INHIBITION OF HIV GENE EXPRESSION BY ADENOVIRUS MUTANTS

腺病毒突变体对 HIV 基因表达的抑制

• 批准号: 3144394
• 负责人: GOVINDASWAMY CHINNADURAI
• 金额: $ 20.9万
• 依托单位: SAINT LOUIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-07-01 至 1995-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3144394
• 关键词: AIDS; Adenoviridae; Alphaherpesvirinae; CD4 molecule; DNA binding protein; antiviral antibody; gel electrophoresis; gene expression; gene induction /repression; genetic enhancer element; genetic manipulation; hepatitis B virus group; human immunodeficiency virus; human immunodeficiency virus 1; human tissue; immunoprecipitation; lymphocyte; molecular cloning; mutant; nucleic acid repetitive sequence; phorbols; simian immunodeficiency virus; virus DNA; virus protein; virus replication

Adenovirus Ela has two different transcriptional regulatory activities. It activates a number of viral and cellular genes and also represses the activity of certain enhancers. We have observed that the Ela proteins coded by a trans-activation defective dominant negative mutant (hr5) of Ad5 suppress basal HIV-1 LTR activity and also inhibit trans-activation of the LTR mediated by a variety of agents including the tat gene of HIV. Hr5 also has a strong inhibitory effect on HIV replication in transient assays. The negative regulatory effect of hr5 appears to be at least partly mediated by the repression of the HIV enhancer. In addition, an effect on other LTR promoter elements is also possible. We propose to investigate the mechanism of enhancer repression in detail. The HIV enhancer region consists of two core enhancers. We will identify which of the two core enhancers is the target for hr5-mediated repression. To elucidate the mechanism of repression, the ability of nuclear factors from cells infected with hr5 to bind to the individual enhancers will be determined by electrophoretic mobility shift assays. Possible association of E1a proteins with known enhancer binding proteins will be investigated by a DNA affinity precipitation assay and by immunoprecipitation analysis using E1a antibodies. The hr5-mediated repression can be relieved by the tax gene of HTLV-II and phorbol esters. The enhancer sequence wherein relief of hr5- mediated repression occurs and nuclear factors whose enhancer binding activity may be affected as a consequence thereof will be identified. We will determine whether herpes simplex virus-1 infection or the hepatitis B virus X gene could also relieve the inhibitory effect of hr5 on LTR expression. In addition to enhancer repression we will also investigate the effect on interaction of various transcription and regulatory factors with promoter elements, including the trans-activation region (TAR) and the negative regulatory element (NRE). To investigate the effect of hr5 on HIV replication in detail, the effect of hr5 viral infection on HIV-1 replication in primary human peripheral blood lymphocytes will be studied. To study the effect of hr5 on the expression LTR of other immunodeficiency viruses and to extend the in vitro results to an animal model int he future, the effect of hr5 on simian immunodeficiency virus (SIV) LTR expression and SIV replication in primary human blood lymphocytes will be examined. We will design a derivative of hr5 specifically suited for inhibiting HIV replication. Such a variant will be constructed by substituting the V1 domain of CD4 for part of adenovirus fiber. The hr5:CD4 recombinant virus is expected to have restricted tropism for only those cells expressing HIV Env protein (gp120) and should also be valuable in specific targeting of Ela and other inhibitory agents to HIV-infected cells.

腺病毒Ela具有两种不同的转录调节活性。 它 激活许多病毒和细胞基因，并抑制 某些增强子的活性。 我们观察到Ela蛋白 由 Ad5 的反式激活缺陷显性失活突变体 (hr5) 编码 抑制 HIV-1 LTR 的基础活性，并抑制 HIV-1 LTR 的反式激活 LTR 由多种因子介导，包括 HIV 的 tat 基因。 小时5 在瞬时检测中对 HIV 复制也有很强的抑制作用。 hr5 的负面调节作用似乎至少部分是 通过抑制 HIV 增强子介导。 此外，还对 其他LTR启动子元件也是可能的。 我们建议调查 增强子抑制机制的详细信息。 HIV增强子区域 由两个核心增强子组成。 我们将确定两个核心中的哪一个 增强子是 hr5 介导的抑制的目标。 为了阐明 抑制机制，感染细胞核因子的能力 hr5 与各个增强子的结合将由下式确定 电泳迁移率变动分析。 E1a 的可能关联 具有已知增强子结合蛋白的蛋白质将通过 DNA 进行研究 亲和沉淀分析和使用 E1a 的免疫沉淀分析 抗体。 hr5介导的抑制可以通过tax基因缓解 HTLV-II 和佛波酯。 增强子序列，其中hr5-的缓解 介导的抑制发生，核因子的增强子结合 活动可能会受到影响，其后果将被确定。 我们 将确定是否感染单纯疱疹病毒 1 或乙型肝炎 病毒X基因也能解除hr5对LTR的抑制作用 表达。 除了增强子抑制之外，我们还将研究 对各种转录和调节因子相互作用的影响 具有启动子元件，包括反式激活区（TAR）和 负调控元件（NRE）。 研究 hr5 对 HIV 的影响 详细复制，hr5病毒感染对HIV-1的影响 将研究原代人外周血淋巴细胞中的复制。 研究hr5对其他免疫缺陷LTR表达的影响 病毒并将体外结果扩展到动物模型 未来，hr5对猿猴免疫缺陷病毒（SIV）LTR的影响 原代人血液淋巴细胞中的表达和 SIV 复制 检查了。 我们将设计一款专门适合 hr5 的衍生产品 抑制HIV复制。 这样的变体将由以下方式构建 用CD4的V1结构域替换部分腺病毒纤维。 这 hr5：CD4重组病毒预计仅具有有限的趋向性 这些表达 HIV 包膜蛋白 (gp120) 的细胞也应该很有价值 Ela 和其他抑制剂专门针对 HIV 感染者 细胞。