转录因子JunD在HIV-1前病毒DNA转录调控中的作用研究
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摘要
人类免疫缺陷病毒1型(HIV-1)潜伏感染的形成与AP-1家族蛋白的转录调节作用密切相关。为研究AP-1家族成员JunD分子在HIV-1潜伏感染形成中的作用,本研究基于可支持HIV-1复制的T细胞系Jurkat,构建敲除JunD和稳定(过)表达JunD的细胞系;拯救HIV-1假病毒并分别感染对照细胞、JunD敲除以及JunD稳定表达的细胞系,通过荧光素酶技术检测HIV-1前病毒DNA转录水平的变化。结果显示,与对照组相比,JunD敲除显著抑制了HIV-1的复制,而过表达JunD则促进了HIV-1的复制。该结果表明,JunD参与了HIV-1前病毒DNA的转录调控,并可能由此影响HIV-1在宿主细胞内的复制。本实验为研究激活HIV-1细胞潜伏感染的靶点提供了实验数据。
HIV-1 latency infection is closely related to the activity of transcription factors from the activating protein-1(AP-1)family. However, whether JunD, a member of AP-1 family, is involved in regulating the transcription of HIV-1 provirus DNA remains unclear. In this study, the JunD knockout and the overexpression Jurkat cell lines were developed. And HIV-1 pseudovirus was used to infect these JunD knockout, overexpressing and control cell lines, respectively. The transcription level of HIV-1 proviral DNA was detected by luciferase assay. The results showed that HIV-1 provirus DNA transcription in the JunD knockout cells was significantly inhibited, compared with that in the control cells, whereas HIV-1 provirus DNA transcription was promoted in the JunD overexpression cells. It suggested that JunD was involved in the activating transcription of HIV-1 provirus DNA and thereby regulating the replication of HIV-1 in host cells. This study uncover a potential target to activate HIV-1 latency infection.
HIV-1 latency infection is closely related to the activity of transcription factors from the activating protein-1(AP-1)family. However, whether JunD, a member of AP-1 family, is involved in regulating the transcription of HIV-1 provirus DNA remains unclear. In this study, the JunD knockout and the overexpression Jurkat cell lines were developed. And HIV-1 pseudovirus was used to infect these JunD knockout, overexpressing and control cell lines, respectively. The transcription level of HIV-1 proviral DNA was detected by luciferase assay. The results showed that HIV-1 provirus DNA transcription in the JunD knockout cells was significantly inhibited, compared with that in the control cells, whereas HIV-1 provirus DNA transcription was promoted in the JunD overexpression cells. It suggested that JunD was involved in the activating transcription of HIV-1 provirus DNA and thereby regulating the replication of HIV-1 in host cells. This study uncover a potential target to activate HIV-1 latency infection.
引文
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[2]Thurtle-Schmidt D M,Lo T W.Molecular biology at the cutting edge:A review on CRISPR/CAS9 gene editing for undergraduates[J].Biochem Mol Biol Educ,2018,46(2):195-205.
[3]Chaudhary K,Chattopadhyay A,Pratap D.The evolution of CRISPR/Cas9 and their cousins:Hope or hype[J].Biotechnol Lett,2018,40(3):465-477.
[4]Venkatachari N J,Zerbato J M,Jain S,et al.Temporal transcriptional response to latency reversing agents identifies specific factors regulating HIV-1 viral transcriptional switch[J].Retrovirology,2015,6(12):85.
[5]Canonne-Hergaux F,Aunis D,Schaeffer E.Interactions of the transcription factor AP-1 with the long terminal repeat of different human immunodeficiency virus type 1 strains in Jurkat,glial,and neuronal cells[J].J Virol,1995,69(11):6634-6642.
[6]van der Sluis R M,Derking R,Breidel S,et al.Interplay between viral Tat protein and c-Jun transcription factor in controlling LTR promoter activity in different human immunodeficiency virus type I subtypes[J].J Gen Virol,2014,95(4):968-979.
[7]Duverger A,Wolschendorf F,Zhang Ming-ce,et al.An AP-1binding site in the enhancer/core element of the HIV-1 promoter controls the ability of HIV-1 to establish latent infection[J].JVirol,2013,87(4):2264-2277.
[8]Zhou Yin,Machius M,Nestler E J,et al.Activator Protein-1:redox switch controlling structure and DNA-binding[J].Nucleic Acids Res,2017,45(19):11425-11436.
[9]Sanjana N E,Shalem O,Zhang Feng.Improved vectors and genome-wide libraries for CRISPR screening[J].Nat Methods,2014,11(8):783-784.
[10]Vemula S V,Veerasamy R,Ragupathy V,et al.HIV-1 induced nuclear factor I-B(NF-IB)expression negatively regulates HIV-1replication through interaction with the long terminal repeat region[J].Viruses,2015,7(2):543-558.
[11]Duverger A,Jones J,May J,et al.Determinants of the establishment of human immunodeficiency virus type 1 latency[J].J Virol,2009,83(7):3078-3093.
[12]McN amara L A,Ganesh J A,Collins K L.Latent HIV-1 infection occurs in multiple subsets of hematopoietic progenitor cells and is reversed by NF-κB activation[J].J Virol,2012,86(17):9337-9350.
[13]Fortin J F,Barat C,Beauséjour Y,et al.Hyper-responsiveness to stimulation of human immunodeficiency virus-infected CD4+T cells requires Nef and Tat virus gene products and results from higher NFAT,NF-kappaB,and AP-1 induction[J].J Biol Chem,2004,279(38):39520-39531.
[14]Roebuck K A,Gu De-sheng,Kagnoff M F.Activating protein-1cooperates with phorbol ester activation signals to increase HIV-1 expression[J].AIDS,1996,10(8):819-826.