HIV-1转录抑制剂的研究
摘要
HIV-1是一种人类免疫缺陷病毒,感染人体后导致艾滋病的发生。目前艾滋病的治疗药物主要为反转录酶以及蛋白酶抑制剂等,副作用多,不能达到根治目的。因此,研究开发新的抗HIV-1药物十分重要。根据HIV-1病毒转录复制过程中,需要HIV-1编码的调节蛋白Tat和LTR启动子中-TAR RNA序列特异性结合,以激活转录。因此,阻断Tat-TAR的结合有可能抑制病毒的转录复制。本课题通过构建两种实验模型,筛选具有能有阻断Tat-TAR结合并具有转录抑制活性的化合物。
通过构建Tat的真核表达载体pCMV-Tat以及LTR荧光素酶报告基因载体pGL3-LTR,将两者共转染HeLa细胞后,检测荧光素酶活性,以观察Tat蛋白对LTR启动子的转录激活作用,建立转录活化细胞模型,用于筛选转录抑制性化合物。
将HIV-1 Tat (1-86aa)编码序列插入原核表达质粒pET28a中,转化E.coli BL21(DE3), IPTG诱导表达,并用Ni+金属螯合层析介质纯化获得Tat蛋白；把编码TARRNA的DNA序列插入含有T7启动子的载体pBluescript II SK(+)中,用T7 RNA多聚酶体外转录合成TAR RNA。用毛细管电泳法分析Tat与TAR的结合活性,以观察活性化合物对Tat-TAR的阻断作用。,
结果显示：通过对68个化合物的筛选,并结合细胞毒性试验,总共筛选出了10个能够有效抑制转录的化合物；制备的Tat和TAR能相互结合,此结合能被转录抑制性化合物AO081/15571098阻断,说明此模型可用于筛选阻断Tat-TAR结合的化合物。
Human immunodeficiency virus type 1(HIV-1) is the cause of AIDS(Acquired Immune Deficiency Syndrome). There are no drugs can cure AIDS completely up to now. So search for new drugs that can cure AIDS has become very important. Because of the transcription of HIV-1 requires specific interaction of Tat with TAR RNA, disruption of Tat-TAR interaction could inhibit HIV-1 transcription and replication. So we construct two models for screening compounds which can inhibit the Tat binding to TAR and the Tat activated transcription of HIV-1.
HIV-1 Tat (1-86aa) coding sequence and LTR sequence was inserted into eukaryotic expression plasmid pCMV-HA and luciferase report gene vector pGL3-Basic, named pCMV-Tat and pGL3-LTR. The two vectors were transfected into HeLa cells, the activity of luciferase were detected with illuminator. This Tat activated transcription experiments was used as cell model for screening transcription inhibitory compounds.
HIV-1 Tat coding sequence was inserted into the prokaryotic expression plasmid pET28a and transformed into E. coli BL21(DE3). Tat protein expression was induced by IPTG and purified with Ni-NTA column affinity chromatography. Then the TAR sequence was inserted into pBluescript II SK(+),which contain T7 promoter. The TAR RNA was synthesized by T7 RNA polymerase transcription in vitro. The binding activity of Tat with TAR was analyzed by Capillary Electrophoresis. This experiment was used as Tat-TAR binding model in vitro to test the binding blocking activity of compounds.
The results show that:we screened 10 active compounds which can inhibit Tat activated transcription through cell model; Tat protein can combine TAR effectively and the compound AO081/15571098 can block the binding of Tat and TAR.
通过构建Tat的真核表达载体pCMV-Tat以及LTR荧光素酶报告基因载体pGL3-LTR,将两者共转染HeLa细胞后,检测荧光素酶活性,以观察Tat蛋白对LTR启动子的转录激活作用,建立转录活化细胞模型,用于筛选转录抑制性化合物。
将HIV-1 Tat (1-86aa)编码序列插入原核表达质粒pET28a中,转化E.coli BL21(DE3), IPTG诱导表达,并用Ni+金属螯合层析介质纯化获得Tat蛋白；把编码TARRNA的DNA序列插入含有T7启动子的载体pBluescript II SK(+)中,用T7 RNA多聚酶体外转录合成TAR RNA。用毛细管电泳法分析Tat与TAR的结合活性,以观察活性化合物对Tat-TAR的阻断作用。,
结果显示：通过对68个化合物的筛选,并结合细胞毒性试验,总共筛选出了10个能够有效抑制转录的化合物；制备的Tat和TAR能相互结合,此结合能被转录抑制性化合物AO081/15571098阻断,说明此模型可用于筛选阻断Tat-TAR结合的化合物。
Human immunodeficiency virus type 1(HIV-1) is the cause of AIDS(Acquired Immune Deficiency Syndrome). There are no drugs can cure AIDS completely up to now. So search for new drugs that can cure AIDS has become very important. Because of the transcription of HIV-1 requires specific interaction of Tat with TAR RNA, disruption of Tat-TAR interaction could inhibit HIV-1 transcription and replication. So we construct two models for screening compounds which can inhibit the Tat binding to TAR and the Tat activated transcription of HIV-1.
HIV-1 Tat (1-86aa) coding sequence and LTR sequence was inserted into eukaryotic expression plasmid pCMV-HA and luciferase report gene vector pGL3-Basic, named pCMV-Tat and pGL3-LTR. The two vectors were transfected into HeLa cells, the activity of luciferase were detected with illuminator. This Tat activated transcription experiments was used as cell model for screening transcription inhibitory compounds.
HIV-1 Tat coding sequence was inserted into the prokaryotic expression plasmid pET28a and transformed into E. coli BL21(DE3). Tat protein expression was induced by IPTG and purified with Ni-NTA column affinity chromatography. Then the TAR sequence was inserted into pBluescript II SK(+),which contain T7 promoter. The TAR RNA was synthesized by T7 RNA polymerase transcription in vitro. The binding activity of Tat with TAR was analyzed by Capillary Electrophoresis. This experiment was used as Tat-TAR binding model in vitro to test the binding blocking activity of compounds.
The results show that:we screened 10 active compounds which can inhibit Tat activated transcription through cell model; Tat protein can combine TAR effectively and the compound AO081/15571098 can block the binding of Tat and TAR.
引文
[1]李朝阳.RNA干扰下调cyclinTl蛋白的表达及其对靶细胞和HIV-1的影响[D].武汉：武汉大学,2005
[2]李太生.HIV/ADIS研究的成功与挑战[J].临床内科杂志.2009,05(1)：293-294
[3]Kittiworakarn J, Lecoq A, Moine G, et al. HIV-1 Tat raisesanadjuvant-free humoral immune response controlled by its core region and its ability to form cysteinemediated oligomers[J]. J Biol Chem.2006,281(6):3105-3115
[4]Brigitic, Giaccam, Noonandm, et al. HIV Tat, its Targets and the control of viral gene expression[J]. FEMS M icrobiol Lett.2003,220(1):57-65
[5]崔凡,刘维达.HIV-1转录调控机制的研究进展[J].国外医学·皮肤性病学分册.2003,29(2)：123-126
[6]张传明,肖志,艳胡春.HIV-1反式激活抑制剂的研究进展[J].中国药物科学杂志.2008,18(6)：461-467
[7]艾青,王丽梅,狄美玉.Tat蛋白结构与功能研究的进展[J].细胞与分子免疫学杂志,2005,21(3)：133-134
[8]白如珺,刘永新.HIV-1反式激活与抑制[J].药学学报,2006,41(4)：289-295
[9]Chao Zhou, Tariq M. Rana. A Bimolecular Mechanism of HIV-1 Tat Protein Interaction with RNA Polymerase II Transcription Elongation Complexes[J]. JMB.2002,320(2): 925-942
[10]Alastair I.H.Murchie, Ben Davis, Mohammad Afshar, et al. Structure-based Drug Design Targeting an Inactive RNA Conformation:Exploiting the Flexibiliy of HIV-1 TAR RNA. J.Mol.Biol.2004,336:625-638
[11]Julie K Jadlowsky, Masanori Nojima, koh Fujinaga. Dominant negative mutant CyclinTl proteins inhibit HIV transcription by specifically degrading Tat. Retrovirology.2008,5: 63
[12]Zhu, Y., Pe'ery, et al. Transcription elongation factor P-TEFb is required for HIV-1Tat transactivation in vitro. Genes Dev.1997,11:2622-2632
[13]Mancebo, H.S.Y., Lee, et al. P-TEFb kinase is required for HIV Tat transcriptional activation in vivo and in vitro. Genes Dev,1997,11:2633-2644
[14]Wei, P., Garber, et al. A novel CDK9-associated C-type cyclin interacts directly with HIV-1 Tat and mediates its high-affinity, loop specific binding to TAR RNA. Cell.1998, 92:451-462
[15]Garber, M.E., Wei, et al. The interaction between HIV-1 Tat and human cyclin T1 requires zinc and a critical cysteine residue that is not conserved in the murine CycTl protein. Genes Dev.1998,12:3512-3527
[16]李淼,黄山.HIV-1进入抑制剂的研究进展[J].药学进展.2007,31(12)：550-560
[17]Biscone M J, Pierson T C, Dows R W. Opportunities and challenges intargeting HIV entry. Curr Opin Pharmacol.2002,2(5):529-533
[18]吉昌华.人免疫缺陷病毒LTR转录调控机制的研究进展[J].国外医学分子生物学分册.1991,13(3)：106-109
[19]欧强,卢洪洲.抗HIV药物研究进展[J].世界感染杂志.2008,8(6)：397-400
[20]孙哲,吕秋君.基于检测报告基因的药物筛选方法研究进展[J].中国药学杂志.2000,35(8)：507-509
[21]Bronstein I, Martin CS, Fortin JJ, et al. Chemiluminescence:sensitive detection technology for repoter gene assays[J]. Clin Chem.1996,42(9):1542
[22]Silverman L, Campbell, Broach JR. New assay technology for highthroughput screening[J]. Curr Opin Chem Biol.1998,2:397
[23]De Wet, J R. Cloning of firefly luciferase cDNA and the expression of active luciferase in E.coil[J]. Proc Natl Acad SCI USA.1985,87:7870-7873.
[24]Jeang KT, Xiao H, Rich EA.Multifaceted activities of the HIV-1 transactivator of transcription[J]. Biol Chem.1999,274(41):28837-28840
[25]Dandekar DH, Ganesh KN, Mitra D. HIV-1 Tat directly binds to NFkappaB enhancer sequence:role in viral and cellular gene expression[J]. Nucleic Acids Res.2004,32(4): 1270-1278
[26]刘定燮,周晓巍,黄培唐.哺乳动物表达系统及其研究进展.生物技术通讯.2003,14(4)：308-310
[27]Xu A, Kudo S, Fukuda M, et al. A novel expression vector composed of a regulatory element of the human leukosialin-encoding gene in different types of mammalian cells. Gene,1995.160:183
[28]Arzumanov, A., Walsh, et al. Inhibition of HIV-1 tat-dependent trans activation by steric block chimeric 2'-O-methyl/LNA oligoribonucleotides[J]. Biochemistry.2001,40: 14645-14654
[29]Mischiati, C., Jeang, et al. Aromatic polyamidines inhibiting the tat-induced HIV-1 transcription recognize structured TAR-RNA[J]. Antisense Nucl. Acid Drug Dev. 2001,11,209-217
[30]Kaushik, N., Basu, et al. Anti-TARpolyamide nucleotide analog conjugated with a membrane-permeating peptide inhibits human immunodeficiency virus type 1 production[J]. Virol.76:3881-3891 [31] Beatrice M, Arzuonanov A, Singh M, et al. Oligonucleotide inhibition of the interaction of HIV-1 Tat protein with the transactivation responsive region (TAR) of HIV RNA. Biochimica et Biophys Acta.1999,1445:86
[32]李在留,刘朝奇,邹坤.HIV-1 Tat蛋白结合Tar的抗艾滋病药物筛选模型的建立研究.时珍国医国药.2008,19(2)：352-354
[33]G. Wang, H.F. Chen, M.H. Huang, N.L. Wang, J.C. Zhang, Y. Zhang, et al., Methyl protodioscin induces G2/M cell cycle arrest and apoptosis in HepG2 liver cancer cells. Cancer Letters.2006,241(1):102-109
[34]Haase-Pettingell, C.A, J.King. Formation of aggregates from a thermolabile in vivo folding intermediate in P22 tailspike maturation:A model for inclusion body formation[J]. Biol.Chem,1988,263:4977-4983
[35]Anna Mitraki, Jonathan King. Protein folding intermediates and infusion body formation[J]. Nature Biotechnology.1989,7:690-697
[36]Catheine H.Schein, Mathieu H.M.Noteborn. Formation of soluble recombinant pretein in E.coil is favored by lower temperation[J]. Nature Biotechnology.1988,6:291-294
[37]Hockney. Recent developments in heterologous protein production in E.coli [J]. Trends Biotechnol.1994,12(11):456-463
[38]Drysdale, M. J., Lentzen, et al. RNA as a drug target [J]. Prog.Med.Chem.2002,9: 73-119
[39]Foreyen, M. &Herdewijn, P. RNA as a target for drug design, the example of Tat-TAR interaction [J]. Curr.Top.Med.Chem.2002,2:1123-1145
[40]杨玉志,唐祖明,陆祖宏.毛细管电泳在微生物分离与检测中的应用[J].微生物学杂志.2005,25(4)：77-81
[41]Mohamed N A. Enantioselective binding analysis of verapamil by capillary electrophoresis-frontal analysis [J]. Journal of Chromatography A.2000,875:447-453
[42]王亮,姚鑫,袁倬斌.毛细管电泳在生物大分子分析中的应用[J].分析试验室,2001,20：28-29
[43]Mucha P. Structural requirements for conserved Agr52 residue for interaction of the human immunodeficiency virus 1 transactivation responsive element with transactivator of transcription protein(49-57). Capillary electrophoresis mobility shift assay [J]. Journal of Chromatography A.2002,968:211-220
[44]Meizi He, Dekai Yuan, Ming yang, et al. Synthesis and assay of isoquinoline derivatives as HIV-1 Tat-TAR interaction inhibitors [J]. Bioorganic & Medicinal Chemistry Letter. 2005,15:3978-3981
[45]Ruifang pang, Chunlei zhang, Ming Yang. Design and SAR of new substituted purines bearing ary groups at N9 position as HIV-1 Tat-TAR interaction inhibitors [J]. Bioorganic & Medicinal Chemistry.2008,16:8178-8186
[46]Min Wang, zhidong Xu, Ming Yang, et al. a,a-Trehalose derivatives bearing guanidine groups as inhibitors to HIV-1 Tat-TAR RNA interaction in human cells [J]. Bioorganic & Medicinal Chemistry Letters.2004,14:2582-2588
[47]Dekai Yuan, Meizi He, Ming Yang, et al. The design, synthesis, and biological evalution of novel substituted purines as HIV-1 Tat-TAR inhibitors [J]. Bioorganic & Medicinal Chemistry.2007,15:265-272
[48]Li Ding, Xin-Xiang Zhang, Ming Yang, et al. Study on the interaction between anti-HIV-1 active compounds with trans-activation response RNA by affinity capillary electrophoresis [J]. Journal of Chromatography B.2005,814:99-104
[49]Shuguang Chen, Ran Chen, Ming Yang, et al. Design, synthesis, and biological evaluation of novel quinoline derivatives as HIV-1 Tat-TAR interaction inhibitors [J]. Bioorganic & Medicinal Chemistry.2009,17:1948-1956
[50]张景霞,苏海霞,赵晓宁.体外转录法制备地高辛标记HCV核心区RNA探针及其初步应用[J], Chin J Dis Control Prev.2006,10(4):324-327
[2]李太生.HIV/ADIS研究的成功与挑战[J].临床内科杂志.2009,05(1)：293-294
[3]Kittiworakarn J, Lecoq A, Moine G, et al. HIV-1 Tat raisesanadjuvant-free humoral immune response controlled by its core region and its ability to form cysteinemediated oligomers[J]. J Biol Chem.2006,281(6):3105-3115
[4]Brigitic, Giaccam, Noonandm, et al. HIV Tat, its Targets and the control of viral gene expression[J]. FEMS M icrobiol Lett.2003,220(1):57-65
[5]崔凡,刘维达.HIV-1转录调控机制的研究进展[J].国外医学·皮肤性病学分册.2003,29(2)：123-126
[6]张传明,肖志,艳胡春.HIV-1反式激活抑制剂的研究进展[J].中国药物科学杂志.2008,18(6)：461-467
[7]艾青,王丽梅,狄美玉.Tat蛋白结构与功能研究的进展[J].细胞与分子免疫学杂志,2005,21(3)：133-134
[8]白如珺,刘永新.HIV-1反式激活与抑制[J].药学学报,2006,41(4)：289-295
[9]Chao Zhou, Tariq M. Rana. A Bimolecular Mechanism of HIV-1 Tat Protein Interaction with RNA Polymerase II Transcription Elongation Complexes[J]. JMB.2002,320(2): 925-942
[10]Alastair I.H.Murchie, Ben Davis, Mohammad Afshar, et al. Structure-based Drug Design Targeting an Inactive RNA Conformation:Exploiting the Flexibiliy of HIV-1 TAR RNA. J.Mol.Biol.2004,336:625-638
[11]Julie K Jadlowsky, Masanori Nojima, koh Fujinaga. Dominant negative mutant CyclinTl proteins inhibit HIV transcription by specifically degrading Tat. Retrovirology.2008,5: 63
[12]Zhu, Y., Pe'ery, et al. Transcription elongation factor P-TEFb is required for HIV-1Tat transactivation in vitro. Genes Dev.1997,11:2622-2632
[13]Mancebo, H.S.Y., Lee, et al. P-TEFb kinase is required for HIV Tat transcriptional activation in vivo and in vitro. Genes Dev,1997,11:2633-2644
[14]Wei, P., Garber, et al. A novel CDK9-associated C-type cyclin interacts directly with HIV-1 Tat and mediates its high-affinity, loop specific binding to TAR RNA. Cell.1998, 92:451-462
[15]Garber, M.E., Wei, et al. The interaction between HIV-1 Tat and human cyclin T1 requires zinc and a critical cysteine residue that is not conserved in the murine CycTl protein. Genes Dev.1998,12:3512-3527
[16]李淼,黄山.HIV-1进入抑制剂的研究进展[J].药学进展.2007,31(12)：550-560
[17]Biscone M J, Pierson T C, Dows R W. Opportunities and challenges intargeting HIV entry. Curr Opin Pharmacol.2002,2(5):529-533
[18]吉昌华.人免疫缺陷病毒LTR转录调控机制的研究进展[J].国外医学分子生物学分册.1991,13(3)：106-109
[19]欧强,卢洪洲.抗HIV药物研究进展[J].世界感染杂志.2008,8(6)：397-400
[20]孙哲,吕秋君.基于检测报告基因的药物筛选方法研究进展[J].中国药学杂志.2000,35(8)：507-509
[21]Bronstein I, Martin CS, Fortin JJ, et al. Chemiluminescence:sensitive detection technology for repoter gene assays[J]. Clin Chem.1996,42(9):1542
[22]Silverman L, Campbell, Broach JR. New assay technology for highthroughput screening[J]. Curr Opin Chem Biol.1998,2:397
[23]De Wet, J R. Cloning of firefly luciferase cDNA and the expression of active luciferase in E.coil[J]. Proc Natl Acad SCI USA.1985,87:7870-7873.
[24]Jeang KT, Xiao H, Rich EA.Multifaceted activities of the HIV-1 transactivator of transcription[J]. Biol Chem.1999,274(41):28837-28840
[25]Dandekar DH, Ganesh KN, Mitra D. HIV-1 Tat directly binds to NFkappaB enhancer sequence:role in viral and cellular gene expression[J]. Nucleic Acids Res.2004,32(4): 1270-1278
[26]刘定燮,周晓巍,黄培唐.哺乳动物表达系统及其研究进展.生物技术通讯.2003,14(4)：308-310
[27]Xu A, Kudo S, Fukuda M, et al. A novel expression vector composed of a regulatory element of the human leukosialin-encoding gene in different types of mammalian cells. Gene,1995.160:183
[28]Arzumanov, A., Walsh, et al. Inhibition of HIV-1 tat-dependent trans activation by steric block chimeric 2'-O-methyl/LNA oligoribonucleotides[J]. Biochemistry.2001,40: 14645-14654
[29]Mischiati, C., Jeang, et al. Aromatic polyamidines inhibiting the tat-induced HIV-1 transcription recognize structured TAR-RNA[J]. Antisense Nucl. Acid Drug Dev. 2001,11,209-217
[30]Kaushik, N., Basu, et al. Anti-TARpolyamide nucleotide analog conjugated with a membrane-permeating peptide inhibits human immunodeficiency virus type 1 production[J]. Virol.76:3881-3891 [31] Beatrice M, Arzuonanov A, Singh M, et al. Oligonucleotide inhibition of the interaction of HIV-1 Tat protein with the transactivation responsive region (TAR) of HIV RNA. Biochimica et Biophys Acta.1999,1445:86
[32]李在留,刘朝奇,邹坤.HIV-1 Tat蛋白结合Tar的抗艾滋病药物筛选模型的建立研究.时珍国医国药.2008,19(2)：352-354
[33]G. Wang, H.F. Chen, M.H. Huang, N.L. Wang, J.C. Zhang, Y. Zhang, et al., Methyl protodioscin induces G2/M cell cycle arrest and apoptosis in HepG2 liver cancer cells. Cancer Letters.2006,241(1):102-109
[34]Haase-Pettingell, C.A, J.King. Formation of aggregates from a thermolabile in vivo folding intermediate in P22 tailspike maturation:A model for inclusion body formation[J]. Biol.Chem,1988,263:4977-4983
[35]Anna Mitraki, Jonathan King. Protein folding intermediates and infusion body formation[J]. Nature Biotechnology.1989,7:690-697
[36]Catheine H.Schein, Mathieu H.M.Noteborn. Formation of soluble recombinant pretein in E.coil is favored by lower temperation[J]. Nature Biotechnology.1988,6:291-294
[37]Hockney. Recent developments in heterologous protein production in E.coli [J]. Trends Biotechnol.1994,12(11):456-463
[38]Drysdale, M. J., Lentzen, et al. RNA as a drug target [J]. Prog.Med.Chem.2002,9: 73-119
[39]Foreyen, M. &Herdewijn, P. RNA as a target for drug design, the example of Tat-TAR interaction [J]. Curr.Top.Med.Chem.2002,2:1123-1145
[40]杨玉志,唐祖明,陆祖宏.毛细管电泳在微生物分离与检测中的应用[J].微生物学杂志.2005,25(4)：77-81
[41]Mohamed N A. Enantioselective binding analysis of verapamil by capillary electrophoresis-frontal analysis [J]. Journal of Chromatography A.2000,875:447-453
[42]王亮,姚鑫,袁倬斌.毛细管电泳在生物大分子分析中的应用[J].分析试验室,2001,20：28-29
[43]Mucha P. Structural requirements for conserved Agr52 residue for interaction of the human immunodeficiency virus 1 transactivation responsive element with transactivator of transcription protein(49-57). Capillary electrophoresis mobility shift assay [J]. Journal of Chromatography A.2002,968:211-220
[44]Meizi He, Dekai Yuan, Ming yang, et al. Synthesis and assay of isoquinoline derivatives as HIV-1 Tat-TAR interaction inhibitors [J]. Bioorganic & Medicinal Chemistry Letter. 2005,15:3978-3981
[45]Ruifang pang, Chunlei zhang, Ming Yang. Design and SAR of new substituted purines bearing ary groups at N9 position as HIV-1 Tat-TAR interaction inhibitors [J]. Bioorganic & Medicinal Chemistry.2008,16:8178-8186
[46]Min Wang, zhidong Xu, Ming Yang, et al. a,a-Trehalose derivatives bearing guanidine groups as inhibitors to HIV-1 Tat-TAR RNA interaction in human cells [J]. Bioorganic & Medicinal Chemistry Letters.2004,14:2582-2588
[47]Dekai Yuan, Meizi He, Ming Yang, et al. The design, synthesis, and biological evalution of novel substituted purines as HIV-1 Tat-TAR inhibitors [J]. Bioorganic & Medicinal Chemistry.2007,15:265-272
[48]Li Ding, Xin-Xiang Zhang, Ming Yang, et al. Study on the interaction between anti-HIV-1 active compounds with trans-activation response RNA by affinity capillary electrophoresis [J]. Journal of Chromatography B.2005,814:99-104
[49]Shuguang Chen, Ran Chen, Ming Yang, et al. Design, synthesis, and biological evaluation of novel quinoline derivatives as HIV-1 Tat-TAR interaction inhibitors [J]. Bioorganic & Medicinal Chemistry.2009,17:1948-1956
[50]张景霞,苏海霞,赵晓宁.体外转录法制备地高辛标记HCV核心区RNA探针及其初步应用[J], Chin J Dis Control Prev.2006,10(4):324-327