Virus Genes

, Volume 53, Issue 6, pp 831–839 | Cite as

The 3D protein of duck hepatitis A virus type 1 binds to a viral genomic 3′ UTR and shows RNA-dependent RNA polymerase activity

  • Yu Zhang
  • Qianda Cao
  • Mingshu Wang
  • Renyong Jia
  • Shun Chen
  • Dekang Zhu
  • Mafeng Liu
  • Kunfeng Sun
  • Qiao Yang
  • Ying Wu
  • Xinxin Zhao
  • Xiaoyue Chen
  • Anchun Cheng
Article
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Abstract

To explore the RNA-dependent RNA polymerase (RdRP) function of the 3D protein of duck hepatitis A virus type 1 (DHAV-1), the gene was cloned into the pET-32a(+) vector for prokaryotic expression. The 3′ untranslated region (3′ UTR) of DHAV-1 together with a T7 promoter was cloned into the pMD19-T vector for in vitro transcription of 3′ UTR RNA, which was further used as a template in RNA-dependent RNA polymerization. In this study, three methods were applied to analyze the RdRP function of the 3D protein: (1) ammonium molybdate spectrophotometry to detect pyrophosphate produced during polymerization; (2) quantitative reverse transcription PCR (RT-qPCR) to investigate the changes in RNA quantity during polymerization; and (3) electrophoresis mobility shift assay to examine the interaction between the 3D protein and 3′ UTR. The results showed the 3D protein was successfully expressed in bacteria culture supernatant in a soluble form, which could be purified by affinity chromatography. In 3D enzymatic activity assays, pyrophosphate and RNA were produced, the amounts of which increased based on approximative kinetics, and binding of the 3D protein to the 3′ UTR was observed. These results indicate that prokaryotically expressed soluble DHAV-13D protein can bind to a viral genomic 3′ UTR and exhibit RdRP activity.

Keywords

DHAV-1 3D protein RdRP activity 3′ UTR 
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Notes

Acknowledgements

The research was supported by the National Natural Science Foundation of China (Grant No. 31472223), the China Agricultural Research System (CARS-43-8), and the Integration and Special Fund for Key Laboratory of Animal Disease and Human Health of Sichuan Province (2016JPT0004).

Statement of author contributions

YZ cloned plasmids, purified the protein, performed the enzymatic activity detection and wrote the manuscript. QC cloned plasmids and expressed and identified the protein. MW and AC designed the experiments. RJ, SC, DZ, ML, KS, QY, YW, XZ, and XC helped with proofreading of the manuscript.

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not include any studies with human participants or animals performed by any of the authors.

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Copyright information

© Springer Science+Business Media, LLC 2017

Authors and Affiliations

  • Yu Zhang
    • 1
    • 2
  • Qianda Cao
    • 1
    • 2
  • Mingshu Wang
    • 1
    • 2
    • 3
  • Renyong Jia
    • 1
    • 2
    • 3
  • Shun Chen
    • 1
    • 2
    • 3
  • Dekang Zhu
    • 2
    • 3
  • Mafeng Liu
    • 1
    • 2
    • 3
  • Kunfeng Sun
    • 1
    • 2
    • 3
  • Qiao Yang
    • 1
    • 2
    • 3
  • Ying Wu
    • 1
    • 2
    • 3
  • Xinxin Zhao
    • 1
    • 2
    • 3
  • Xiaoyue Chen
    • 1
    • 2
  • Anchun Cheng
    • 1
    • 2
    • 3
  1. 1.Institute of Preventive Veterinary MedicineSichuan Agricultural UniversityChengduPeople’s Republic of China
  2. 2.Key Laboratory of Animal Disease and Human Health of Sichuan ProvinceSichuan Agricultural UniversityChengduPeople’s Republic of China
  3. 3.Research Center of Avian Disease, College of Veterinary MedicineSichuan Agricultural UniversityChengduPeople’s Republic of China

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