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Peer-reviewed

RNA-dependent RNA Polymerase Assay for Hepatitis E Virus

戊型肝炎病毒依赖于RNA的RNA聚合酶活性测定

VN Vidya P. Nair
SA Saumya Anang
AS Akriti Srivastava
MS Milan Surjit

发布: 2017年04月05日第7卷第7期 DOI: 10.21769/BioProtoc.2199 浏览次数: 10581

评审: Yannick DebingVamseedhar RayaproluDavid Paul

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参见作者原研究论文

The authors used this protocol in:

Cover of PLOS Pathogens, featuring study using the protocol.

Apr 2016

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Abstract

RNA-dependent RNA polymerase (RdRp) is essential for the replication of viral RNA for RNA viruses. It synthesizes the complementary strand of viral genomic RNA, which is used subsequently as a template to generate more copies of viral genome. This assay measures activity of the hepatitis E virus (HEV) RdRp. In contrast to protocols available to assay the RdRp activity of many other viruses, this assay utilizes DIG-11-UTP as a nonradioactive alternative to 32P-UTP, thereby increasing the convenience of performing the assay.

Keywords: Hepatitis E virus (戊型肝炎病毒) search RNA virus (RNA病毒) search RdRp (RdRp) search RdRp assay (RdRp测定) search Viral replication (病毒复制) search Nonradioactive RdRp assay (非放射性RdRp测定) search

Background

No assay was available to measure the activity of HEV RdRp. RdRp activity has been measured in few other viruses such as hepatitis C virus using a radiolabeled nucleotide (Behrens et al., 1996). We have adapted the protocol described by Behrens et al. (1996) and modified it to establish a non-radioactive assay protocol, which is dependent on incorporation of DIG-11-UTP into the antisense RNA strand as a measure of the activity of HEV RdRp. This assay utilizes an in vitro synthesized viral RNA fragment as a template to measure the activity of HEV RdRp protein purified from human hepatoma cells using a chemiluminescence-based strategy.


Materials and Reagents

  1. 60 mm plates
  2. 1.6 ml RNase free microcentrifuge tube
  3. PVDF (Polyvinylidene fluoride) membrane (Pall, catalog number: BSP0149 )
  4. Nylon membrane (Pall, catalog number: 60207 )
  5. Whatman filter paper (Sigma-Aldrich, catalog number: WHA10426972 )
  6. Cling film wrap/plastic food wrap (available in general stores/supermarkets)
  7. Blotting paper (Praveen Scientific, catalog number: PSC 006 )
    Note: It is general blotting paper, used in labs in routine application, similar to but cheaper than Whatman filter paper.
  8. Mammalian expression plasmids (pUNO RdRp-flag [RdRp sequence with C-terminal flag tag was PCR amplified, digested with AgeI, NheI and ligated into pUNO vector digested with same], pUNO ORF2-flag [ORF2 with C-terminal flag tag was PCR amplified with primers: pUNO ORF2-flag FP, pUNO ORF2-flag RP; digested with BglII and ligated into pUNO vector digested with NheI [blunted] and BamHI); Nair et al. [2016])
  9. Huh7 (human hepatoma cells, obtained from Dr. C. M. Rice; Blight et al. [2000])
  10. pSKHEV2 RdRp template (pSK HRt) plasmid (Genbank No. AF444002.1, Emerson et al., 2004)
  11. Lipofectamine 2000 (Thermo Fisher Scientific, InvitrogenTM, catalog number: 11668019 )
  12. DMEM
  13. 10% fetal bovine serum (FBS)
  14. Phosphate-buffered saline (PBS) (Bio Basic, catalog number: PD0100 )
  15. Flag M2 agarose resin (Sigma-Aldrich, catalog number: A2220 )
  16. Flag peptide (Sigma-Aldrich, catalog number: F4799 )
  17. Octa-probe antibody (Santa Cruz Biotechnology, catalog number: sc-807 )
  18. Skimmed milk powder (Sigma-Aldrich, catalog number: 70166 )
    Note: This product has been discontinued.
  19. Anti-rabbit IgG Horseradish peroxidase(HRPO) (Santa Cruz Biotechnology, catalog number: sc-2004 )
  20. Clarity Western ECL blotting substrate (Bio-Rad Laboratories, catalog number: 1705061 )
  21. Silver stain kit (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 24612 )
  22. Bradford assay reagent (Bio-Rad Laboratories, catalog number: 5000002 )
  23. StuI, NheI and Tth111I restriction enzymes
  24. BglII (New England Biolabs, catalog number: R0144L )
  25. PCR purification kit (Agilent Technologies, catalog number: 400771 )
  26. mMessage mMachine T7 kit (Thermo Fisher Scientific, AmbionTM, catalog number: AM1344 )
  27. RNAsin
  28. ATP
  29. CTP
  30. GTP
  31. UTP
  32. DIG-UTP
  33. Actinomycin D (Sigma-Aldrich, catalog number: A1410 )
  34. DMSO
  35. RNase A
  36. Proteinase K (Sigma-Aldrich, catalog number: P2308 )
  37. Glycogen (Sigma-Aldrich, catalog number: G0885 )
  38. Ethanol (EMD Millipore, catalog number: 100983 )
  39. Nuclease free water (Thermo Fisher Scientific, AmbionTM, catalog number: AM9937 )
  40. Agarose (Bio Basic, catalog number: AB0014 )
  41. DEPC-treated water (Thermo Fisher Scientific, AmbionTM, catalog number: AM9922 )
  42. 37% formaldehyde (Sigma-Aldrich, catalog number: F8775 )
  43. Formamide (Sigma-Aldrich, catalog number: F9037 )
  44. Gel loading dye
  45. Millennium marker (Thermo Fisher Scientific, AmbionTM, catalog number: AM7151 )
  46. Ethidium bromide (Sigma-Aldrich, catalog number: E1510 )
  47. DIG Northern Starter Kit (Roche Diagnostics, catalog number: 12039672910 )
  48. Tris (Sigma-Aldrich, catalog number: T6066 )
  49. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
  50. Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E6758 )
  51. Ethylene glycol-bis(2-aminoethylether)-N,N,N’,N’,-tetraacetic acid (EGTA) (Sigma-Aldrich, catalog number: 03777 )
  52. Triton X-100 (Sigma-Aldrich, catalog number: 93418 )
    Note: This product has been discontinued.
  53. Sodium pyrophosphate tetrabasic decahydrate (Na4P2O4·10H2O) (Sigma-Aldrich, catalog number: S6422 )
  54. β-glycerol phosphate (Sigma-Aldrich, catalog number: 50020 )
  55. Sodium orthovanadate (Na3VO4) (Sigma-Aldrich, catalog number: S6508 )
  56. Protease inhibitor cocktail (Roche Diagnostics, catalog number: 04693132001 )
  57. Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M8266 )
  58. DL-dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D0632 )
  59. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333 )
  60. SDS
  61. MOPS (Sigma-Aldrich, catalog number: M5162 )
  62. Sodium acetate (Sigma-Aldrich, catalog number: S2889 )
  63. Maleic acid (Sigma-Aldrich, catalog number: M0375 )
  64. Tween 20 (Sigma-Aldrich, catalog number: P9416 )
  65. Tri-sodium citrate dihydrate (Himedia, catalog number: RM1415 )
  66. IP (Immunoprecipitation) buffer (see Recipes)
  67. 5x assay buffer (see Recipes)
  68. 2x proteinase K buffer (see Recipes)
  69. 10x MOPS (pH 7.0) (see Recipes)
  70. 20x SSC buffer (see Recipes)
  71. Maleic acid buffer (pH 7.5) (see Recipes)
  72. 1x blocking solution (see Recipes)
  73. Washing buffer (pH 7.5) (see Recipes)
  74. Detection buffer (pH 9.5) (see Recipes)
  75. Phosphate buffered saline with 0.1% Tween 20 (PBST, see Recipes)


Equipment

  1. 5% CO2 incubator
  2. Centrifuge
  3. Flip-flop rocker
  4. Gel documentation system (Bio-Rad Laboratories, model: ChemiDocTM MP )
  5. Chemical fume hood
  6. Conical flask
  7. Standard horizontal agarose gel electrophoresis apparatus
  8. Rectangular glass tray
  9. Glass plates
  10. Glass jar
  11. UV cross linker (UVP, model: CL-1000 )


Procedure

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文章信息

版权信息

© 2017 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

  1. Nair, V. P., Anang, S., Srivastava, A. and Surjit, M. (2017). RNA-dependent RNA Polymerase Assay for Hepatitis E Virus. Bio-protocol 7(7): e2199. DOI: 10.21769/BioProtoc.2199.
  2. Nair, V. P., Anang, S., Subramani, C., Madhvi, A., Bakshi, K., Srivastava, A., Shalimar, Nayak, B., Ct, R. K. and Surjit, M. (2016). Endoplasmic reticulum stress induced synthesis of a novel viral factor mediates efficient replication of genotype-1 hepatitis E virus. PLoS Pathog 12(4): e1005521.

    3. ris ris Download Citation in RIS Format

分类

微生物学 > 微生物生物化学 > RNA

生物化学 > RNA > RNA-蛋白质相互作用

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