Intra- and interspecific variation of the CCR5 gene in higher primates

We have evaluated the molecular evolution of the chemokine receptor CCR5 in primates. The chemokine receptor CCR5 serves as a major co-receptor for human immunodeficiency virus/simian immunodeficiency virus (HIV/SIV) infection. Knowledge of evolution of the CCR5 molecule and selection on the CCR5 gene may shed light on its functional role. The comparison of differences between intraspecific polymorphisms and interspecific fixed substitutions provides useful information regarding modes of selection during the course of evolution. There is marked polymorphism in the CCR5 gene sequence within different primate species, whereas sequence divergence between different species is small. By using contingency tests, we compared synonymous (SS) and nonsynonymous (NS) CCR5 mutations occurring within and between a broad range of primates. Our results demonstrate that CCR5 evolution did not follow expectations, of strict neutrality at the level of the whole gene. The proportion of NS to SS at the intraspecific level was significantly higher than that observed at the interspecific level. These results suggest that most CCR5 NS polymorphisms are slightly deleterious. However, at domains more closely correlated with its known biological functions, there was no obvious evidence to support deviation from neutrality.

树突状细胞与 HIV-1 辅助蛋白相互作用的体外研究——细胞系模型研究价值的验证和新的研究方向的确立

树突状细胞（Dendritic cells，DCs）作为人类免疫缺陷病毒（Human Immunodeficiency Virus，HIV）感染的第一靶细胞和第一道防线， 在HIV-1感染 和传播过程当中发挥重要的功能。DC的免疫功能主要包括抗原的捕获、加工、 递呈并激活T细胞对HIV-1作出免疫反应，这些功能的发挥依赖于其自身接受刺 激有效地分化和成熟。 与其它慢病毒（lentivirus）相同，HIV-1所具有的6种辅助蛋白（Nef，Rev， Tat，Vif，Vpr和Vpu），决定着病毒自身的复制增殖和对机体的感染和致病力。 目前，HIV-1辅助蛋白对CD4+ T细胞影响的研究较为深入，是否影响和调节DC 的分化和成熟尚不够清楚，现有的文献报道很少，且相互不一致甚至矛盾。因此， 建立合适的体外研究体系和细胞模型，有针对性地进行研究DC与HIV-1之间的相 互作用，将有助于加深对HIV/AIDS致病和发病机理的理解，具有重要的生物医 学意义。 本实验选择了可被HIV-1感染的白血病细胞系THP-1为实验模型，首先评价 了THP-1作为DC前体在研究DC分化、成熟中的可用性，特别是判定DC分化成熟 和功能状态的主要细胞表面标记的动态变化和规律。进而在相同条件下分析了6 个辅助蛋白基因对THP-1的凋亡诱导作用，证实了Nef和Tat确可诱导转染细胞自 身凋亡，而Rev和Vpr可在THP-1细胞中持续表达，形成了稳定的细胞系，为进一 步研究和比较Rev、Vpr对DC的分化、成熟的影响奠定了实验基础。更重要的是， 我们发现，Vif和Vpu不能在THP-1中有效表达，其原因可能直接与限制性因子 APOBEC3G的存在有关，提示Vpu与APOBEC3G可能存在着新的相互作用——这 一线索已作为实验室新的研究方向，进一步深入的研究可能为HIV/AIDS致病、 发病机理和机体的抗病机制提供新的科学依据。

稳定表达HIV-1辅助蛋白Rev的THP-1细胞模型的建立和鉴定

Human immunodeficiency virus-1(HIV-1)辅助蛋白在其感染和艾滋病发病过程中起着非常重要的作用.Regulator of expression of virion proteins(Rev)作为HIV-1辅助蛋白之一,可以调节病毒结构蛋白mRNA出核转运和蛋白表达,对于病毒的复制至关重要.为研究Rev蛋白对靶细胞表犁和功能的影响,本实验采用电穿孔的方法,将HIV-1的rev基因导入THP-1细胞,通过流式分选结合G418筛选的方法建立稳定表达Rev蛋白的细胞模型;并通过RT-PCR、荧光观察及流式检测的方法,在mRNA和蛋白两个水平对所建立的细胞模犁进行鉴定.结果证实rev基凶成功导入了THP-1细胞并稳定表达,为后续rev基因产物与细胞相互作用的研究提供了平台.

光动力疗法抑制人免疫缺陷病毒复制的实验研究

通过对人免疫缺陷病毒复制过程的抑制作用研究，探索光动力疗法(PDT)在艾滋病防治中的潜在价值。使 用不同稀释浓度的光敏剂血卟啉单甲醚(HMME)和亚甲蓝(MB)分别与人免疫缺陷病毒HIV一1Ⅲs或宿主细胞 MT4，C8166或H9／HIV-IⅢB孵育2 h。以波长630 nm能量密度0．3 J／cm2的半导体激光进行照射。继续孵育若干 小时后，用噻唑蓝(MTT)比色法检测细胞存活率或合胞体计数，同时收集培养上清液用ELISA法检测HIV-I p24 抗原。结果表明，光动力疗法能显著抑制人免疫缺陷病毒诱导的细胞一细胞融合(HMME-PDT抑制率64．68％， MB-PDT抑制率61．56％)和病毒一细胞融合(HMME—PDT抑制率85％，Mt}PDT抑制率73．64％)，并对游离病毒 有很强的杀伤作用，最高可达到100％。光动力疗法不能抑制慢性感染期和急性感染2 h后病毒的复制过程。可 见光动力疗法对游离病毒和病毒感染诱导的膜融合有显著抑制作用，有可能为艾滋病的防治提供一种新的方法。

恒河猴TRIM5α的组织分布及不同刺激促进PBMC中TRIM5α转录水平的上调

TRIM5α(tripartite motif protein 5-alpha)蛋白是恒河猴体内一种非常重要的限制因子,能抑制人免疫缺陷病毒(HIV-1,human immunodeficiency virus type 1)、马感染性贫血病毒(EIAV, equine infectious anemia virus)和猫免疫缺陷病毒(FIV, feline immunodeficiencyvirus)等逆转录病毒的复制.恒河猴TRIM5α的组织分布以及在受到外界刺激时TRIM5α mRNA表达量的变化研究还未见报道.本研究从中国恒河猴的各组织中提取总RNA,以β-actin基因作为内参照,通过半定量RT-PCR检测各组织中TRIMSα mRNA的表达.选择HIV-GFP-VSVG假病毒感染外周血单核细胞(peripheral blood mononuclear cell,PBMC),非特异性刺激剂--佛波脂(Phorbol myfismte acetate,PMA)+离子霉素(ionomycin,Ion)及CD28抗体+CD49d抗体分别共刺激恒河猴PBMC,研究不同刺激对恒河猴TRIM5α mRNA表达水平的影响.结果表明:TRIM5α mRNA表达于所研究的恒河猴21种组织中,免疫系统和泌尿生殖系统组织中表达量最高,而神经系统组织,如大脑、脊髓中表达较少,其他组织中未见明显的表达差异;HIV-GFP-VSVG感染和用PMA+Ion、CD28抗体+CD49d抗体分别共刺激PBMC能促进PBMC中TRIM5α mRNA的转录水平的上调.

贻贝凝集素抗-HIV活性研究

分离提取海洋无脊椎动物贻贝(Crenomytilus grayanus)凝集素,考察其抗HIV活性。采用半乳糖-Sepharose 6B亲和层析和Sephacryl S-200层析分离提取贻贝凝集素(Crenomytilus grayanus lectin,CGL),以光镜检查合胞体抑制试验,以ELSA测定HIVp24抗原表达水平。从海洋无脊椎动物贻贝中分离出的凝集素(CGL),为N-乙酰半乳糖胺/半乳糖(GalNAc/Gal)特异性的凝集素。CGL在27.88mg.L-1浓度时,对HIV诱导细胞病变的抑制达50%;在45.70mg.L-1时,对HIV-1复制的抑制达50%;同时在35.12mg.L-1浓度时,对HIV感染细胞融合的阻断达50%。

顶体反应抑制剂4'- 乙酰胺苯基4-胍基苯甲酸酯(AGB)具有阻断HIV-1进入细胞的作用

】目的: 研究4'- 乙酰胺苯基4- 胍基苯甲酸酯(AGB)抗 HIV-1 活性及作用靶点。方法: 通过 AGB对宿主细胞的毒性实验、合胞体抑制实验、融合阻断实验、对HIV-1感染细胞的保护作用实 验和对HIV-1急性感染细胞p24抗原产生的抑制作用等试验，观察AGB对HIV-1复制的影响和作 关键词: AGB; HIV-1; 病毒进入; 杀微生物剂; 杀精子活性 中图分类号: R967; 文献标识码: A 文章编号: 0253-357X(2005)11-0660-05 本研究为国家高技术研究发展计划(2003AA219142)、国 家科技攻关计划 (2004BA719A14)、中国科学院知识创新 工程重要方向(KSCX2-SW-216; KSCX12-SW-11)、云南 省科技攻关计划(2004NG12)和云南省生育调节与少数民 族优生研究重点实验室资助项目 通讯作者: 郑永唐; Tel: +86-871-5195684; Fax:+86-871-5191823; E-mail: zhengyt@mail.kiz.ac.cn 在我国, 人免疫缺陷病毒(human immunodeficiency virus, HIV)危害日趋严重，处在全国低流行 与局部地区及特定人群高流行并存的态势。卫生部 的数据显示，截止2005 年3 月底，全国累计报告 HIV 感染者114 703 例。专家估计我国实际HIV 感 染者超过100 万人。预计到2010 年, 全国HIV感染 者将突破千万。截止2004 年底, 云南省累计报告的 HIV 感染者已达28 391 人, 是全国流行最严重的地 区。艾滋病流行正由高危人群向一般人群传播。 新的证据显示近年来由性传播途径感染的比例有所 上升, 女性感染者的比例有较大幅度的上升, 迫切需 要发展一种女性可自主控制的方法 [1]。杀微生物剂 是可以局部用药于阴道和宫颈、能够杀灭或抑制 包括HIV等病毒性和细菌性病原体、人工合成或天 然的药物。具有避孕作用的杀微生物剂更是近年 来的研究热点，也具有广阔的应用前景[2,3]。 顶体酶是存在于精子顶体内的一种类胰蛋白 酶, 它是受精过程中的一种重要的蛋白水解酶, 此酶 能水解卵细胞的透明带, 使精子能够与卵细胞相融 合; 顶体酶还能促进生殖系统中激肽的释放, 后者能 够增强精子的活力和促进精子的运动, 顶体酶的失 活将导致不孕[4]。AGB(4'- 乙酰胺苯基 4- 胍基苯甲 酸酯)是顶体酶的抑制剂, 实验表明在多种动物中有 很好的杀精子作用[5-7]。Bourimbaiar等[8]曾报道AGB 还具有体外抗HIV-1的作用, 活性较N-9高, 且毒性 较小。在本实验中, 我们发现AGB 的体外抗HIV-1 活性主要是阻断HIV-1 进入细胞。 用机制。结果: AGB抑制HIV-1IIIB诱导C8166细胞形成合胞体, EC50为39.5 μg/ml; 抑制HIV-1感染 细胞上清中HIV-1 p24抗原的表达, EC50为33.36 μg/ml; 阻断HIV-1慢性感染H9细胞与正常C8166 细胞间融合的作用。结论: AGB具有阻断HIV-1 进入宿主细胞的作用，是一种有前景的具杀精子 作用的杀微生物剂。

我国中药来源的抗HIV天然化合物研究进展

由于迄今仍无艾滋病(acquired immunodeficiency syndrome,AIDS)疫苗问世,抗人类免疫缺陷病毒(human immunodeficiency virus,HIV)药物仍然是艾滋病治疗的主要手段。传统中药和药用植物来源的天然化合物具有结构多样性、毒性较低、来源广泛等特点,因而在防治艾滋病方面有着独特的优势和巨大的潜力。研究者已经对天然化合物抗HIV作用进行了大量研究,并取得了可喜的成绩,发现了一些生物碱、香豆素、木脂素、黄酮类、萜类、鞣质类、多糖类、蛋白质和多肽类等天然化合物具有抗HIV的活性。然而,多数研究都是在体外试验完成的,大多数天然化合物体外抗HIV活性偏低,而且抗HIV的靶点仍不十分清楚。本文结合笔者实验室研究工作,重点介绍近年来我国传统中药来源的抗HIV活性较强的天然化合物研究进展。

Anti-HIV-1 activity of trichobitacin, a novel ribosome-inactivating protein

AIM: To determine whether trichobitacin, a novel ribosome-inactivating protein purified from the root tubers of Trichosanthes kirilowii, possesses the anti-HIV activity. METHODS: The inhibition of syncytial cell formation induced by human immunodeficiency virus type 1 (HIV-1),was determined under microscope, reduction of HIV-1 p24 antigen expression level was measured by ELISA, and decrease in numbers of HIV-1 antigen positive cells in acutely and-chronically infected cultures were detected by indirect immunofluorescence assay. RESULTS: Trichobitacin Was-found to greatly suppress syncytial cell formation induced by HIV-1 and to markedly reduce both expression of HIV-1 p24 antigen and the number of HIV antigen positive cells in acutely but not chronically HIV-1 infected culture. The median inhibitory concentration (IC50) in inhibition of syncytial cell formation and HIV antigen positive cells were 5 mu g.L-1 (95 % confidence limits: 1.3 - 20 mu g.L-1) and 0.09 mg.L-1 (95 % confidence limits: 0.011 - 0.755 mg.L-1), respectively. CONCLUSION: Trichobitacin is a novel ribosome-inactivating protein with anti-HIV-l activity.

Anti-HIV-1 property of trichosanthin correlates with its ribosome inactivating activity

Trichosanthin (TCS) is a type I ribosome inactivating (RI) protein possessing anti-tumor and antiviral activity, including human immunodeficiency virus (HIV). The mechanism of these actions is not entirely clear, but is generally attributed to its RI property. In order to study the relationship between the anti-HIV-1 activity of TCS and its RI activity, three TCS mutants with different RI activities were constructed by using site-directed mutagenesis. The anti-HIV-1 activities of the three mutants were tested in vitro. Results showed that two TCS mutants, namely TCSM((120-123)), TCSE160A/E189A, with the greatest decrease in RI activity, lost almost all of the anti-HIV activity and cytopathic effect. Another mutant TCSR122G, which exhibited a 160-fold decrease in RI activity, retained some anti-HIV activity. The results from this study suggested that RI activity of TCS may have significant contribution to its anti-HIV-1 property. (C) 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

Independency of anti-HIV-1 activity from ribosome-inactivating activity of trichosanthin

Trichosanthin (TCS) is a type I ribosome-inactivating (RI) protein possessing multiple biological and pharmacological activities. Its major action is inhibition of human immunodeficiency virus (HIV) replication but the mechanism is still elusive. All evidences showed that this action is related to its RI activity. Previous studies found that TCS mutants with reduced RI activity simultaneously lost some anti-HIV activity. In this study, an exception was demonstrated by two TCS mutants retaining almost all RI activity but were devoid of anti-HIV-1 activity. Five mutants were constructed by using site-directed mutagenesis with either deletion or addition of amino acids to the C-terminal sequence. Results showed that the RI activity of mutants with C-terminal deletion mutants (TCSC2, TCSC4, and TCSC14) decreased by 1.2-3.3-fold with parallel downshifting of its anti-HIV-1 activity (1.4-4.8-fold). Another two mutants, TCSC19aa and TCSKDEL having 19 amino acid extension and a KDEL signal sequence added to the C-terminal sequence, retained all RI activity but subsequently lost most of the anti-HIV-1 activity. These findings suggested that ribosome inactivation alone might not be adequate to explain the anti-HIV action of TCS. (C) 2003 Elsevier Science (USA). All rights reserved.

Pharmacokinetics of sifuvirtide, a novel anti-HIV-1 peptide, in monkeys and its inhibitory concentration in vitro

Aim: To study the pharmacokinetics of sifuvirtide, a novel anti-human immunodeficiency virus (HIV) peptide, in monkeys and to compare the inhibitory concentrations of sifuvirtide and enfuvirtide on HIV-1-infected-cell fusion. Methods: Monkeys received 1.2 mg/kg iv or sc of sifuvirtide. An on-line solid-phase extraction procedure combined with liquid chromatography tandem mass spectrometry (SPELC/MS/MS) was established and applied to determine the concentration of sifuvirtide in monkey plasma. A four-I-127 iodinated peptide was used as an internal standard. Fifty percent inhibitory concentration (IC50) of sifuvirtide on cell fusion was determined by co-cultivation assay. Results: The assay was validated with good precision and accuracy. The calibration curve for sifuvirtide in plasma was linear over a range of 4.88-5000 mu g/L, with correlation coefficients above 0.9923. After iv or sc administration, the observed peak concentrations of sifuvirtide were 10626 +/- 2886 mu g/L and 528 +/- 191 mu g/L, and the terminal elimination half-lives (T,12) were 6.3 +/- 0.9 h and 5.5 +/- 1.0 h, respectively. After sc, T-max was 0.25-2 h, and the absolute bioavailability was 49% +/- 13%. Sifuvirtide inhibited the syncytium formation between HIV-1 chronically infected cells and uninfected cells with an IC50 of 0.33 mu g/L. Conclusion: An on-line SPE-LC/MS/MS approach was established for peptide pharmacokinetic studies. Sifuvirtide was rapidly absorbed subcutaneously into the blood circulation. The T-1/2 of sifuvirtide was remarkably longer than that of its analog, enfuvirtide, reported in healthy monkeys and it conferred a long-term plasma concentration level which was higher than its IC50 in vitro.

The anti-HIV-1 effect of scutellarin

Scutellarin was purified from the plant Erigeron breuiscapus (Vant.) Hand.-Mazz. The activity against 3 strains of human immunodeficiency virus (HIV) was determined in vitro in this study. These were laboratory-derived virus (HIV-I-IIIB), drug-resistant virus (HIV-I-74V), and low-passage clinical isolated virus (HIV-1(KM018)). From syncytia inhibition study, the EC50 of scutellarin against HIV-I-IIB direct infection in C8166 cells was 26 mu M with a therapeutic index of 36. When the mode of infection changed from acute infection to cell-to-cell infection, this compound became even more potent and the EC50 reduced to 15 mu M. This suggested that cell fusion might be affected by this compound. By comparing the inhibitory effects on p24 antigen, scutellarin was also found to be active against HIV-1(74V) (EC50 253 mu M) and HIV-1(KM018) (EC50 136 mu M) infection with significant difference in potency. The mechanism of its action was also explored in this study. At a concentration of 433 mu M, scutellarin inhibited 48% of the cell free recombinant HIV-1 RT activity. It also caused 82% inhibition of HIV-1 particle attachment and 45% inhibition of fusion at the concentrations of 54 mu M. In summary, scutellarin was found to inhibit several strains of HIV-1 replication with different potencies. It appeared to inhibit HIV-1 RT activity, HIV-1 particle attachment and cell fusion. These are essential activities for viral transmission and replication. (c) 2005 Elsevier Inc. All rights reserved.

An inhibitor of c-Jun N-terminal kinases (CEP-11004) counteracts the anti-HIV-1 action of trichosanthin

Trichosanthin (TCS) is a type I ribosome-inactivating protein possessing multiple biological and pharmacological activities. One of its major actions is inhibition of human immunodeficiency virus (HIV) replication. The mechanism is still not clear. It is

Synthesis and HIV-1 integrase inhibition activity of some N-arylindoles

Eight simple N-arylindoles were designed, synthesized and evaluated as human immunodeficiency virus (HIV)-1 integrase inhibitors in vitro for the first time. Among these compounds, 3b, 3e and 3g demonstrated significant anti-HIV-1 integrase activity. Espe