Identification of Nonstructural Protein 8 as the N-Terminus of the RNA-Dependent RNA Polymerase of Porcine Reproductive and Respiratory Syndrome Virus
- 157 Downloads
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.
KeywordsPorcine reproductive and respiratory syndrome virus (PRRSV) Nsp8 Nsp9
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.
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.
- den Boon JA, Snijder EJ, Chirnside ED, de Vries AA, Horzinek MC, Spaan WJ (1991) Equine arteritis virus is not a togavirus but belongs to the coronavirus like superfamily. J Virol 65:2910–2920Google Scholar
- den Boon JA, Faaberg KS, Meulenberg JJ, Wassenaar AL, Plagemann PG, Gorbalenya AE, Snijder EJ (1995) Processing and evolution of the N-terminal region of the arterivirus replicase ORF1a protein: identification of two papain like cysteine proteases. J Virol 69:4500–4505Google Scholar
- Kuhn JH, Lauck M, Bailey AL, Shchetinin AM, Vishnevskaya TV, Bao Y, Ng TF, LeBreton M, Schneider BS, Gillis A, Tamoufe U, Diffo Jle D, Takuo JM, Kondov NO, Coffey LL, Wolfe ND, Delwart E, Clawson AN, Postnikova E, Bollinger L, Lackemeyer MG, Radoshitzky SR, Palacios G, Wada J, Shevtsova ZV, Jahrling PB, Lapin BA, Deriabin PG, Dunowska M, Alkhovsky SV, Rogers J, Friedrich TC, O’Connor DH, Goldberg TL (2016) Reorganization and expansion of the nidoviral family Arteriviridae. Arch Virol 161:755–768CrossRefGoogle Scholar
- Lehmann KC, Gulyaeva A, Zevenhoven-Dobbe JC, Janssen GM, Ruben M, Overkleeft HS, van Veelen PA, Samborskiy DV, Kravchenko AA, Leontovich AM, Sidorov IA, Snijder EJ, Posthuma CC, Gorbalenya AE (2015) Discovery of an essential nucleotidylating activity associated with a newly delineated conserved domain in the RNA polymerase-containing protein of all nidoviruses. Nucl Acids Res 43:8416–8434CrossRefGoogle Scholar
- Tian K, Yu X, Zhao T, Feng Y, Cao Z, Wang C, Hu Y, Chen X, Hu D, Tian X, Liu D, Zhang S, Deng X, Ding Y, Yang L, Zhang Y, Xiao H, Qiao M, Wang B, Hou L, Wang X, Yang X, Kang L, Sun M, Jin P, Wang S, Kitamura Y, Yan J, Gao GF (2007) Emergence of fatal PRRSV variants: unparalleled outbreaks of atypical PRRS in China and molecular dissection of the unique hallmark. PLoS ONE 2:e526CrossRefGoogle Scholar
- Xu L, Zhou L, Sun W, Zhang P, Ge X, Guo X, Han J, Yang H (2018) Nonstructural protein 9 residues 586 and 592 are critical sites in determining the replication efficiency and fatal virulence of the Chinese highly pathogenic porcine reproductive and respiratory syndrome virus. Virology 517:135–147CrossRefGoogle Scholar
- Zhao K, Gao JC, Xiong JY, Guo JC, Yang YB, Jiang CG, Tang YD, Tian ZJ, Cai XH, Tong GZ, An TQ (2018) Two residues in NSP9 contribute to the enhanced replication and pathogenicity of highly pathogenic porcine reproductive and respiratory syndrome virus. J Virol 92:e02209–17PubMedPubMedCentralGoogle Scholar