibition efficiency by fluorescence analysis, RT-qPCR and western blot assays. By using this approach, we have identified two siRNA molecules (siRNA-469 and siRNA-1802) that have a significant impact on ORF2 fusion gene expression. This provides a quick approach to select effective siRNA in the study of gene expression and function. In this study, we first used BHK21 cells for screening the efficient siRNA, because BHK cells exhibit high transfection efficiency and can support HAstV replication when transfected with an infectious in vitro-transcribed astrovirus genomic RNA [18]. To further study the effects of selected siRNA molecules on HAstV gene expression in other cell models, we used human epithelial colorectal adenocarcinoma cells, Caco-2, which are known to support all of the human astrovirus types that are adaptable to cultivation and are used as an in vitro model for HAstV replication [19, 20]. We designed 59-nt primers that contain a specific 19-nt target sequence from the HAstV genome to create recombinant pGPU6/
siRNA/shRNA inhibiting human astrovirus ORF2 gene expression
GFP/Neo-shRNA plasmid (shRNA-469 and shRNA-1802). All of the results indicated that shRNA-469 and shRNA1802 also inhibit HAstV capsid protein expression in Caco2 cells. HAstV capsid protein shows the largest sequence variability among astrovirus proteins. The N-terminal half of the capsid protein is more conserved than the C-terminal domain. The conserved N-terminal domain of the structural protein forms the capsid core of the particle, whereas the hypervariable C-terminal domain forms the spikes of the virion. Thus, the C-terminal domain is predicted to participate in the early interactions of the virus with the host cell [19, 20]. In this study, we demonstrated that siRNA/ shRNA-469 and siRNA/shRNA-1802, corresponding to the N-terminal and C-terminal domains, respectively, of the capsid protein can effectively inhibit capsid protein expression in host cells, and thus have potential applications for inhibition of different forms of the virion or the interaction of virus with host cells. In future studies, they can be used to study HAstV capsid protein function or HAstV replication in cell culture. In conclusion, we have shown that sequence-specific ORF2 siRNA/shRNA-mediated expression could be used to interfere with HAstV capsid protein expression and therefore has a potential application in protein function analysis or virus replication. To our knowledge, this is the first report siRNA/shRNA inhibition of human astrovirus ORF2 gene expression in different cultured cells. Further studies are required to determine whether the siRNA/ shRNA can inhibit HAstV replication in vivo or in vitro, as well as to interfere with HAstV infection. Acknowledgments This work was supported by National Nature Science Foundation of China (project no. 81201285), Education Department Foundation of Liaoning Province (project no. L2011142), and College Student’s Innovative and Business Project Foundation of Liaoning Province (project no.201210160005).
References 1. De Benedictis P, Schultz-Cherry S, Burnham A, Cattoli G (2011) Astrovirus infections in humans and animals - molecular biology, genetic diversity, and interspecies transmissions. Infect Genet Evol 11(7):1529–1544 2. Kirkwood CD, Clark R, Bogdanovic-Sakran N, Bishop RF (2005) A 5-year study of the prevalence and genetic diversity of human caliciviruses associated with sporadic cases of acute gastroenteritis in young children admitted to hospital in Melbourne, Australia (1998–2002). J Med Virol 77:96–101
3. Quan PL, Wagner TA, Briese T, Torgerson TR, Hornig M, Tashmukhamedova A, Firth C, Palacios G, Baisre-De-Leon A, Paddock CD, Hutchison SK, Egholm M, Zaki SR, Goldman JE, Ochs HD, Lipkin WI (2010) Astrovirus encephalitis in boy with X-linked agammaglobulinemia. Emerg Infect Dis 16:918–925 4. Holtz LR, Wylie KM, Sodergren E, Jiang Y, Franz CJ, Weinstock GM, Storch GA, Wang D (2011) Astrovirus MLB2 viremia in febrile child. Emerg Infect Dis 17:2050–2052 5. Matsui SM, Greenberg HB (1996) Astroviruses. In: Fields BN, Knipe DM, Howley PM, Chanock RM, Melnick JL, Monath TP, Roizman B, Straus SE (eds) Fields virology, 3 edn. LippincottRaven Publishers, Philadelphia, pp 811–824 6. Hair PS, Gronemus JQ, Crawford KB, Salvi VP, Cunnion KM, Thielens NM (2010) Human astrovirus coat protein binds C1q and MBL and inhibits the classical and lectin pathways of complement activation. Mol Immunol 47(4):792–798 7. Moser LA, Carter M, Schultz-Cherry S (2007) Astrovirus increases epithelial barrier permeability independently of viral replication. J Virol 81(21):11937–11945 8. Bass DM, Qiu S (2000) Proteolytic processing of the astrovirus capsid. J Virol 74(4):1810–1814 9. Hannon GJ (2002) RNA interference. Nature 418:244–251 10. McCaffrey AP, Meuse L, Pham TT, Conklin DS, Hannon GJ, Kay MA (2002) RNA interference in adult mice. Nature 18:38–39 11. Adelman ZN, Sanchez-Vargas I, Travanty EA, Carlson JO, Beaty BJ, Blair CD, Olson KE (2002) RNA silencing of dengue virus type 2 replication in transformed C6/36 mosquito cells transcribing an inverted-repeat RNA derived from the virus genome. J Virol 76:12925–12933 12. Coburn GA, Cullen BR (2002) Potent and specific inhibition of human immunodeficiency virus type 1 replication by RNA interference. J Virol 76:9225–9231 13. Hamasaki K, Nakao K, Matsumoto K, Ichikawa T, Ishikawa H, Eguchi K (2003) Short interfering RNA-directed inhibition of hepatitis B virus replication. FEBS Lett 543:51–54 14. Jacque JM, Triques K, Stevensom M (2002) Modulation of HIV1 replication by RNA interference. Nature 418:435–538 15. Jiang M, Milner J (2002) Selective silencing of viral gene expression in HPV-positive human cervical carcinoma cells treated with siRNA, a primer of RNA interference. Oncogene 21:6041–6048 16. Kapadia SB, Brideau-Andersen A, Chisari FV (2003) Interference of hepatitis C virus RNA replication by short interfering RNAs. Proc Natl Acad Sci USA 100:2014–2018 17. Brinker JP, Blacklow NR, Herrmann JE (2000) Human astrovirus isolation and propagation in multiple cell lines. Arch Virol 145:1847–1856 18. Geigenmuller U, Ginzton NH, Matsui SM (1997) Construction of a genome-length cDNA clone for human astrovirus serotype 1 and synthesis of infectious RNA transcripts. J Virol 71(2): 1713–1717 19. Dong J, Dong L, Me´ndez E, Tao Y (2011) Crystal structure of the human astrovirus capsid spike. Proc Natl Acad Sci USA 108(31):12681–12686 20. Krishna NK (2005) Identification of structural domains involved in astrovirus capsid biology. Viral Immunol 18(1):17–26
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BRIEF REPORT
Screening effective short interfering RNA/short hairpin RNA for inhibition of human astrovirus ORF2 gene expression in cultured cells Wei Zhao • Ke Niu • Wen-Hui Liu • Jian Zhao • Yi-Ming Jin • Ting-Ting Sui
Received: 31 July 2013 / Accepted: 15 October 2013 Ó Springer-Verlag Wien 2013
Abstract In this study, we have evaluated four different 21-nt duplexes of small interfering RNA (siRNA-469, siRNA-852, siRNA-1802 and siRNA-1806) that specifically target the ORF2 gene of human astrovirus (HAstV) in inhibiting HAstV capsid protein expression in transfected BHK-21 cells. Furthermore, fluorescence analysis, realtime quantitative PCR (RT-qPCR) and western blot assays showed that pGPU6/GFP/Neo-shRNA inhibits ORF2 gene expression in Caco2 cells. The results indicate that siRNA/ shRNA-469 and siRNA/shRNA-1802 can interfere with capsid protein expression in cell culture, and this provides a powerful tool for the study of HAstV gene functions and the biological properties of the capsid protein. Keywords Human astrovirus siRNA Capsid protein Gene expression shRNA
Astroviruses are recognized as important viral etiological agents, and infections are a major public health concern. They are known to cause sporadic diarrhea or large outbreaks of gastroenteritis in human and animals [1]. Human astrovirus (HAstV) is the main causative agent of diarrhea in young children [2]. In recent reports, it has also been associated with encephalitis and viremia in
W. Zhao (&) School of Basic Medical Sciences, Liaoning Medical University, No. 40, The Third Section of SongPo Rd, Jinzhou 121200, Liaoning, People’s Republic of China e-mail: [email protected] K. Niu W.-H. Liu J. Zhao Y.-M. Jin T.-T. Sui Department of Laboratory Animals of Basic Medical Sciences, Liaoning Medical University, Jinzhou 121200, Liaoning, People’s Republic of China
immunocompromised patients [3, 4]. HAstV is a small, single-stranded, positive-sense RNA virus. The 5’-terminal portion of the genome encodes two major nonstructural proteins, ORF1a and ORF1b. The 3’-terminal one-third of the genome (ORF2) encodes the structural protein (capsid protein) [5]. The astrovirus capsid protein has several unique biological properties. It has been shown to be a unique inhibitor of the complement pathway [6], and its potential action as an enterotoxin increases epithelial barrier permeability by disrupting cellular tight junction complexes [7] and may contribute to diarrhea in vivo. The C-terminal domain of the capsid protein may include a cell-attachment and receptor-binding domain, and it forms the spikes of the virion. This domain is predicted to participate in the early interactions of the virus with the host cell [8]. Small interfering RNAs (siRNAs) are a powerful tool for sequence-specific post-transcriptional gene silencing [9]. It has been utilized successfully in cultured mammalian cells to reduce the expression of specific genes [10]. siRNA or short hairpin RNA (shRNA) has been used recently to interfere with the replication of a number of viruses [11–16]. To investigate whether siRNA can be used to inhibit HAstV capsid protein expression, we designed four siRNAs targeting the ORF2 gene and tested their ability to inhibit human astrovirus ORF2 gene expression in BHK-21 cells. Additionally, an shRNA expression vector was also constructed to detect inhibition of ORF2 expression in Caco2 cells. Four 21-nt siRNAs directed against the sequence of the HAstV ORF2 gene were designed according to QIAGEN’s guidelines (http://www.qiagen.com) and chemically synthesized (Invitrogen, Life Technologies, China). The sequences are shown in Table 1. In order to choose the most effective siRNA, we screened siRNA by fusing the
123
W. Zhao et al. Table 1 Four siRNA sequences targeting the ORF2 gene Location
Sense
ORF2-469
5’ CCUCAGAGUUUCACUUAAUTT 3’
5’ AUUAAGUGAAACUCUGAGGTT 3’
ORF2-852
5’ CGCCAUUAAUAAUGAAUGUTT 3’
5’ ACAUUCAUUAUUAAUGGCGTT 3’
ORF2-1802
5’ CUGGCUGGAUCUGGAAUAATT 3’
5’ UUAUUCCAGAUCCAGCCAGTT 3’
ORF2-1862
5’ GCCUGAUUCACCUGAUAAUTT 3’
5’ AUUAUCAGGUGAAUCAGGCTT 3’
targeted sequence and the reporter EGFP gene together to produce a recombinant plasmid that could be used for expression of ORF2-EGFP fusion mRNAs. The positive HAstV I (GenBank ID: KF211475), isolated from human feces from the city of Jinzhou, China, was used as template. The primer 5’-CCGctcgagGGATGGCTAGCAAGT CCAATAAGCAGGTAACT-3’ (sense; lower-case letters indicate an XhoI recognition site) and 5’-CGGggatccC TCGGCGTGGCCGCGGCTTCCAGAAGT-3’ (antisense; lower-case letters indicate a KpnI recognition site) were used to amplify the HAstV ORF2 gene. RT-PCR was performed according to a procedure that was described previously (REFERENCE?). The purified RT-PCR product was then cloned into the XhoI and KpnI sites of pEGFP-N3 (Clontech) to generate the plasmid pEGFP-N3ORF2. To determine whether these ORF2 gene-specific siRNA can suppress the expression of capsid protein, BHK-21 cells were cotransfected with pEGFP-N3-ORF2 and siRNA. The BHK-21 cells were maintained in Dulbecco’s modified Eagle medium (GIBCO/BRL) supplemented with 10 % fetal bovine serum at 37°C in a 5 % CO2 atmosphere. Cells were seeded into a 6-well plate and grown to reach approximately 70 % confluence at the time of transfection. Cells were transfected with 4 lg of pEGFP-N3-ORF2 plasmid together with 20 lM of siRNA, using Lipofectamine 2000 transfected reagent (Invitrogen, Life Technologies, China) according to the manufacturer’s protocol. The cells were harvested 48 h after transfection for EGFP fluorescence analysis and measurement of mRNA and protein expression. One-step SYBR Green RT-qPCR using a SYBR PrimerScriptTM RT-PCR Kit II (TaKaRa, Japan) was carried out using a real-time thermal cycler (Bio-Rad Laboratories, Inc., USA) to measure the effect of siRNA inhibition on the mRNA expression level. The 2-DDCT method was used to evaluate the relative expression level of ORF2 gene. The ORF2-specific primers were 5’TTAATAATGAATGTGCCAGAGGGAAG-3’ (antisense) and 5’-AGCACCTGCCATACTGTGTCTGAG-3’ (antisense). The b-actin F primer was 5’-GCATCCACGAAAC TACATTCAACTC-3’, and the R primer was 5’-CACTG TGTTGGCATAGAGGTCTTTG-3’. BHK-21 cells were harvested 48 h post-transfection. Equal amounts of total protein were separated by 10 % sodium dodecyl sulfate
123
Antisense
polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred to PVDF membranes (BioRad, USA). Protein was detected using Anti-GFP Tag Monoclonal Antibody (Abmart, Shanghai, China). A goatmouse IgG conjugated with HRP was used as secondary antibody (Promega; 1:1000 dilution). The b-actin protein of transfected cells served as a loading control. A gel image scan was performed to find the bands and to measure the grey values of bands using Quantity One software (BioRad, USA). To further study the effects of selected siRNA molecules on ORF2 gene expression in other cell culture models, we constructed a hairpin siRNA expression cassette. The following DNA oligonucleotides were synthesized: ORF21802 (5’-CACCgctggctggatctggaataaTTCAAGAGAttatt ccagatccagccagTTTTTTG-3’), ORF2-469 (5’-CACCgcc tcagagttcacttaatTTCAAGAGAattaagtgaaactctgaggtTTTTT G-3’; lower-case letters indicate the ORF2 sequence, and the stretch of five thymidines at the 3’ end [pol III terminator] are underlined). Primers were annealed and cloned into the BbsI and BamHI sites of the pGPU6/GFP/Neo vector. The corresponding shRNA expression vector pGPU6 contained a human U6 polymerase III (pol-III) promoter and a neomycin resistance gene (NEOr). After cotransfection of BHK-21 cells with pEGFP-N3ORF2 and siRNA, EGFP marker protein was quantified by fluorescence assay. The results show that EGFP protein expression 48 after transfection decreased 50 %, 70 %, 50 % and 60 % when cells were cotransfected with siRNA469, siRNA-852, siRNA-1802 and siRNA-1863, respectively (Fig. 1). To determine whether ORF2 gene siRNA-expressing cells suppressed capsid protein expression, mRNA expression of the HAstV ORF2 gene was measured by RTqPCR 48 hours post-transfection and found to be decreased about 50 %, 59 %, 50 % and 39 % in cells co-transfected with HAstV-specific siRNA-469, siRNA-852, siRNA1802, siRNA-1863, respectively. No significant change was observed, however, in the blank or NC controls (Fig. 2). The HAstV ORF2 gene encodes a protein with an approximate molecular mass of 79 kDa, and the fusion protein ORF2-EGFP has an approximate molecular mass of 110 kDa (79 kDa ? 27 kDa of EGFP protein). To analyze
siRNA/shRNA inhibiting human astrovirus ORF2 gene expression
Fig. 1 Inhibition of EGFP reporter protein expression by siRNA in normal BHK-21 cells (1003). BHK-21 cells were co-transfected with the pEGFP-N3- ORF2 plasmid and various siRNAs. After 48 hours, EGFP marker protein was quantified by fluorescence microscopy. The results show that EGFP protein expression 48 h after transfection decreased 50 %, 70 %, 50 % and 60 % when cells were
Fig. 2 Inhibition of HAstV ORF2 mRNA expression detected by RT-qPCR 48 hours post-transfection. The transcription level of the ORF2 gene 48 hours post-transfection was decreased about 50 %, 59 %, 50 % and 39 % in cells co-transfected with HAstVspecific siRNA-469, siRNA852, siRNA-1802, siRNA-1863, respectively, but no significant change was observed in the blank or NC controls. The 2-DDCT and confidence interval were both derived from the DDCT value. All procedures were performed in triplicate, and data are expressed as means ±S.D.
cotransfected with siRNA-469, siRNA-852, siRNA-1802 and siRNA1863. Normal, BHK-21 cells under white light; Blank, pEGFP-N3ORF2 expression under fluorescence light; Mock, Lipofectamine 2000 transfection reagent control; ORF2-NC, siRNA-NC-FAM control
siRNA-469
siRNA-852
siRNA-1802
ΔCT
2.06 ± 0.27
2.35 ± 0.13
2.07 ± 0.16
ΔΔCT
1.00 ± 0.28
1.29 ± 0.15
0.50
0.41
2-
ΔΔC
T
NC
Blank
1.77 ± 0.23
1.06 ± 0.08
0.96 ± 0.23
1.01 ± 0.18
0.71 ± 0.24
0.00 ± 0.11
-0.10 ± 0.24
0.50
0.61
1.00
1.07
siRNA-1862
123
W. Zhao et al. Table 2 shRNA for inhibiting human astrovirus ORF2 gene expression in Caco2 cells
A
B
C ORF2
NC Blank siRNA-469 siRNA-852 siRNA-1802 siRNA-1862
after 48h post-transfection Relative interference expression efficiency 1 1.03273 0.294506 0.758165 0.376226 1.136378
70% 24% 62% 0%
Fig. 3 Inhibition of HAstV ORF2 fusion protein expression detected by western blot analysis 48 hours post-transfection. BHK-21 cell monolayers were co-transfected with pEGFP-N3-ORF2 plasmid expressing HAstV capsid protein and four siRNA molecules. At 48 h post-transfection, lysates were collected, and western blotting was performed to detect ORF2-EGFP fusion protein. b-actin was used as a loading control. The protein expression level is given as a ratio, relative to the amount of GAPDH present in the sample. A, inhibition of protein expression measured by SDS-PAGE; B, histogram of ORF2 expression relative to GAPDH for the ORF2-NC sample; C, semiquantitative measurement of the gray values of bands
the suppression of the ORF2 gene at the protein level, BHK-21 cells were collected for western blot analysis 48 h post-transfection. Western blot analysis showed that ORF2 protein expression was significantly reduced in cells cotransfected with HAstV-specific siRNAs-469 (70 %) and siRNA-1802 (62 %) compared with cells transfected with the blank. siRNA-852 treatment yielded only 24 % inhibition, and treatment with siRNA-1862 did not result in any inhibition (Fig. 3). The data showed that siRNA-469 and siRNA-1802 were more efficient at inhibiting human astrovirus ORF2 gene expression in BHK-21cells. To further study the effects of selected siRNA molecules on ORF2 gene expression in HAstV host cell models, we constructed the pGPU6/GFP/ Neo-shRNA plasmid (shRNA-469 and shRNA-1802). We used the Caco2 cell line, which is stably infected with
123
ORF2
Relative expression of mRNA
NC
1.00
Interference efficiency
Relative expression of protein
Interference efficiency
1
Blank
0.93
shRNA469
0.5
50 %
1.022 0.27
73 %
shRNA1802
0.41
59 %
0.35
65 %
HAstV and is used as a host and in vitro and in vivo model for HAstV infection and replication [17]. The effects of shRNA on ORF2 gene expression was also studied thoroughly by analyzing the levels of mRNA and protein by RT-qPCR analysis and western blot, respectively. The results indicated that treatment with shRNA-469 and shRNA-1802 significantly reduced viral mRNA and protein expression in Caco2 cell culture (Table 2). All the result suggested that a high inhibition efficiency and specificity induced by siRNAs/shRNA had been achieved, especially by siRNA/shRNA-469 and siRNA/shRNA1802. Here, we report the first use of siRNA/shRNA to suppress expression of the HAstV capsid protein in cell culture. We identified four different siRNA target sequences in the HAstV capsid protein. In order to choose the most effective siRNA molecules, we produced recombinant ORF2-EGFP plasmid by fusing the targeted sequence and reporter EGFP gene together, resulting in the expression of ORF2 and EGFP fusion mRNAs. Therefore, we can initially select suitable siRNA duplexes by cotransfection and analysis of siRNA inhibition efficiency by fluorescence analysis, RT-qPCR and western blot assays. By using this approach, we have identified two siRNA molecules (siRNA-469 and siRNA-1802) that have a significant impact on ORF2 fusion gene expression. This provides a quick approach to select effective siRNA in the study of gene expression and function. In this study, we first used BHK21 cells for screening the efficient siRNA, because BHK cells exhibit high transfection efficiency and can support HAstV replication when transfected with an infectious in vitro-transcribed astrovirus genomic RNA [18]. To further study the effects of selected siRNA molecules on HAstV gene expression in other cell models, we used human epithelial colorectal adenocarcinoma cells, Caco-2, which are known to support all of the human astrovirus types that are adaptable to cultivation and are used as an in vitro model for HAstV replication [19, 20]. We designed 59-nt primers that contain a specific 19-nt target sequence from the HAstV genome to create recombinant pGPU6/
siRNA/shRNA inhibiting human astrovirus ORF2 gene expression
GFP/Neo-shRNA plasmid (shRNA-469 and shRNA-1802). All of the results indicated that shRNA-469 and shRNA1802 also inhibit HAstV capsid protein expression in Caco2 cells. HAstV capsid protein shows the largest sequence variability among astrovirus proteins. The N-terminal half of the capsid protein is more conserved than the C-terminal domain. The conserved N-terminal domain of the structural protein forms the capsid core of the particle, whereas the hypervariable C-terminal domain forms the spikes of the virion. Thus, the C-terminal domain is predicted to participate in the early interactions of the virus with the host cell [19, 20]. In this study, we demonstrated that siRNA/ shRNA-469 and siRNA/shRNA-1802, corresponding to the N-terminal and C-terminal domains, respectively, of the capsid protein can effectively inhibit capsid protein expression in host cells, and thus have potential applications for inhibition of different forms of the virion or the interaction of virus with host cells. In future studies, they can be used to study HAstV capsid protein function or HAstV replication in cell culture. In conclusion, we have shown that sequence-specific ORF2 siRNA/shRNA-mediated expression could be used to interfere with HAstV capsid protein expression and therefore has a potential application in protein function analysis or virus replication. To our knowledge, this is the first report siRNA/shRNA inhibition of human astrovirus ORF2 gene expression in different cultured cells. Further studies are required to determine whether the siRNA/ shRNA can inhibit HAstV replication in vivo or in vitro, as well as to interfere with HAstV infection. Acknowledgments This work was supported by National Nature Science Foundation of China (project no. 81201285), Education Department Foundation of Liaoning Province (project no. L2011142), and College Student’s Innovative and Business Project Foundation of Liaoning Province (project no.201210160005).
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