非洲猪瘟病毒解旋酶D1133L基因序列分析、蛋白结构预测及亚细胞定位

doi:10.11843/j.issn.0366-6964.2021.07.017

摘要/Abstract

摘要： 旨在通过预测分析和亚细胞定位研究非洲猪瘟病毒（ASFV） D1133L基因结构、亚细胞定位与功能间的关系。作者使用Mega6.0软件绘制了7株不同基因型的D1133L解旋酶和ASFV编码的其他5个解旋酶基因的系统进化树，使用EXPASY、PRABI和SWISS-MODEL软件预测了该基因所编码蛋白序列的二、三级结构；根据GenBank公布的ASFV序列（LR743116.1），合成D1133L基因并构建其重组真核表达质粒pCMV-D1133L，蛋白免疫印迹（Western blot，WB）验证该基因表达后，将重组质粒转染至PK-15细胞，应用间接免疫荧光试验（indirect immunofluorescence assay，IFA）研究了该蛋白的亚细胞定位。结果表明，通过ASFV解旋酶的基因系统进化树，发现5种解旋酶基因均相对保守。D1133L基因全长3 402 bp，表达的蛋白为124.63 ku，G+C含量为6.1%，A+T含量为10.6%；二级结构分析表明α螺旋占48.90%，扩展链占13.50％，无规卷曲为33.27%；三级结构分析表明六自由度结构（six degree of freedom，QMQE）为0.20，覆盖率84%，且预测以α螺旋为主的高级结构；通过LOCSVMPSI分析预测，显示D1133L蛋白定位于细胞核内与细胞质内的概率是相同的。WB结果显示pCMV-D1133L质粒正常表达，IFA试验证明ASFV编码的解旋酶D1133L同时分布于细胞核和细胞质。本研究通过对D1133L基因序列、结构功能预测，并通过IFA验证了D1133L亚细胞分布，为进一步揭示D1133L基因功能奠定了一定基础。

关键词: 非洲猪瘟病毒, 解旋酶, D1133L基因, 序列分析, 结构预测, 亚细胞定位

Abstract: The relationships between the structure, subcellular localization and function of D1133L gene of African swine fever virus (ASFV) were explored by predictive analysis and subcellular localization. Mega6.0 software was used to make phylogenetic trees of five helicase genes encoded by ASFV, and Swiss-Model software was used to predict the secondary and tertiary structures of GenBank D1133L gene; according to the ASFV (LR743116.1) sequence published in GenBank (LR743116.1), the D1133L gene was synthesized and its recombinant eukaryotic expression plasmid pCMV-D1133L was constructed. After expression, the gene was verified by Western blot (WB), the recombinant plasmid was transfected into PK-15 cells, and the subcellular localization of the protein was observed with indirect immunofluorescence assay (IFA). The results showed that the 5 ASFV helicases were relatively conservative. The total length of D1133L gene was 3 402 bp, G+C content was 6.1%, and A+T content was 10.6%; the expressed protein was 124.36 ku, and the secondary structure indicated that helix accounted for 48.90%, the extended chain accounted for 13.50%, random curl accounted for 33.27%; the tertiary structure indicated that QMQE was 0.20, and the coverage rate was 84%, it has a helix-dominated advanced structure; using LOCSVMPSI analysis and prediction, the probability of D1133L protein localization in the nucleus is the same as that in the cytoplasm. The WB results verified the expression of pCMV-D1133L, and the result of IFA proves that the helicase D1133L encoded by ASFV was localized in the nucleus and cytoplasm at the same time. These results of this study have accumulated data for further study of D1133L protein inhibiting immune response and clarifying the pathogenic mechanism of helicase proteins of ASFV.

Key words: African swine fever virus, helicase, D1133L genes, sequence analysis, structural prediction, subcellular localization

中图分类号:

S852.659.1

侯景, 申超超, 张大俊, 杨博, 史喜绢, 张婷, 崔卉梅, 袁兴国, 赵登率, 陈学辉, 张克山, 郑海学, 刘湘涛. 非洲猪瘟病毒解旋酶D1133L基因序列分析、蛋白结构预测及亚细胞定位[J]. 畜牧兽医学报, 2021, 52(7): 1953-1962.

HOU Jing, SHEN Caocao, ZHANG Dajun, YANG Bo, SHI Xijuan, ZHANG Ting, CUI Huimei, YUAN Xingguo, ZHAO Dengshuai, CHEN Xuehui, ZHANG Keshan, ZHENG Haixue, LIU Xiangtao. Gene Sequence Analysis, Protein Structure Prediction and Subcellular Localization of African Swine Fever Virus Helicase D1133L[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(7): 1953-1962.

参考文献 42

[1]	MONTGOMERY R E. On a form of swine fever occurring in British east Africa (Kenya Colony)[J]. J Comp Pathol Ther, 1921, 34:159-191.
[2]	BURMAKINA G, BLIZNETSOV K, MALOGOLOVKIN A. Real-time analysis of the Cytopathic effect of African swine fever virus[J]. J Virol Methods, 2018, 257:58-61.
[3]	申超超, 李国丽, 张大俊, 等. 非洲猪瘟病毒MGF 360-9L基因序列分析、蛋白结构预测及亚细胞定位[J]. 畜牧兽医学报, 2020, 51(6):1371-1381.SHEN C C, LI G L, ZHANG D J, et al. Gene Sequence analysis, protein structure prediction and subcellular localization of MGF 360-9L from African swine fever virus[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(6):1371-1381. (in Chinese)
[4]	ARIAS M, JURADO C, GALLARDO C, et al. Gaps in African swine fever:analysis and priorities[J]. Transbound Emerg Dis, 2018, 65(S1):235-247.
[5]	GALINDO I, ALONSO C. African swine fever virus:a review[J]. Viruses, 2017, 9(5):103.
[6]	KIM S H, KIM J, SON K, et al. Wild boar harbouring African swine fever virus in the demilitarized zone in South Korea, 2019[J]. Emerg Microbes Infect, 2020, 9(1):628-639.
[7]	SÁNCHEZ-CORDÓN P J, MONTOYA M, REIS A L, et al. African swine fever:a re-emerging viral disease threatening the global pig industry[J]. Vet J, 2018, 233:41-48.
[8]	TATOYAN M R, IZMAILYAN R A, SEMERJYAN A B, et al. Patterns of alveolar macrophage activation upon attenuated and virulent African swine fever viruses in vitro[J]. Comp Immunol Microbiol Infect Dis, 2020, 72:101513.
[9]	MIMA K A, KATORKINA E I, KATORKIN S A, et al. In silico prediction of B- and T-cell epitopes in the CD2v protein of African swine fever virus (African swine fever virus, Asfivirus, Asfarviridae)[J]. Vopr Virusol, 2020, 65(2):103-112. (in Russian)
[10]	LI C F, HE X L, YANG Y, et al. Rapid and visual detection of African swine fever virus antibody by using fluorescent immunochromatography test strip[J]. Talanta, 2020, 219:121284.
[11]	FREITAS F B, FROUCO G, MARTINS C, et al. The QP509L and Q706L superfamily II RNA helicases of African swine fever virus are required for viral replication, having non-redundant activities[J]. Emerg Microbes Infect, 2019, 8(1):291-302.
[12]	FAZIO N T, HAO L X, LOHMAN T M. The nuclease domain of RecBCD influences DNA binding and helicase activity[J]. Biophys J, 2020, 118(3):72A.
[13]	GAO J, BYRD A K, ZYBAILOV B L, et al. DEAD-box RNA helicases Dbp2, Ded1 and Mss116 bind to G-quadruplex nucleic acids and destabilize G-quadruplex RNA[J]. Chem Commun, 2019, 55(31):4467-4470.
[14]	DONG Y S, ZHUANG H H, HAO Y, et al. Poly(N-Isopropyl-Acrylamide)/Poly(γ-Glutamic Acid) thermo-sensitive hydrogels loaded with superoxide dismutase for wound dressing application[J]. Int J Nanomedicine, 2020, 15:1939-1950.
[15]	YÁÑEZ R J, RODRÍGUEZ J M, BOURSNELL M, et al. Two putative African swine fever virus helicases similar to yeast ‘DEAH’ pre-mRNA processing proteins and vaccinia virus ATPases D11L and D6R[J]. Gene, 1993, 134(2):161-174.
[16]	EL JAMAL S M, ALAMODI A, WAHL R U, et al. Melanoma stem cell maintenance and chemo-resistance are mediated by CD133 signal to PI3K-dependent pathways[J]. Oncogene, 2020, 39(32):5468-5478.
[17]	LOlO R. Molecular characterisation of HELQ helicase's role in DNA repair and genome stability[D]. London:University College, 2020.
[18]	MARGADANT C. Positive and negative feedback mechanisms controlling tip/stalk cell identity during sprouting angiogenesis[J]. Angiogenesis, 2020, 23(2):75-77.
[19]	PATNAMSETTY M, SOMANI M C, GHOSH S, et al. Processing map for controlling microstructure and unraveling various deformation mechanisms during hot working of CoCrFeMnNi high entropy alloy[J]. Mater Sci Eng A, 2020, 793:139840.
[20]	ZENG J, HUYNH N, PHELPS B, et al. Snail synchronizes endocycling in a TOR-dependent manner to coordinate entry and escape from endoreplication pausing during the Drosophila critical weight checkpoint[J]. PLoS Biol, 2020, 18(2):e3000609.
[21]	OSTASZEWSKI M, MAZEIN A, GILLESPIE M E, et al. COVID-19 Disease Map, building a computational repository of SARS-CoV-2 virus-host interaction mechanisms[J]. Sci Data, 2020, 7(1):136.
[22]	LU S J, DONG L D, FANG C, et al. Stepwise selection on homeologous PRR genes controlling flowering and maturity during soybean domestication[J]. Nat Genet, 2020, 52(4):428-436.
[23]	LYONS L, BRASOF M. Building the capacity for student leadership in high school:a review of organizational mechanisms from the field of student voice[J]. J Educat Adm, 2020, 58(3):357-372.
[24]	FRANT M P, LYJAK M, BOCIAN L, et al. African swine fever virus (ASFV) in Poland:prevalence in a wild boar population (2017-2018)[J]. Vet Med, 2020, 65(4):143-158.
[25]	AMPARO C, CLARK J, BEDELL V, et al. Duplex DNA from sites of helicase-polymerase uncoupling links Non-B DNA structure formation to replicative stress[J]. Cancer Genomics Proteomics, 2020, 17(2):101-115.
[26]	DIXON L K, SÁNCHEZ-CORDÓN P J, GALINDO I, et al. Investigations of pro- and anti-apoptotic factors affecting African swine fever virus replication and pathogenesis[J]. Viruses, 2017, 9(9):241.
[27]	DIXON L K, ISLAM M, NASH R, et al. African swine fever virus evasion of host defences[J]. Virus Res, 2019, 266:25-33.
[28]	ZAHER N H, MOSTAFA M I, ALTAHER A Y. Design, synthesis and molecular docking of novel triazole derivatives as potential CoV helicase inhibitors[J]. Acta Pharm, 2020, 70(2):145-159.
[29]	COHEN M, LICHTEN M. C-terminal HA tags compromise function and exacerbate phenotypes of Saccharomyces cerevisiae bloom's helicase homolog sgs1 SUMOylation-Associated mutants[J]. G3(Bethesda), 2020, 10(8):2811-2818.
[30]	KOBAYASHI T, MURAKAMI T, HIROSE Y, et al. Purification and characterization of double-stranded nucleic acid-dependent ATPase activities of tagged dicer-related helicase 1 and its short isoform in Caenorhabditis elegans[J]. Genes, 2020, 11(7):734.
[31]	MARCON B H, REBELATTO C K, COFRÉ A R, et al. DDX6 helicase behavior and protein partners in human adipose tissue-derived stem cells during early Adipogenesis and Osteogenesis[J]. Int J Mol Sci, 2020, 21(7):2607.
[32]	THOMAS C A, CRAIG J M, LASZLO A H, et al. SV40 large t antigen helicase unwinds double-stranded DNA in single-nucleotide steps as revealed by nanopore tweezers[J]. Biophys J, 2020, 118(3):72-99.
[33]	AN Y, ZHANG L N, LIU W W, et al. De novo variants in the Helicase-C domain of CHD8 are associated with severe phenotypes including autism, language disability and overgrowth[J]. Hum Genet, 2020, 139(4):499-512.
[34]	NATH S, NAGARAJU G. FANCJ helicase promotes DNA end resection by facilitating CtIP recruitment to DNA double-strand breaks[J]. PLoS Genet, 2020, 16(4):e1008701.
[35]	LU Y, TAN F, ZHAO Y, et al. A chromodomain-helicase-DNA-binding factor functions in chromatin modification and gene regulation[J]. Plant Physiol, 2020, 183(3):1035-1046.
[36]	KOJIMA K K. AcademH, a lineage of Academ DNA transposons encoding helicase found in animals and fungi[J]. Mob DNA, 2020, 11:15.
[37]	ZHANG H, WU Z Y, LU J Y, et al. DEAD-Box helicase 18 counteracts PRC2 to safeguard ribosomal DNA in pluripotency regulation[J]. Cell Rep, 2020, 30(1):81-97.e7.
[38]	ANDRS M, HASANOVA Z, ORAVETZOVA A, et al. RECQ5:a mysterious helicase at the interface of DNA replication and transcription[J]. Genes, 2020, 11(2):232.
[39]	KAUR P, LONGLEY M J, PAN H, et al. Single-molecule level structural dynamics of DNA unwinding by human mitochondrial Twinkle helicase[J]. J Biol Chem, 2020, 295(17):5564-5576.
[40]	CHEN C, HAN X, CHEN C, et al. Crystal structure of the NS3 helicase of tick-borne encephalitis virus[J]. Biochem Biophys Res Commun, 2020, 528(3):601-606.
[41]	GULEVICH E P, KUZNETSOVA L V, KIL Y V, et al. Features of DNA helicase encoded by the uvrD gene of Deinococcus radiodurans R1 in Escherichia coli K-12 cells[J]. Mol Genet Microbiol Virol, 2020, 35(1):32-37.
[42]	IRETON R C, GALE JR M. RIG-I like receptors in antiviral immunity and therapeutic applications[J]. Viruses, 2011, 3(6):906-919.