Eukaryotic Expression and Bioinformatics Analysis of RNA-dependent RNA Polymerase of Duck tembusu virus

doi:10.7668/hbnxb.20192082

Abstract

Abstract: The purpose of this study is to express the RdRp of DTMUV in eukaryotic cells and to analyze its structure and function by bioinformatics. Firstly,specific primers were designed and synthesized based on the sequence of the RdRp gene of DTMUV JS804 strain in GenBank,and then the gene was amplified by RT-PCR,subcloned into the pCMV-N-Flag eukaryotic expression vector,transfected into BHK-21 cells. Next,the expression of RdRp was determined by immunofluorescence observation and Western Blot. After that,bioinformatics software was used to analyze the sequence,structure and function of RdRp. RdRp gene was successfully amplified by PCR,and the constructed eukaryotic expression plasmid PCMV-Flag-RdRp was verified to be correct by double enzyme digestion and identification. Further,indirect immunofluorescence and Western Blot detection showed that the recombinant plasmid PCMV-Flag-RdRp was normally expressed in BHK-21 cells. Bioinformatics analysis found that,DTMUV RdRp consisted of 606 amino acids,with a formula of C₃₀₉₁H₄₈₁₀N₈₇₀O₈₉₄S₄₁,an average hydropathicity of-0.536,and an instability index of 42.15. The RdRp was rich in glycosylation and phosphorylation sites. The ratios of α-helix,random coil and extending strand in the secondary structure were 46.37%,35.31%,12.54%,respectively. All of these results provided supporting data for the further research on biological functions of DTMUV.

Key words: Duck tembusu virus, RNA-dependent RNA polymerase, Bioinformatics

CLC Number:

Q78
S432.4

HAN Kaikai, YAN Ruofeng, LIU Qingtao, LIU Yuzhuo, LI Yin, ZHAO Dongmin, HUANG Xinmei, ZHANG Lijiao, YANG Jing, FU Chen. Eukaryotic Expression and Bioinformatics Analysis of RNA-dependent RNA Polymerase of Duck tembusu virus[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2021, 36(6): 204-210. doi: 10.7668/hbnxb.20192082.

/ / Recommend / Download Citations

URL: http://www.hbnxb.net/EN/10.7668/hbnxb.20192082

http://www.hbnxb.net/EN/Y2021/V36/I6/204

References

[1] Li N,Zhao J,Yang Y D,Zeng Y Q,Liu S D. Innate immune responses to duck Tembusu virus infection[J]. Veterinary Research,2020,51(1):87.doi:10.1186/s13567-020-00814-9.
[2] Zhao D M,Han K K,Huang X M,Zhang L J,Wang H L,Liu N,Tian Y J,Liu Q T,Yang J,Liu Y Z,Li Y. Screening and identification of B-cell epitopes within envelope protein of Tembusu virus[J]. Virology Journal,2018,15(1):142.doi:10.1186/s12985-018-1052-1.
[3] Wang X L,Yang Z Y,Wang X Q,Duan H J,Liu L X,Cheng H M,Yang C H,Hou L D,Pan J,Zhao J C,Liu Y H,Lin J. Development of a hemagglutination inhibition assay for Duck tembusu virus[J]. Avian Diseases,2019,63(2):298-301.doi:10.1637/11954-082018-Reg.1.
[4] Zhang W,Chen S,Mahalingam S,Wang M S,Cheng A C. An updated review of avian-origin Tembusu virus:A newly emerging avian Flavivirus[J]. The Journal of General Virology,2017,98(10):2413-2420.doi:10.1099/jgv.0.000908.
[5] Zhang L J,Zhao D M,Han K K,Huang X M,Liu Y Z,Liu Q T,Yang J,Li S,Li Y. Tembusu virus enters BHK-21 cells through a cholesterol-dependent and clathrin-mediated endocytosis pathway[J]. Microbial Pathogenesis,2020,147:104242.doi:10.1016/j.micpath.2020.104242.
[6] Sun X J,Liu E X,Iqbal A,Wang T Z,Wang X D,Haseeb A,Ahmed N,Yang P,Chen Q S. The dynamic distribution of Duck tembusu virus in the spleen of infected shelducks[J]. BMC Veterinary Research,2019,15(1):112.doi:10.1186/s12917-019-1860-6.
[7] Li L L,Zhang Y,Dong J W,Zhang J Q,Zhang C H,Qin J R,Sun M H,Xu Z H. Development of chimeric virus-like particles containing the E glycoprotein of Duck tembusu virus[J]. Veterinary Microbiology,2019,238:108425.doi:10.1016/j.vetmic.2019.108425.
[8] Lü J,Yang L X,Qu S H,Meng R Z,Li Q,Liu H C,Wang X Y,Zhang D B. Detection of neutralizing antibodies to Tembusu virus:Implications for infection and immunity[J]. Frontiers in Veterinary Science,2019,6:442.doi:10.3389/fvets.2019.00442.
[9] Zhao D M,Yang J,Han K K,Liu Q T,Wang H L,Liu Y Z,Huang X M,Zhang L J,Li Y. The unfolded protein response induced by Tembusu virus infection[J]. BMC Veterinary Research,2019,15(1):34.doi:10.1186/s12917-019-1781-4.
[10] Chen L,Yu B,Hua J G,Ni Z,Ye W C,Yun T,Zhang C. Optimized expression of Duck tembusu virus E gene delivered by a vectored Duck enteritis virus in vitro[J]. Molecular Biotechnology,2019,61(10):783-790.doi:10.1007/s12033-019-00206-1.
[11] Wang Y W,Zhang S,Tang Y,Diao Y X. Screening of Duck tembusu virus NS3 interacting host proteins and identification of its specific interplay domains[J]. Viruses,2019,11(8):740.doi:10.3390/v11080740.
[12] Peng S H,Su C L,Chang M C,Hu H C,Yang S L,Shu P Y. Genome analysis of a novel Tembusu virus in Taiwan[J]. Viruses,2020,12(5):567.doi:10.3390/v12050567.
[13] Han K K,Zhao D M,Liu Y Z,Liu Q T,Huang X M,Yang J,Zhang L J,Li Y. The ubiquitin-proteasome system is necessary for the replication of Duck tembusu virus[J]. Microbial Pathogenesis,2019,132:362-368.doi:10.1016/j.micpath.2019.04.044.
[14] 刘强强,王明连,李艳. 登革病毒非结构蛋白NS5研究进展[J].中国病毒病杂志,2016,6(4):317-321.doi:10.16505/j.2095-0136.2016.04.015.
Liu Q Q,Wang M L,Li Y. Research progress on Dengue virus nonstructural protein 5[J]. Chin J Viral Dis,2016,6(4):317-321.
[15] Selisko B,Dutartre H,de Guillemot J C,Debarnot C,Benarroch D,Khromykh A,de Desprès P,Egloff M P,Canard B. Comparative mechanistic studies of de novo RNA synthesis by Flavivirus RNA-dependent RNA polymerases[J]. Virology,2006,351(1):145-158.doi:10.1016/j.virol.2006.03.026.
[16] 王晓龙,王秀然,卢天成. 蛋白质糖基化修饰的研究进展[J].基因组学与应用生物学,2017,36(10):4380-4384.doi:10.13417/j.gab.036.004380.
Wang X L,Wang X R,Lu T C. The research progress of protein glycosylation modification[J]. Genomics and Applied Biology,2017,36(10):4380-4384.
[17] Korkuć P,Walther D. Spatial proximity statistics suggest a regulatory role of protein phosphorylation on compound binding[J]. Proteins,2016,84(5):565-579.doi:10.1002/prot.25001.
[18] 周希珍,赵慧,高岚,秦成峰. 虫媒黄病毒NS5蛋白的生物学功能研究进展[J].军事医学,2012,36(1):70-72.doi:10.3969/j.issn.1674-9960.2012.01.018.
Zhou X Z,Zhao H,Gao L,Qin C F. Advance in research on biological functions of nonstructural protein 5 of arthropod-borne Flavivirus[J]. Military Medical Sciences,2012,36(1):70-72.
[19] Best S M,Morris K L,Shannon J G,Robertson S J,Mitzel D N,Park G S,Boer E,Wolfinbarger J B,Bloom M E. Inhibition of interferon-stimulated JAK-STAT signaling by a tick-borne flavivirus and identification of NS5 as an interferon antagonist[J]. Journal of Virology, 2005,79(20):12828-12839.doi:10.1128/jvi.79.20.12828-12839.2005.
[20] Lu G F,Bluemling G R,Collop P,Hager M,Kuiper D,Gurale B P,Painter G R,Rosa A D L,Kolykhalov A A. Analysis of ribonucleotide 5'-triphosphate analogs as potential inhibitors of Zika virus RNA-dependent RNA polymerase by using nonradioactive polymerase assays[J]. Antimicrob Agents Chemother,2017,61(3):e01967.doi:10.1128/AAC.01967-16.
[21] Reznik S E,Ashby C R. Sofosbuvir:an antiviral drug with potential efficacy against Zika infection[J]. International Journal of Infectious Diseases,2017,55:29-30.doi:10.1016/j.ijid.2016.12.011.

Please choose a citation manager

Content to export