HIV-1整合酶C末端及N末端结构域共结合多肽候选药物的筛选

doi:10.3877/cma.j.issn.2095-1221.2022.05.001

目的

利用双分子荧光互补技术筛选人类免疫缺陷病毒1 （HIV-1）的整合酶（IN）C末端结构域（CTD）及N末端结构域（NTD）多肽类药物。

方法

构建用于双分子荧光互补技术筛选的HIV-1整合酶CTD及NTD结构域的重组质粒pBiFc-VN173-Flag-IN-CTD及pBiFc-VN173-Flag-IN-NTD，并进行限制性内切酶酶切及测序鉴定。将重组质粒pBiFc-VN173-Flag-IN-CTD、pBiFc-VN173-Flag-IN-NTD及对照质粒pBiFC-VN173-Flag分别转染HEK293T细胞，使用蛋白免疫印迹法和Flag抗体检测2种重组质粒的表达。将3组质粒分别与多肽药物表达文库pBiFc-VC155-TrxA-11AA-TrxA共转染HEK293T细胞，在荧光显微镜下观察绿色荧光强度。将筛选到的多肽表达载体pcDNA-cmyc-11AA及对照pcDNA-cmyc分别转染HEK293T细胞，24 h后再用表达绿色荧光蛋白的慢病毒感染，36 h后观察绿色荧光强度。多组间比较用单因素方差分析，多个组与同一对照组比较采用Dunnett-t检验。

结果

经酶切及测序结果确认，重组质粒pBiFc-VN173-Flag-IN-CTD及pBiFc-VN173-Flag-IN-NTD构建成功，用Flag抗体可检测到两种质粒表达的重组蛋白。多肽文库中peptide 4和peptide 192与重组质粒分别共转染HEK293T细胞后，在荧光显微镜下可观察到绿色荧光的出现。经慢病毒感染后，与对照比较，转染多肽表达载体pcDNA-cmyc-peptide4和pcDNA-cmyc-peptide192的HEK293T细胞中荧光细胞数[（78.33±5.81）、（29.33±2.96）比（350.30±8.67）个]减少，差异有统计学意义（P < 0.05）。

结论

用于双分子荧光互补技术筛选的HIV-1整合酶CTD及NTD表达载体构建成功，并筛选获得靶向2个结构域的多肽peptide 4和peptide 192，其可能通过和整合酶的结合而抑制慢病毒的整合。

关键词: HIV-1整合酶, NTD, CTD, 多肽类药物, 双分子荧光互补技术

Objective

To screen human immunodeficiency virus 1 (HIV-1) integrase (IN) C-terminal domain (CTD) and N-terminal domain (NTD) peptide drugs using bimolecular fluorescence complementation (BiFC) technology.

Methods

The recombinant plasmids pBiFc-VN173-Flag-IN-CTD and pBiFc-VN173-Flag-IN-NTD of HIV-1 integrase CTD and NTD were constructed for the screening of HIV-1 integrase by BiFC technology and identified by restriction endonuclease enzyme and sequencing. The recombinant plasmids (pBiFc-VN173-Flag-IN-CTD and pBiFc-VN173-Flag-IN-NTD) and the control plasmid pBiFC-VN173-Flag were transfected into HEK293T cells, respectively, and the expression levels of fused proteins were identified by Western blotting with anti-Flag antibody. The two recombinant plasmids and the control plasmid were co-transfected into HEK293T cells with the peptide library pBiFc-VC155-TrxA-11AA-TrxA, respectively, then the intensity of green fluorescence was observed under a fluorescence microscope. The selected polypeptide expression vector pcDNA3.1-cmyc-11AA and the control vector pcDNA3.1-cmyc were transfected into HEK293T cells, then after 24 hours the cells were infected with lentivirus expressing green fluorescence protein, and the green fluorescence intensity was observed 36 hours later.

Results

The recombinant plasmids pBiFc-VN173-Flag-IN-CTD and pBiFc-VN173-Flag-IN-NTD were confirmed by restriction endonuclease enzyme digestion and sequencing, and the fused proteins expressed by these two plasmids could be detected with anti-Flag antibody. In addition, peptide 4 and peptide 192 were screened from the peptide library after co-transfection with recombinant plasmids in HEK293T cells based on the occurrence of green fluorescence. After lentivirus infection, the fluorescence intensity of HEK293T cells transfected with peptide expression vectors pcDNA3.1-cmyc-peptide 4 and pcDNA3.1-cmyc-peptide 192 was significantly decreased compared with the control.

Conclusions

Two plasmids expressing Flag-fused HIV-1 integrase CTD and NTD were successfully constructed and confirmed. Two peptides including peptide 4 and peptide 192 could target these two specific domains within HIV-1 integrase, were obtained based on BiFC technology. They maybe inhibit lentiviral integration though binding to integrase.

Key words: HIV-1 integrase, NTD, CTD, Peptide drugs, Bimolecular fluorescence complementation technology

表1 引物序列表

基因	序列（5'→3'）	预期产物扩增长度（bp）
IN-NTD	上游CGGAATTCATTTTTAGATGGAATAGAT	150
	下游GGGGTACCGACATGGCTTCCCCTTTTAG
IN-CTD	上游CGGAATTCATTACAAAAACAAATTACA	228
	下游GGGGTACCGAATCCTCATCCTGTCTACT

表1 引物序列表

基因	序列（5'→3'）	预期产物扩增长度（bp）
IN-NTD	上游CGGAATTCATTTTTAGATGGAATAGAT	150
	下游GGGGTACCGACATGGCTTCCCCTTTTAG
IN-CTD	上游CGGAATTCATTACAAAAACAAATTACA	228
	下游GGGGTACCGAATCCTCATCCTGTCTACT

图1 IN-NTD及IN-CTD结构域基因扩增产物及重组质粒的酶切鉴定注：1为pBiFc-VN173-Flag-IN-CTD酶切产物鉴定；2为IN-CTD结构域基因扩增产物；3为pBiFc-VN173-Flag-IN-NTD酶切产物鉴定；4为IN-NTD结构域基因扩增产物；5为Genestar 2000 plus DNAmarker

图2 IN-NTD及IN-CTD结构域基因扩增测序结果和HIV的gag-pol基因序列比对结果注：a为扩增的NTD测序结果和HIV的gag-pol基因序列比对的结果；b为扩增的CTD测序结果和HIV的gag-pol基因序列比对的结果

图3 Flag抗体检测重组质粒蛋白表达情况注：1为重组质粒pBiFc-VN173-Flag-IN-CTD表达IN-CTD结构域蛋白；2为重组质粒pBiFc-VN173-Flag-IN-NTD表达IN-NTD结构域蛋白；3为对照质粒pBiFc-VN173-Flag表达VN-173-Flag

图4 荧光显微镜下观察pBiFc-VC155-TrxA-11AA-TrxA和2种重组质粒共转染HEK293T细胞荧光表达情况（阳性组）（×200）注：a ~ f图分别为pBiFc-VC155-TrxA-peptide 4-TrxA与pBiFc-VN173-Flag、pBiFc-VN173-Flag-IN-CTD、pBiFc-VN173-Flag-IN-NTD共转染荧光表达情况；g ~ l图分别为pBiFc-VC155-TrxA-peptide 192-TrxA与pBiFc-VN173-Flag、pBiFc-VN173-Flag-IN-CTD、pBiFc-VN173-Flag-IN-NTD共转染荧光表达情况

图5 荧光显微镜下观察pBiFc-VC155-TrxA-11AA-TrxA和2种重组质粒共转染HEK293T细胞荧光表达情况（阴性组）（×200）注：a ~ f图分别为pBiFc-VC155-TrxA-peptide12-TrxA与pBiFc-VN173-Flag、pBiFc-VN173-Flag-IN-CTD、pBiFc-VN173-Flag-IN-NTD共转染荧光表达情况；g ~ l图分别为pBiFc-VC155-TrxA-peptide 15-TrxA与pBiFc-VN173-Flag、pBiFc-VN173-Flag-IN-CTD、pBiFc-VN173-Flag-IN-NTD共转染荧光表达情况

表2 肽适配子12、15、4和192的核苷酸序列及氨基酸序列

Peptide序号	核苷酸序列	氨基酸序列
12	TTTTTTTTCATTCTCCAGTTTGTCTCTACCTTG	FFFILQFVSTL
15	TGTTGTTTAGTATTTTTTTTGTTTCTATTTTTG	CCLVFFLFLFL
4	GTTGTTGCATTTGAATTTGGCTTTGTATTTCTG	VVAFEFGFVFL
192	TTTTCTGTGTCTTTTTTTTCTTATTGGTTTCTG	FSVSFFSYWFL

表2 肽适配子12、15、4和192的核苷酸序列及氨基酸序列

Peptide序号	核苷酸序列	氨基酸序列
12	TTTTTTTTCATTCTCCAGTTTGTCTCTACCTTG	FFFILQFVSTL
15	TGTTGTTTAGTATTTTTTTTGTTTCTATTTTTG	CCLVFFLFLFL
4	GTTGTTGCATTTGAATTTGGCTTTGTATTTCTG	VVAFEFGFVFL
192	TTTTCTGTGTCTTTTTTTTCTTATTGGTTTCTG	FSVSFFSYWFL

图6 荧光显微镜下观察pcDNA3.1-cmyc-peptide 4、pcDNA3.1-cmyc-peptide 192、pcDNA3.1-cmyc和表达红色荧光质粒dsred分别转染HEK293T细胞（×100）注：a、e图为pcDNA3.1-cmyc-peptide 4转染；b、f图为pcDNA3.1-cmyc-peptide 192转染；c、g图为pcDNA3.1-cmyc-转染；d、h图为表达红色荧光质粒dsred转染

图7 荧光显微镜下观察表达绿色荧光蛋白PCDH-IRES-GFP的慢病毒表达绿色荧光情况（×40）

图8 慢病毒感染转染多肽表达质粒的HEK293T细胞数注：与慢病毒感染pcDNA3.1-cmyc-转染比较，^aP < 0.001，n = 3

图9 荧光显微镜下慢病毒感染转染多肽表达质粒的HEK293T细胞及对照组荧光表达情况（×100）注：a、d图为慢病毒感染pcDNA3.1-cmyc-peptide 4转染；b、e图为病毒感染pcDNA3.1-cmyc-peptide 192转染；c、f图为慢病毒感染pcDNA3.1-cmyc-转染

1	Barre-Sinoussi F, Chermann JC, Rey F, et al. Isolation of a T-lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (AIDS)[J]. Science, 1983, 220(4599):868-871.
2	Hu WS, Hughes SH. HIV-1 reverse transcription[J]. Cold Spring Harb Perspect Med, 2012, 2(10):a006882. doi: 10.1101/cshperspect.a006882.
3	Engelman A, Mizuuchi K, Craigie R. HIV-1 DNA integration: mechanism of viral DNA cleavage and DNA strand transfer[J]. Cell, 1991, 67(6):1211-1221.
4	Brown PO, Bowerman B, Varmus HE, et al. Retroviral integration: structure of the initial covalent product and its precursor, and a role for the viral IN protein[J]. Proc Natl Acad Sci U S A, 1989, 86(8):2525-2529.
5	Krishnan L, Li X, Naraharisetty HL, et al. Structure-based modeling of the functional HIV-1 intasome and its inhibition[J]. Proc Natl Acad Sci U S A, 2010, 107(36):15910-15915.
6	Hare S, Gupta SS, Valkov E, et al. Retroviral intasome assembly and inhibition of DNA strand transfer[J]. Nature, 2010, 464(7286):232-236.
7	Maertens G, Cherepanov P, Pluymers W, et al. LEDGF/p75 is essential for nuclear and chromosomal targeting of HIV-1 integrase in human cells[J]. J Biol Chem, 2003, 278(35):33528-33539.
8	Ciuffi A, Llano M, Poeschla E, et al. A role for LEDGF/p75 in targeting HIV DNA integration[J]. Nat Med, 2005, 11(12):1287-1289.
9	Lapaillerie D, Lelandais B, Mauro E, et al. Modulation of the intrinsic chromatin binding property of HIV-1 integrase by LEDGF/p75[J]. Nucleic Acids Res, 2021, 49(19):11241-11256.
10	Kessl JJ, Kutluay SB, Townsend D, et al. HIV-1 integrase binds the viral RNA genome and is essential during virion morphogenesis[J]. Cell, 2016, 166(5):1257-1268.e1212.
11	Elliott JL, Eschbach JE, Koneru PC, et al. Integrase-RNA interactions underscore the critical role of integrase in HIV-1 virion morphogenesis[J]. Elife, 2020, 9:e54311. doi: 10.7554/eLife.54311.
12	Elliott JL, Kutluay SB. Going beyond integration: the emerging role of HIV-1 integrase in virion morphogenesis[J]. Viruses, 2020, 12(9):1005. doi: 10.3390/v12091005.
13	Summa V, Petrocchi A, Bonelli F, et al. Discovery of raltegravir, a potent, selective orally bioavailable HIV-integrase inhibitor for the treatment of HIV-AIDS infection[J]. J Med Chem, 2008, 51(18):5843-5855.
14	Pommier Y, Johnson AA, Marchand C. Integrase inhibitors to treat HIV/AIDS[J]. Nat Rev Drug Discov, 2005, 4(3):236-248.
15	Johnson AA, Marchand C, Pommier Y. HIV-1 integrase inhibitors: a decade of research and two drugs in clinical trial[J]. Curr Top Med Chem, 2004, 4(10):1059-1077.
16	Wang Y, Gu S X, He Q, et al. Advances in the development of HIV integrase strand transfer inhibitors[J]. Eur J Med Chem, 2021, 225:113787. doi: 10.1016/j.ejmech.2021.113787.
17	Smith SJ, Zhao XZ, Passos DO, et al. Integrase strand transfer inhibitors are effective Anti-HIV drugs[J]. Viruses, 2021, 13(2):205. doi: 10.3390/v13020205.
18	Mbhele N, Chimukangara B, Gordon M. HIV-1 integrase strand transfer inhibitors: a review of current drugs, recent advances and drug resistance[J]. Int J Antimicrob Agents, 2021, 57(5):106343. doi: 10.1016/j.ijantimicag.2021.106343.
19	Karimi N, Roudsari RV, Hajimahdi Z, et al. Design, synthesis, and docking studies of thioimidazolyl diketoacid derivatives targeting HIV- 1 integrase[J]. Med Chem, 2022, 18(5):616-628.
20	Adu-Ampratwum D, Pan Y, Koneru PC, et al. Identification and optimization of a Novel HIV-1 integrase inhibitor[J]. ACS Omega, 2022, 7(5):4482-4491.
21	Parvez MK, Al-Dosari MS, Sinha GP. Machine learning-based predictive models for identifying high active compounds against HIV-1 integrase[J]. SAR QSAR Environ Res, 2022, 33(5):387-402.
22	Zhang C, Xie Q, Wan CC, et al. Recent advances in small-molecule HIV-1 integrase inhibitors[J]. Curr Med Chem, 2021, 28(24):4910-4934.
23	Ohata Y, Tomonaga M, Watanabe Y, et al. Antiviral activity and resistance profile of the novel HIV-1 non-catalytic site integrase inhibitor JTP-0157602[J]. J Virol, 2022, 96(6):e0184321.doi: 10.1128/JVI.01843-21.
24	Taoda Y, Akiyama T, Tomita K, et al. Discovery of tricyclic HIV-1 integrase-LEDGF/p75 allosteric inhibitors by intramolecular direct arylation reaction[J]. Bioorg Med Chem Lett, 2022, 64:128664. doi: 10.1016/j.bmcl.2022.128664.
25	Sugiyama S, Akiyama T, Taoda Y, et al. Discovery of novel HIV-1 integrase-LEDGF/p75 allosteric inhibitors based on a pyridine scaffold forming an intramolecular hydrogen bond[J]. Bioorg Med Chem Lett, 2021, 33:127742. doi: 10.1016/j.bmcl.2020.127742.
26	Debyser Z, Bruggemans A, Van Belle S, et al. LEDGINs, inhibitors of the interaction between HIV-1 integrase and LEDGF/p75, are potent antivirals with a potential to cure HIV infection[J]. Adv Exp Med Biol, 2021, 1322:97-114.
27	Maehigashi T, Ahn S, Kim UI, et al. A highly potent and safe pyrrolopyridine-based allosteric HIV-1 integrase inhibitor targeting host LEDGF/p75-integrase interaction site[J]. PLoS Pathog, 2021, 17(7):e1009671.doi: 10.1371/journal.ppat.1009671.
28	Rashamuse TJ, Fish MQ, Coyanis EM, et al. Studies towards the design and synthesis of novel 1,5-Diaryl-1H-imidazole-4-carboxylic acids and 1,5-Diaryl-1H-imidazole-4-carbohydrazides as host LEDGF/p75 and HIV-1 integrase interaction inhibitors[J]. Molecules, 2021, 26(20):6203. doi: 10.3390/molecules26206203.
29	Panwar U, Singh SK. In silico virtual screening of potent inhibitor to hamper the interaction between HIV-1 integrase and LEDGF/p75 interaction using E-pharmacophore modeling, molecular docking, and dynamics simulations[J]. Comput Biol Chem, 2021, 93:107509. doi: 10.1016/j.compbiolchem.2021.107509.
30	Kessl JJ, Jena N, Koh Y, et al. Multimode, cooperative mechanism of action of allosteric HIV-1 integrase inhibitors[J]. J Biol Chem, 2012, 287(20):16801-16811.
31	Tsiang M, Jones GS, Niedziela-Majka A, et al. New class of HIV-1 integrase (IN) inhibitors with a dual mode of action[J]. J Biol Chem, 2012, 287(25):21189-21203.
32	Balakrishnan M, Yant SR, Tsai L, et al. Non-catalytic site HIV-1 integrase inhibitors disrupt core maturation and induce a reverse transcription block in target cells[J]. PLoS One, 2013, 8(9):e74163. doi: 10.1371/journal.pone.0074163.
33	Jurado KA, Wang H, Slaughter A, et al. Allosteric integrase inhibitor potency is determined through the inhibition of HIV-1 particle maturation[J]. Proc Natl Acad Sci U S A, 2013, 110(21):8690-8695.
34	Eijkelenboom AP, van den Ent FM, Vos A, et al. The solution structure of the amino-terminal HHCC domain of HIV-2 integrase: a three-helix bundle stabilized by zinc[J]. Curr Biol, 1997, 7(10):739-746.
35	Goldgur Y, Dyda F, Hickman AB, et al. Three new structures of the core domain of HIV-1 integrase: an active site that binds magnesium[J]. Proc Natl Acad Sci U S A, 1998, 95(16):9150-9154.
36	Sangeetha B, Muthukumaran R, Amutha R. The dynamics of interconverting D- and E-forms of the HIV-1 integrase N-terminal domain[J]. Eur Biophys J, 2014, 43(10-11):485-498.
37	Zheng R, Jenkins TM, Craigie R. Zinc folds the N-terminal domain of HIV-1 integrase, promotes multimerization, and enhances catalytic activity[J]. Proc Natl Acad Sci U S A, 1996, 93(24):13659-13664.
38	Cherepanov P, Ambrosio AL, Rahman S, et al. Structural basis for the recognition between HIV-1 integrase and transcriptional coactivator p75[J]. Proc Natl Acad Sci U S A, 2005, 102(48):17308-17313.
39	Hare S, Shun MC, Gupta SS, et al. A novel co-crystal structure affords the design of gain-of-function lentiviral integrase mutants in the presence of modified PSIP1/LEDGF/p75[J]. PLoS Pathog, 2009, 5(1):e1000259. doi: 10.1371/journal.ppat.1000259.
40	Knyazhanskaya E, Anisenko A, Shadrina O, et al. NHEJ pathway is involved in post-integrational DNA repair due to Ku70 binding to HIV-1 integrase[J]. Retrovirology, 2019, 16(1):30. doi: 10.1186/s12977-019-0492-z.
41	Cereseto A, Manganaro L, Gutierrez MI, et al. Acetylation of HIV-1 integrase by p300 regulates viral integration[J]. EMBO J, 2005, 24(17):3070-3081.
42	Terreni M, Valentini P, Liverani V, et al. GCN5-dependent acetylation of HIV-1 integrase enhances viral integration[J]. Retrovirology, 2010, 7:18. doi: 10.1186/1742-4690-7-18.
43	Winans S, Goff S P. Mutations altering acetylated residues in the CTD of HIV-1 integrase cause defects in proviral transcription at early times after integration of viral DNA[J]. PLoS Pathog, 2020, 16(12):e1009147. doi: 10.1371/journal.ppat.1009147.
44	Schneider W M, Wu DT, Amin V, et al. MuLV IN mutants responsive to HDAC inhibitors enhance transcription from unintegrated retroviral DNA[J]. Virology, 2012, 426(2):188-196.

[1]	褚依然, 岳麓. 液体活检技术在肺癌中的应用进展[J]. 中华肺部疾病杂志(电子版), 2023, 16(01): 125-127.
[2]	刘骞, 崔巍, 高亦博, 张泽跃, 李典格, 王治杰, 王洁, 赫捷, 中国结直肠癌筛查与早诊早治指南制定专家组. 结直肠肿瘤早诊筛查新策略：基于液体活检技术的筛查方案[J]. 中华结直肠疾病电子杂志, 2023, 12(01): 17-20.

阅读次数

全文

摘要

选择文件类型/文献管理软件名称

选择包含的内容

Screening of HIV-1 integrase CTD and NTD co-binding candidate peptide drugs

模态框（Modal）标题

选择文件类型/文献管理软件名称

选择包含的内容

Screening of HIV-1 integrase CTD and NTD co-binding candidate peptide drugs