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Virologica Sinica

, Volume 33, Issue 5, pp 429–439 | Cite as

Identification of Nonstructural Protein 8 as the N-Terminus of the RNA-Dependent RNA Polymerase of Porcine Reproductive and Respiratory Syndrome Virus

  • Yuanyuan Liu
  • Yunhao Hu
  • Yue Chai
  • Liping Liu
  • Jiangwei Song
  • Shaochuan Zhou
  • Jia Su
  • Lei Zhou
  • Xinna Ge
  • Xin Guo
  • Jun Han
  • Hanchun Yang
Research Article
  • 157 Downloads

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) is a member within the family Arteriviridae of the order Nidovirales. Replication of this positive-stranded RNA virus within the host cell involves expression of viral replicase proteins encoded by two ORFs, namely ORF1a and ORF1b. In particular, translation of ORF1b depends on a -1-ribosomal frameshift strategy. Thus, nonstructural protein 9 (nsp9), the first protein within ORF1b that specifies the function of the viral RNA-dependent RNA polymerase, is expressed as the C-terminal extension of nsp8, a small nsp that is encoded by ORF1a. However, it has remained unclear whether the mature form of nsp9 in virus-infected cells still retains nsp8, addressing which is clearly critical to understand the biological function of nsp9. By taking advantage of specific antibodies to both nsp8 and nsp9, we report the following findings. (1) In infected cells, PRRSV nsp9 was identified as a major product with a size between 72 and 95 kDa (72–95 KDa form), which exhibited the similar mobility on the gel to the in vitro expressed nsp8–9ORF1b, but not the ORF1b-coded portion (nsp9ORF1b). (2) The antibodies to nsp8, but not to nsp7 or nsp10, could detect a major product that had the similar mobility to the 72–95 KDa form of nsp9. Moreover, nsp9 could be co-immunoprecipitated by antibodies to nsp8, and vice versa. (3) Neither nsp4 nor nsp2 PLP2 was able to cleave nsp8–nsp9 in vitro. Together, our studies provide experimental evidence to suggest that nsp8 is an N-terminal extension of nsp9. Our findings here paves way for further charactering the biological function of PRRSV nsp9.

Keywords

Porcine reproductive and respiratory syndrome virus (PRRSV) Nsp8 Nsp9 

Notes

Acknowledgements

This work was supported by the National Key Basic Research Plan Grant from the Chinese Ministry of Science and Technology (2014CB542700), the China National Thousand Youth Talents program (1051-21986001), and the earmarked fund for China Agriculture Research System (CARS-35) from the Chinese Ministry of Agriculture.

Author Contributions

HY, JH and YL conceptualized and designed the study. YL and JS performed the experiments in the study. YH, YC, LPL, JS, SZ and JS contributed reagents to this study. JH and YL analyzed the data. ZL, XNG and XG contributed to the study design. JH, YL and HY wrote the manuscript. All authors read and approved the final manuscript.

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no conflict of interest.

Animal and Human Rights Statement

The animal experiments in this study were approved by The Laboratory Animal Ethical Committee of China Agricultural University. All institutional and national guidelines for the care and use of animals were followed.

Supplementary material

12250_2018_54_MOESM1_ESM.pdf (368 kb)
Supplementary material 1 (PDF 369 kb)

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Copyright information

© Wuhan Institute of Virology, CAS and Springer Nature Singapore Pte Ltd. 2018

Authors and Affiliations

  1. 1.Key Laboratory of Animal Epidemiology of the Ministry of Agriculture, College of Veterinary Medicine and State Key Laboratory of AgrobiotechnologyChina Agricultural UniversityBeijingChina

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