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: 3144398
• 负责人: GOVINDASWAMY CHINNADURAI
• 金额: $ 22.1万
• 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 1995-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3144398
• 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; transcription factor; 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动。 它 激活许多病毒和细胞基因，也抑制 某些增强剂的活动。 我们观察到ELA蛋白 通过AD5的反式激活有缺陷的显性阴性突变体（HR5）编码 抑制基础HIV-1 LTR活性，并抑制跨激活 LTR由包括HIV的TAT基因在内的多种药物介导。 HR5 对瞬态测定中的HIV复制也有很强的抑制作用。 HR5的负调控作用似乎至少部分是 通过抑制艾滋病毒增强剂的介导。 另外，对 其他LTR启动子元素也是可能的。 我们建议调查 增强子抑制的机制详细抑制。 HIV增强子区域 由两个核心增强剂组成。 我们将确定两个核心中的哪个 增强剂是HR5介导的抑制的目标。 阐明 抑制机制，感染细胞的核因子的能力 使用HR5绑定到单个增强子将由 电泳移动性转移测定法。 E1A可能的关联 具有已知增强剂结合蛋白的蛋白质将通过DNA研究 亲和力沉淀测定和通过E1A进行免疫沉淀分析 抗体。 HR5介导的抑制可以通过 HTLV-II和Phorbol酯。 增强子序列，其中hr5-缓解 发生介导的抑制作用和增强子结合的核因子 结果可能会影响活动。 我们 将确定单纯疱疹病毒1感染还是肝炎 病毒X基因还可以缓解HR5对LTR的抑制作用 表达。 除了增强剂抑制外，我们还将调查 对各种转录和调节因素相互作用的影响 具有启动子元素，包括反式激活区域（TAR）和 负调节元件（NRE）。 研究HR5对HIV的影响 详细复制，HR5病毒感染对HIV-1的影响 将研究原发性人外周血淋巴细胞中的复制。 研究HR5对其他免疫缺陷的表达LTR的影响 病毒并将体外结果扩展到动物模型 Future，HR5对SIMIAN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复制将是 检查。 我们将设计专门适合的HR5的衍生物 抑制HIV复制。 这样的变体将由 将CD4的V1结构域取代腺病毒纤维的一部分。 这 HR5：CD4重组病毒预计仅对托管 那些表达HIV Env蛋白（GP120）的细胞，也应该很有价值 在特定靶向ELA和其他抑制剂对HIV感染的靶向 细胞。