条件性敲除D1133L基因的重组非洲猪瘟病毒的构建及增殖特性

doi:10.11843/j.issn.0366-6964.2023.02.027

Abstract

Abstract: Previous studies have shown that the D1133L gene encoded by African swine fever virus (ASFV) is crucial to ASFV replication. In order to further explore the role of D1133L in ASFV replication, in this study, we used homologous recombination technology and Escherichia coli lac repression operating system to knockout D1133L gene conditionally, constructed recombinant transfer vector ASFV Δi130 using pUC118 as vector, transfected BMDM cells with recombinant transfer vector, and infected BMDM cells with ASFV CN/GS/2018 wild type strain. In the presence of isopropyl-β-D-thiogalactoside (IPTG), conditional knockout D1133L recombinant strain vD1133Li was identified by green fluorescence and qPCR. The replication of the recombinant virus strain and the parent virus strain in PAM cells was observed with fluorescence microscope. Differences between vD1133Li and the parent virus strain were analyzed by qPCR. The replication difference between conditionally knockout D1133L recombinant strain with or without IPTG was also further investigated. The vD1133Li with conditional deletion of D1133L was constructed successfully in this study. Replication level of vD1133Li was significantly lower than that of the wild type virus strain. In MA-104 cell lines that overexpressing stably D1133L, the replication ability of vD1133Li was recovered. In summary, D1133L is crucial for ASFV replication. These results provide a basis for further research on the function of D1133L in ASFV replication, the pathogenic mechanism and the development of D1133L targeted drugs.

Key words: African swine fever virus, conditional D1133L gene knockout recombinant virus, proliferation characterizations

CLC Number:

S852.659.1

ZHANG Ting, FENG Tao, YANG Jinke, HAO Yu, YANG Xing, ZHANG Dajun, SHI Xijuan, YAN Wenqian, CHEN Lingling, LIU Xiangtao, ZHENG Haixue, ZHANG Keshan. Construction and Growth Characteristics of Recombinant African Swine Fever Virus with Conditional Deletion of D1133L Gene[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(2): 706-714.

References 30

[1]	PENRITH M L, BASTOS A D, ETTER E M C, et al. Epidemiology of African swine fever in Africa today:Sylvatic cycle versus socio-economic imperatives[J]. Transbound Emerg Dis, 2019, 66(2):672-686.
[2]	GAUDREAULT N N, RICHT J A. Subunit vaccine approaches for African swine fever virus[J]. Vaccines, 2019, 7(2):56.
[3]	KARGER A, PÉREZ-NÚÑEZ D, URQUIZA J, et al. An update on African swine fever virology[J]. Viruses, 2019, 11(9):864.
[4]	DIXON L K, SUN H, ROBERTS H. African swine fever[J]. Antiviral Res, 2019, 165:34-41.
[5]	DIXON L K, STAHL K, JORI F, et al. African swine fever epidemiology and control[J]. Annu Rev Anim Biosci, 2020, 8:221-246.
[6]	DIXON L K, CHAPMAN D A G, NETHERTON C L, et al. African swine fever virus replication and genomics[J]. Virus Res, 2013, 173(1):3-14.
[7]	KOONIN E V, YUTIN N. Evolution of the large nucleocytoplasmic DNA viruses of eukaryotes and convergent origins of viral gigantism[J]. Adv Virus Res, 2019, 103:167-202.
[8]	ANDRÉS G, CHARRO D, MATAMOROS T, et al. The cryo-EM structure of African swine fever virus unravels a unique architecture comprising two icosahedral protein capsids and two lipoprotein membranes[J]. J Biol Chem, 2020, 295(1):1-12.
[9]	WANG N, ZHAO D M, WANG J L, et al. Architecture of African swine fever virus and implications for viral assembly[J]. Science, 2019, 366(6465):640-644.
[10]	ALEJO A, MATAMOROS T, GUERRA M, et al. A proteomic atlas of the African swine fever virus particle[J]. J Virol, 2018, 92(23):e01293-18.
[11]	RODRÍGUEZ J M, SALAS M L. African swine fever virus transcription[J]. Virus Res, 2013, 173(1):15-28.
[12]	CACKETT G, SYKORA M, WERNER F. Transcriptome view of a killer:African swine fever virus[J]. Biochem Soc Trans, 2020, 48(4):1569-1581.
[13]	SUAREZ C, ANDRES G, KOLOVOU A, et al. African swine fever virus assembles a single membrane derived from rupture of the endoplasmic reticulum[J]. Cell Microbiol, 2015, 17(11):1683-1698.
[14]	SALAS M L, ANDRÉS G. African swine fever virus morphogenesis[J]. Virus Res, 2013, 173(1):29-41.
[15]	张婷, 申超超, 杨博, 等. 非洲猪瘟病毒解旋酶结构与功能研究进展[J]. 中国兽医科学, 2021, 51(2):135-143.ZHANG T, SHEN C C, YANG B, et al. Research advances on the structure and function of African swine fever virus helicases[J]. Chinese Veterinary Science, 2021, 51(2):135-143. (in Chinese)
[16]	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.
[17]	侯景, 申超超, 张大俊, 等. 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 J, SHEN C C, ZHANG D J, et al. 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. (in Chinese)
[18]	MENG X Z, EMBRY A, ROSE L, et al. Vaccinia virus A6 is essential for virion membrane biogenesis and localization of virion membrane proteins to sites of virion assembly[J]. J Virol, 2012, 86(10):5603-5613.
[19]	GARCÍA-ESCUDERO R, ANDRÉS G, ALMAZÁN F, et al. Inducible gene expression from African swine fever virus recombinants:analysis of the major capsid protein p72[J]. J Virol, 1998, 72(4):3185-3195.
[20]	ANDRÉS G, GARCÍA-ESCUDERO R, VIÑUELA E, et al. African swine fever virus structural protein pE120R is essential for virus transport from assembly sites to plasma membrane but not for infectivity[J]. J Virol, 2001, 75(15):6758-6768.
[21]	MATAMOROS T, ALEJO A, RODRÍGUEZ J M, et al. African swine fever virus protein pe199l mediates virus entry by enabling membrane fusion and core penetration[J]. mBio, 2020, 11(4):e00789-20.
[22]	PORTUGAL R S, BAUER A, KEIL G M. Selection of differently temporally regulated African swine fever virus promoters with variable expression activities and their application for transient and recombinant virus mediated gene expression[J]. Virology, 2017, 508:70-80.
[23]	RATHAKRISHNAN A, CONNELL S, PETROVAN V, et al. Differential effect of deleting members of African swine fever virus multigene families 360 and 505 from the genotype II Georgia 2007/1 isolate on virus replication, virulence, and induction of protection[J]. J Virol, 2022, 96(6):e01899-21.
[24]	RATHAKRISHNAN A, REIS A L, GOATLEY L C, et al. Deletion of the K145R and DP148R genes from the virulent ASFV Georgia 2007/1 isolate delays the onset, but does not reduce severity, of clinical signs in infected pigs[J]. Viruses, 2021, 13(8):1473.
[25]	RODRÍGUEZ I, NOGAL M L, REDREJO-RODRíGUEZ M, et al. The African swine fever virus virion membrane protein pE248R is required for virus infectivity and an early postentry event[J]. J Virol, 2009, 83(23):12290-12300.
[26]	TURLEWICZ-PODBIELSKA H, KURIGA A, NIEMYJSKI R, et al. African swine fever virus as a difficult opponent in the fight for a vaccine-current data[J]. Viruses, 2021, 13(7):1212.
[27]	TEKLUE T, SUN Y, ABID M, et al. Current status and evolving approaches to African swine fever vaccine development[J]. Transbound Emerg Dis, 2020, 67(2):529-542.
[28]	URBANO A C, FERREIRA F. Role of the DNA-binding protein pA104R in ASFV genome packaging and as a novel target for vaccine and drug development[J]. Vaccines, 2020, 8(4):585.
[29]	LIU R L, SUN Y P, CHAI Y, et al. The structural basis of African swine fever virus pA104R binding to DNA and its inhibition by stilbene derivatives[J]. Proc Natl Acad Sci U S A, 2020, 117(20):11000-11009.
[30]	LIU B Z, CUI Y S, LU G, et al. Small molecule inhibitor E-64 exhibiting the activity against African swine fever virus pS273R[J]. Bioorg Med Chem, 2021, 35:116055.

Construction and Growth Characteristics of Recombinant African Swine Fever Virus with Conditional Deletion of D1133L Gene

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