HIV-1 mediated membrane fusion as target for anti-viral

内容摘要：（1）项目目标： - 建立HIV-1病毒介导的细胞融合及gp 120/CD 4/辅助受体三分子复合物形成的生物学和生化检测方法。 - 研究HIV-1辅助受体在已知为HIV-1感染靶点的原代人细胞上的表达和功能，并研究促炎细胞因子对HIV-1辅助受体功能和原代人细胞感染性的影响。 - 开发能够阻断无细胞或细胞相关HIV-1感染的药物。 (2)实验方法： - 开发了生物学（Ca++通量、趋化性、HIV融合）和生物化学测定，以测量人细胞上的CD 4/gp 120复合物与T嗜性和M嗜性包膜的HIV-1共受体（CXCR 4和CCR 5）的相关性。它们包括来自细胞系和来自原代人细胞的CD 4/共受体的免疫共沉淀（即，单核细胞和巨噬细胞），以及使用我们的兔抗CXCR 4和抗CCR 5试剂的全细胞和膜提取物的Western印迹。 - 对多种细胞类型进行了研究：朗格汉斯细胞（LC）、树突状细胞（DC）、胸腺细胞亚群、CD 34+祖细胞、外周血T细胞、单核细胞（MO）和巨噬细胞（MDM）。 - 开发新试剂用于HIV-1关键共受体（CXCR 4、CCR 5）的免疫沉淀和Western印迹分析。 - 应用新的生物化学检测方法，包括“蛋白质组学”，以确定HIV-1辅助受体的翻译后修饰，影响其在原代人类细胞中的功能。 (3)主要发现： I. 趋化因子受体CXCR 4是HIV-1病毒的主要辅助受体。糖基化CXCR 4的预测分子量为45-47 kDa。然而，来自人淋巴细胞、单核细胞、巨噬细胞和Jurkat T淋巴细胞系的全细胞裂解物的免疫印迹揭示了CXCR 4的多种分子量同种型。其中三条条带可被抗CXCR 4单克隆抗体（101和47 kDa）沉淀或与CD 4（62 kDa）共沉淀。通过用编码CXCR 4的重组牛痘病毒感染来增强这些同种型的表达。在二维凝胶的免疫印迹中，抗泛素抗体与来自单核细胞的62 kDa CXCR 4物质反应，随后与抗CD 4抗体共沉淀。用lactacystin培养单核细胞和淋巴细胞，免疫印迹中101 kDa CXCR 4亚型的量增加了3 - 7倍。在淋巴细胞中，lactacystin也增加了CXCR 4的细胞表面表达，这与增强与HIV-1表达细胞的融合有关。在用溶酶体抑制剂莫能菌素和氯化铵处理后，观察到101 kDa同种型的强度的类似增加。抗泛素抗体与用抗CXCR 4抗体沉淀的62 kDa以上的多种蛋白质反应。我们的数据表明，泛素化可能有助于CXCR 4的异质性，并建议在原代细胞中CXCR 4的组成性营业额的蛋白酶体和溶酶体的作用。 CXCR 4的表达和构象的变化可以影响给定细胞对T嗜性HIV-1株感染的易感性，T嗜性HIV-1株仅使用CXCR 4作为辅助受体。 这样的变化也可能间接影响嗜M性HIV-1的感染，其使用CCR 5作为辅助受体，因为两种辅助受体彼此竞争与CD 4的相互作用（J.Virol. 74：5016-5023，2000）。 此外，构象的变化可能会改变靶向辅助受体的疫苗和小药物的有效性。因此，了解CXCR 4和其他主要辅助受体CCR 5的异质性和功能多样性至关重要。 我们和其他人最近观察到一个主要的CCR 5 62 kDa的物种。泛素化可能在HIV-1辅助受体的组成性细胞内转运、周转和构象中发挥作用。 我们早期的研究结果和目前的数据表明，单泛素化可能通过诱导单核细胞中与CD 4的组成性结合，可能通过改变它们的构象，来增强CXCR 4和CCR 5作为HIV-1辅助受体的作用。另一方面，CXCR 4的多聚泛素化可能降低其在静息细胞中的稳态表面表达。 这些研究已经扩展到比较未活化和活化的胸腺细胞和T细胞。在一项初步研究中，我们发现，在人胸腺细胞和外周T细胞中，通过蛋白质印迹中主要MW物质的变化确定的翻译后修饰的变化在细胞活化后可见。这些变化与增强的融合活性和增加与CD 4分子的关联性相关，而没有转录或共受体的细胞表面密度的平行增加。 二. 在研究对原生动物弓形虫的细胞介导免疫的诱导中，我们发现来自寄生虫的蛋白质C-18（亲环素）与鼠和人CCR 5结合。体外研究表明，重组C-18可有效中和来自不同进化枝的实验室适应性和多种主要HIV R5分离株的感染性，但对X4病毒无活性。与先前描述的用作共受体拮抗剂的趋化因子类似物不同，C-18似乎不触发受体内化，并且作为外来分子不太可能诱导自身免疫。因此，它可以为开发一种新的微生物衍生的拮抗剂提供基础，而这种拮抗剂没有这些有害的副作用。

Summary: (1) Goals of project: - To develop biological and biochemical assays for monitoring HIV-1 envelope-mediated cell fusion and formation of the tri-molecular complex between gp120/CD4/co-receptor. - To study the expression and function of HIV-1 co-receptors on primary human cells known to be targets for HIV-1 infection, and to study the effects of pro-inflammatory cytokines on the function of the HIV-1 co-receptors and infectivity of primary human cells. - Development of agents capable of blocking infection by cell-free or cell-associated HIV-1. (2) Experimental approach: - Biological (Ca++ flux, chemotaxis, HIV-fusion) and biochemical assays were developed to measure the association of the CD4/gp120 complex on human cells with the HIV-1 co-receptors for T-tropic and M-tropic envelopes (CXCR4 and CCR5). They included co-immunoprecipitations of CD4/co-receptors from cell lines and from primary human cells (i.e., monocytes and macrophages), and Western blots of whole cell and membrane extracts using our rabbit anti-CXCR4 and anti-CCR5 reagents. - Studies were conducted on multiple cell types: Langerhans cells (LC), dendritic cells (DC), Thymocyte subsets, CD34+ progenitors, peripheral blood T cells, monocytes (MO) and macrophages (MDM). - Develop new reagents for immunoprecipitation and Western blot analyses of the key HIV-1 co-receptors (CXCR4, CCR5). - Apply new biochemical assays including "Proteomics" to identifying the post-translational modifications of HIV-1 co-receptors that affect their function in primary human cells. (3) Major Findings: I. The chemokine receptor CXCR4 is a primary coreceptor for the HIV -1 virus. The predicted MW of glycosylated CXCR4 is 45-47 kDa. However, immunoblots of whole cell lysates from human lymphocytes, monocytes, macrophages, and the Jurkat T lymphocyte line revealed multiple molecular weight isoforms of CXCR4. Three of the bands could either be precipitated by anti-CXCR4 monoclonal antibodies (101 and 47 kDa) or co-precipitated with CD4 (62 kDa). Expression of these isoforms was enhanced by infection with a recombinant vaccinia virus encoding CXCR4. In immunoblots of two-dimensional gels, anti-ubiquitin antibodies reacted with the 62 kDa CXCR4 species from monocytes subsequent to coprecipitation with anti-CD4 antibodies. Culturing of monocytes and lymphocytes with lactacystin enhanced the amount of the 101 kDa CXCR4 isoform in immunoblots by three- to seven-fold. In lymphocytes, lactacystin also increased cell-surface expression of CXCR4, which correlated with enhanced fusion with HIV -1 envelope-expressing cells. Similar increases in the intensity of the 101 kDa isoform were seen after treatment with the lysosomal inhibitors monensin and ammonium chloride. Anti-ubiquitin antibodies reacted with multiple proteins above 62 kDa that were precipitated with anti-CXCR4 antibodies. Our data indicate that ubiquitination may contribute to CXCR4 heterogeneity and suggest roles for both proteasomes and lysosomes in the constitutive turnover of CXCR4 in primary cells. Changes in expression and conformation of CXCR4 could affect the susceptibility of a given cell to infection by T-tropic HIV-1 strains, which only use CXCR4 as a coreceptor. Such changes may also indirectly influence infection by M-tropic HIV-1, which uses CCR5 as a coreceptor, since the two coreceptors compete with each other for interaction with CD4 (J. Virol. 74:5016-5023, 2000). In addition, changes in conformation may alter the effectiveness of vaccines and small drugs that target the coreceptors. Thus, it is crucial to understand the heterogeneity and functional diversity of CXCR4 and the other principal coreceptor, CCR5. We and others have recently observed a predominant CCR5 62 kDa species. Ubiquitination may play a role in the constitutive intracellular transport, turnover, and conformation of the HIV-1 coreceptors. Our earlier findings together with the current data suggest that mono-ubiquitination may enhance the role of CXCR4 and CCR5 as HIV-1 coreceptors by inducing constitutive association with CD4 in monocytes, perhaps by altering their conformation. On the other hand, polyubiquitination of CXCR4 may decrease its steady state surface expression in resting cells. These studies have been extended to compare unactivated and activated thymocytes and T cells. In a preliminary study we found that in human thymocytes and peripheral T cells changes in post-translational modifications as determined by changes in the predominant MW species in Western blots, are seen following cell activation. These changes correlated with enhanced fusion activity and increased association with CD4 molecules without a parallel increase in transcription or in the cell-surface density of the coreceptors. II. In studying the induction of cell mediated immunity to the protozoan Toxoplasma gondii we have discovered that a protein, C-18 (cyclophilin) from the parasite binds to both murine and human CCR5. In vitro studies demonstrated that recombinant C-18 is effective in neutralizing infectivity of both lab-adapted and multiple primary HIV R5-isolates from different clades but was inactive against X4 viruses. Unlike previously described chemokine analogs used as co-receptor antagonists, C-18 does not appear to trigger receptor internalization and as foreign molecule is unlikely to induce autoimmunity. It therefore could provide the basis for the development of a new microbially derived antagonist lacking these deleterious side effects.