- INTRODUCTION
- MATERIALS AND METHODS
- Cells and virus.
- Plasmids and VIRG construction.
- RNA transcription and cell transfection.
- Deoxyribozyme reactions in vitro.
- Northern blot assay.
- Deoxyribozyme assays in BHK21 cells.
- Luciferase activity assay.
- Enzyme-linked immunosorbent assay.
- Computer-assisted secondary structure prediction.
- Statistical analysis.
- RESULTS
- DISCUSSION
ProCite
RefWorks
Reference Manager
BibTeX
Zotero
EndNote
-
1
Gould EA, Solomon T, 2008. Pathogenic flaviviruses. Lancet 371 :500–509.
-
2
Rico-Hesse R, 2003. Microevolution and virulence of dengue viruses. Adv Virus Res 59 :315–341.
-
3
Kyle JL, Harris E, 2008. Global spread and persistence of dengue. Annu Rev Microbiol 62 :71–92.
-
4
Harris E, Holden KL, Edgil D, Polacek C, Clyde K, 2006. Molecular biology of flaviviruses. Novartis Found Symp 277 :23–39.
-
5
Markoff L, 2003. 5′ and 3′-nocoding regions in flavivirus RNA. Adv Virus Res 59 :177–228.
-
6
Hahn CS, Hahn YS, Rice CM, Lee E, Dalgarno L, Strauss EG, Strauss JH, 1987. Conserved elements in the 3′ untranslated region of flavivirus RNAs and potential cyclization sequences. J Mol Biol 198 :33–41.
-
7
Alvarez DE, Lodeiro MF, Filomatori CV, Fucito S, Mondotte JA, Gamarnik AV, 2006. Structural and functional analysis of dengue virus RNA. Novartis Found Symp 277 :120–132.
-
8
Zhang B, Dong H, Stein DA, Iversen PL, Shi PY, 2008. West Nile virus genome cyclization and RNA replication require two pairs of long-distance RNA interactions. Virology 373 :1–13.
-
9
Holden KL, Harri E, 2004. Enhancement of dengue virus translation: role of the 3′ untranslated region and the terminal 3′ stem-loop domain. Virology 329 :119–133.
-
10
Li W, Brinton MA, 2001. The 3′ stem loop of the West Nile virus genomic RNA can suppress translation of chimeric mRNAs. Virology 287 :49–61.
-
11
Alvarez DE, De Lella Ezcurra AE, Fucito S, Gamarnik AV, 2005. Role of RNA structures present at the 3′ UTR of dengue virus on translation, RNA synthesis, and viral replication. Virology 339 :200–212.
-
12
Khromykh AA, Meka H, Guyatt KJ, Westaway EG, 2001. Essential role of cyclization sequences in flavivirus RNA replication. J Virol 75 :6719–6728.
-
13
Lo MK, Tilgner M, Bernard KA, Shi PY, 2003. Functional analysis of mosquito-borne flavivirus conserved sequence elements within 3′ untranslated region of West Nile virus by use of a reporting replicon that differentiates between viral translation and RNA replication. J Virol 77 :10004–10014.
-
14
Gritsun TS, Venugopal K, Zanotto PM, Mikhailov MV, Sall AA, Holmes EC, Polkinghorne I, Frolova TV, Pogodina VV, Lashkevich VA, Gould EA, 1997. Complete sequence of two tick-borne flaviviruses isolated from Siberia and the UK: analysis and significance of the 5′ and 3′UTRs. Virus Res 49 :27–39.
-
15
Men R, Bray M, Clark D, Chanock RM, Lai CJ, 1996. Dengue type 4 virus mutants containing deletions in the 3′ noncoding region of the RNA genome: analysis of growth restriction in cell culture and altered viremia pattern and immunogenicity in rhesus monkeys. J Virol 70 :3930–3937.
-
16
Nam JH, Chae SL, Won SY, Kim EJ, Yoon KS, Kim BI, Jeong YS, Cho HW, 2001. Short report: genetic heterogeneity of Japanese encephalitis virus assessed via analysis of the full-length genome sequence of a Korean isolate. Am J Trop Med Hyg 65 :388–392.
-
17
Tajima S, Nukui Y, Ito M, Takasaki T, Kurane I, 2006. Nineteen nucleotides in the variable region of 3′ non-translated region are dispensable for the replication of dengue type 1 virus in vitro. Virus Res 116 :38–44.
-
18
Tajima S, Nukui Y, Takasaki T, Kurane I, 2007. Characterization of the variable region in the 3′ non-translated region of dengue type 1 virus. J Gen Virol 88 :2214–2222.
-
19
Yang DK, Kim BH, Kweon CH, Kwon JH, Lim SI, Han HR, 2004. Molecular characterization of full-length genome of Japanese encephalitis virus (KV1899) isolated from pigs in Korea. J Vet Sci 5 :197–205.
-
20
Alvarez DE, Lodeiro MF, Luduena SJ, Pietrasanta LI, Gamarnik AV, 2005. Long-range RNA-RNA interactions circularize the dengue virus genome. J Virol 79 :6631–6643.
-
21
Chiu WW, Kinney RM, Dreher TW, 2005. Control of translation by the 5′ and 3′ terminal regions of the dengue virus genome. J Virol 79 :8303–8315.
-
22
Edgil D, Diamond MS, Holden KL, Paranjape SM, Harris E, 2003. Translation efficiency determines differences in cellular infection among dengue virus type 2 strains. Virology 317 :275–290.
-
23
Holden KL, Stein DA, Pierson TC, Ahmed AA, Clyde K, Iversen PL, Harris E, 2006. Inhibition of dengue virus translation and RNA synthesis by a morpholino oligomer targeted to the top of the terminal 3′ stem-loop structure. Virology 344 :439–452.
-
24
Zhu W, Qin C, Chen S, Jiang T, Yu M, Yu X, Qin E, 2007. Attenuated dengue 2 viruses with deletions in capsid protein derived from an infectious full-length cDNA clone. Virus Res 126 :226–232.
-
25
Qin CF, Qin ED, Yu M, Chen SP, Jiang T, Deng YQ, Duan HY, Zhao H, 2005. Therapeutic effects of dengue 2 virus capsid protein and staphylococcal nuclease fusion protein on dengue-infected cell cultures. Arch Virol 150 :659–669.
-
26
Baum DA, Silverman SK, 2008. Deoxyribozymes: useful DNA catalysts in vitro and in vivo. Cell Mol Life Sci 3 :29–32.
-
27
Santoro SW, Joyce GF, 1997. A general purpose RNA-cleaving DNA enzyme. Proc Natl Acad Sci USA 94 :4262–4266.
-
28
Santoro SW, Joyce GF, 1998. Mechanism and utility of an RNA-cleaving enzyme. Biochemistry 37 :13330–13342.
-
29
Schubert S, Furste JP, Werk D, Grunert HP, Zeichhardt H, Erdmann VA, Kurrech J, 2004. Gaining target access for deoxyribozymes. J Mol Biol 339 :355–363.
-
30
Cairns MJ, Hopkins TM, Witherington C, Wang L, Sun LQ, 1999. Target-site selection for an RNA-cleaving catalytic DNA. Nat Biotechnol 17 :480–486.
-
31
Dass CR, Choong PF, Khachigian LM, 2008. DNAzyme technology and cancer therapy: cleave and let die. Mol Cancer Ther 7 :243–251.
-
32
Pyle AM, Chu VT, Jankowsky E, Boudvillain M, 2000. Using DNAzymes to cut, process, and map RNA molecules for structural studies or modification. Methods Enzymol 317 :140–146.
-
33
Mathews DH, Turner DH, Zuker M, 2007. RNA secondary structure prediction. Curr Protoc Nucleic Acid Chem 11 : Unit 11.2.
-
34
Zuker M, 2003. mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31 :3406–3415.
-
35
Wei Y, Jiang T, Li X, Zhao H, Liu Z, Deng Y, Liu R, Qin C, Qin E, 2008. Effects of the RNA elements within 3′ untranslated region on dengue virus translation. Wei Sheng Wu Xue Bao 48 :583–588.
-
36
Leitmeyer KC, Vaughn DW, Watts DM, Salas R, Villalobos I, de Chacon Ramos C, Rico-Hesse R, 1999. Dengue virus structural differences that correlate with pathogenesis. J Virol 73 :4738–4747.
-
37
Tilgner M, Deas TS, Shi PY, 2005. The flavivirus-conserved penta-nucleotide in the 3′ stem–loop of West Nile virus genome requires a specific sequence and structure for RNA synthesis, but not for viral translation. Virology 331 :375–386.
-
38
Romero TA, Tumban E, Jun J, Lott WB, Hanley KA, 2006. Secondary structure of dengue virus type 4 3′ untranslated region: impact of deletion and substitution mutations. J Gen Virol 87 :3291–3296.
| Past two years | Past Year | Past 30 Days | |
|---|---|---|---|
| Abstract Views | 408 | 324 | 11 |
| Full Text Views | 800 | 9 | 0 |
| PDF Downloads | 323 | 7 | 0 |
Translational Regulation by the 3′ Untranslated Region of the Dengue Type 2 Virus Genome
Yan Wei
Yan Wei
State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, People’s Republic of China
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Chengfeng Qin
Chengfeng Qin
State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, People’s Republic of China
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Tao Jiang
Tao Jiang
State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, People’s Republic of China
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Xiaofeng Li
Xiaofeng Li
State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, People’s Republic of China
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Hui Zhao
Hui Zhao
State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, People’s Republic of China
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Zhongyu Liu
Zhongyu Liu
State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, People’s Republic of China
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Yongqiang Deng
Yongqiang Deng
State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, People’s Republic of China
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Ran Liu
Ran Liu
State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, People’s Republic of China
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Shuiping Chen
Shuiping Chen
State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, People’s Republic of China
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Man Yu
Man Yu
State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, People’s Republic of China
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Ede Qin
Ede Qin
State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, People’s Republic of China
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The role of the 3′untranslated region (UTR) of the dengue virus (DENV) genome during viral translation remains to be elucidated. We assessed the contribution of well-defined RNA elements in the 3′UTR of DENV-2 to viral translation using a virus-induced reporting gene system and deoxyribozymes (DRzs) targeting the 3′UTR of the DENV-2 genome. Results show that mRNAs carrying a deletion of repeated conserved sequence (RCS2)-CS2 are translated less efficiently than wild type mRNAs. However, mRNAs with a deletion of CS1-stem loop (SL) are translated more efficiently. Thus, CS1-SL and RCS2-CS2 may have different effects on translational regulation. Additionally, the translation-suppressing effect of CS1-SL or the SL element is further confirmed in DENV-2-infected cells using DRzs. Mutagenesis studies show that, rather than the secondary structure, nucleotides 10663–10677 and 10709–10723 are responsible for translational suppression of SL. Overall, our results demonstrate that sequences and elements within the DENV-2 3′UTR regulate viral translation.
Author Notes
- INTRODUCTION
- MATERIALS AND METHODS
- Cells and virus.
- Plasmids and VIRG construction.
- RNA transcription and cell transfection.
- Deoxyribozyme reactions in vitro.
- Northern blot assay.
- Deoxyribozyme assays in BHK21 cells.
- Luciferase activity assay.
- Enzyme-linked immunosorbent assay.
- Computer-assisted secondary structure prediction.
- Statistical analysis.
- RESULTS
- DISCUSSION
ProCite
RefWorks
Reference Manager
BibTeX
Zotero
EndNote
-
1
Gould EA, Solomon T, 2008. Pathogenic flaviviruses. Lancet 371 :500–509.
-
2
Rico-Hesse R, 2003. Microevolution and virulence of dengue viruses. Adv Virus Res 59 :315–341.
-
3
Kyle JL, Harris E, 2008. Global spread and persistence of dengue. Annu Rev Microbiol 62 :71–92.
-
4
Harris E, Holden KL, Edgil D, Polacek C, Clyde K, 2006. Molecular biology of flaviviruses. Novartis Found Symp 277 :23–39.
-
5
Markoff L, 2003. 5′ and 3′-nocoding regions in flavivirus RNA. Adv Virus Res 59 :177–228.
-
6
Hahn CS, Hahn YS, Rice CM, Lee E, Dalgarno L, Strauss EG, Strauss JH, 1987. Conserved elements in the 3′ untranslated region of flavivirus RNAs and potential cyclization sequences. J Mol Biol 198 :33–41.
-
7
Alvarez DE, Lodeiro MF, Filomatori CV, Fucito S, Mondotte JA, Gamarnik AV, 2006. Structural and functional analysis of dengue virus RNA. Novartis Found Symp 277 :120–132.
-
8
Zhang B, Dong H, Stein DA, Iversen PL, Shi PY, 2008. West Nile virus genome cyclization and RNA replication require two pairs of long-distance RNA interactions. Virology 373 :1–13.
-
9
Holden KL, Harri E, 2004. Enhancement of dengue virus translation: role of the 3′ untranslated region and the terminal 3′ stem-loop domain. Virology 329 :119–133.
-
10
Li W, Brinton MA, 2001. The 3′ stem loop of the West Nile virus genomic RNA can suppress translation of chimeric mRNAs. Virology 287 :49–61.
-
11
Alvarez DE, De Lella Ezcurra AE, Fucito S, Gamarnik AV, 2005. Role of RNA structures present at the 3′ UTR of dengue virus on translation, RNA synthesis, and viral replication. Virology 339 :200–212.
-
12
Khromykh AA, Meka H, Guyatt KJ, Westaway EG, 2001. Essential role of cyclization sequences in flavivirus RNA replication. J Virol 75 :6719–6728.
-
13
Lo MK, Tilgner M, Bernard KA, Shi PY, 2003. Functional analysis of mosquito-borne flavivirus conserved sequence elements within 3′ untranslated region of West Nile virus by use of a reporting replicon that differentiates between viral translation and RNA replication. J Virol 77 :10004–10014.
-
14
Gritsun TS, Venugopal K, Zanotto PM, Mikhailov MV, Sall AA, Holmes EC, Polkinghorne I, Frolova TV, Pogodina VV, Lashkevich VA, Gould EA, 1997. Complete sequence of two tick-borne flaviviruses isolated from Siberia and the UK: analysis and significance of the 5′ and 3′UTRs. Virus Res 49 :27–39.
-
15
Men R, Bray M, Clark D, Chanock RM, Lai CJ, 1996. Dengue type 4 virus mutants containing deletions in the 3′ noncoding region of the RNA genome: analysis of growth restriction in cell culture and altered viremia pattern and immunogenicity in rhesus monkeys. J Virol 70 :3930–3937.
-
16
Nam JH, Chae SL, Won SY, Kim EJ, Yoon KS, Kim BI, Jeong YS, Cho HW, 2001. Short report: genetic heterogeneity of Japanese encephalitis virus assessed via analysis of the full-length genome sequence of a Korean isolate. Am J Trop Med Hyg 65 :388–392.
-
17
Tajima S, Nukui Y, Ito M, Takasaki T, Kurane I, 2006. Nineteen nucleotides in the variable region of 3′ non-translated region are dispensable for the replication of dengue type 1 virus in vitro. Virus Res 116 :38–44.
-
18
Tajima S, Nukui Y, Takasaki T, Kurane I, 2007. Characterization of the variable region in the 3′ non-translated region of dengue type 1 virus. J Gen Virol 88 :2214–2222.
-
19
Yang DK, Kim BH, Kweon CH, Kwon JH, Lim SI, Han HR, 2004. Molecular characterization of full-length genome of Japanese encephalitis virus (KV1899) isolated from pigs in Korea. J Vet Sci 5 :197–205.
-
20
Alvarez DE, Lodeiro MF, Luduena SJ, Pietrasanta LI, Gamarnik AV, 2005. Long-range RNA-RNA interactions circularize the dengue virus genome. J Virol 79 :6631–6643.
-
21
Chiu WW, Kinney RM, Dreher TW, 2005. Control of translation by the 5′ and 3′ terminal regions of the dengue virus genome. J Virol 79 :8303–8315.
-
22
Edgil D, Diamond MS, Holden KL, Paranjape SM, Harris E, 2003. Translation efficiency determines differences in cellular infection among dengue virus type 2 strains. Virology 317 :275–290.
-
23
Holden KL, Stein DA, Pierson TC, Ahmed AA, Clyde K, Iversen PL, Harris E, 2006. Inhibition of dengue virus translation and RNA synthesis by a morpholino oligomer targeted to the top of the terminal 3′ stem-loop structure. Virology 344 :439–452.
-
24
Zhu W, Qin C, Chen S, Jiang T, Yu M, Yu X, Qin E, 2007. Attenuated dengue 2 viruses with deletions in capsid protein derived from an infectious full-length cDNA clone. Virus Res 126 :226–232.
-
25
Qin CF, Qin ED, Yu M, Chen SP, Jiang T, Deng YQ, Duan HY, Zhao H, 2005. Therapeutic effects of dengue 2 virus capsid protein and staphylococcal nuclease fusion protein on dengue-infected cell cultures. Arch Virol 150 :659–669.
-
26
Baum DA, Silverman SK, 2008. Deoxyribozymes: useful DNA catalysts in vitro and in vivo. Cell Mol Life Sci 3 :29–32.
-
27
Santoro SW, Joyce GF, 1997. A general purpose RNA-cleaving DNA enzyme. Proc Natl Acad Sci USA 94 :4262–4266.
-
28
Santoro SW, Joyce GF, 1998. Mechanism and utility of an RNA-cleaving enzyme. Biochemistry 37 :13330–13342.
-
29
Schubert S, Furste JP, Werk D, Grunert HP, Zeichhardt H, Erdmann VA, Kurrech J, 2004. Gaining target access for deoxyribozymes. J Mol Biol 339 :355–363.
-
30
Cairns MJ, Hopkins TM, Witherington C, Wang L, Sun LQ, 1999. Target-site selection for an RNA-cleaving catalytic DNA. Nat Biotechnol 17 :480–486.
-
31
Dass CR, Choong PF, Khachigian LM, 2008. DNAzyme technology and cancer therapy: cleave and let die. Mol Cancer Ther 7 :243–251.
-
32
Pyle AM, Chu VT, Jankowsky E, Boudvillain M, 2000. Using DNAzymes to cut, process, and map RNA molecules for structural studies or modification. Methods Enzymol 317 :140–146.
-
33
Mathews DH, Turner DH, Zuker M, 2007. RNA secondary structure prediction. Curr Protoc Nucleic Acid Chem 11 : Unit 11.2.
-
34
Zuker M, 2003. mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31 :3406–3415.
-
35
Wei Y, Jiang T, Li X, Zhao H, Liu Z, Deng Y, Liu R, Qin C, Qin E, 2008. Effects of the RNA elements within 3′ untranslated region on dengue virus translation. Wei Sheng Wu Xue Bao 48 :583–588.
-
36
Leitmeyer KC, Vaughn DW, Watts DM, Salas R, Villalobos I, de Chacon Ramos C, Rico-Hesse R, 1999. Dengue virus structural differences that correlate with pathogenesis. J Virol 73 :4738–4747.
-
37
Tilgner M, Deas TS, Shi PY, 2005. The flavivirus-conserved penta-nucleotide in the 3′ stem–loop of West Nile virus genome requires a specific sequence and structure for RNA synthesis, but not for viral translation. Virology 331 :375–386.
-
38
Romero TA, Tumban E, Jun J, Lott WB, Hanley KA, 2006. Secondary structure of dengue virus type 4 3′ untranslated region: impact of deletion and substitution mutations. J Gen Virol 87 :3291–3296.
| Past two years | Past Year | Past 30 Days | |
|---|---|---|---|
| Abstract Views | 408 | 324 | 11 |
| Full Text Views | 800 | 9 | 0 |
| PDF Downloads | 323 | 7 | 0 |