INTERFERON'S ROLE IN ANTIVIRAL ACTIVITIES

干扰素在抗病毒活性中的作用

• 批准号: 3263784
• 负责人: Jerry L Taylor
• 金额: $ 19.59万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-05-01 至 1995-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3263784
• 关键词: Alphaherpesvirinae; antiviral agents; combination chemotherapy; corneal stroma; cytomegalovirus; drug interactions; enzyme linked immunosorbent assay; enzyme mechanism; gel electrophoresis; gene expression; genetic transcription; genetic translation; human tissue; interferon alpha; keratitis; messenger RNA; molecular cloning; nucleic acid metabolism; nucleic acid sequence; nucleobase; nucleoside analog; thymidine kinase; tissue /cell culture; transcription factor; varicella zoster virus; virus RNA; virus protein; virus replication; western blottings

Ocular infections caused by herpesviruses are important clinical problems. Keratitis caused by herpes simplex virus (HSV) is a leading cause of blindness in the U.S. and sometimes require corneal transplantation to restore vision. Treatment of HSV corneal infections with topical nucleoside analogs has been efficacious in treating acute disease, but therapy for other forms of HSV-induced corneal disease and ocular disease caused by other human herpesviruses, including varicella-zoster virus (VZV) and cytomegalovirus (CMV), has not proven very successful. Treatment of HSV keratitis with combinations of nucleoside analogs like acycloguanosine (ACV) and human interferon-alpha(IFN) has been shown to produce better therapeutic response than either antiviral agents alone. We and others have shown that this combined effect reflects true synergism between these two agents at the cellular level. Synergistic antiviral activity between nucleoside analogs and IFN has been demonstrated against other members of the herpesvirus family as well. We have found evidence that IFN treatment alters the metabolism of nucleosides in HSV-infected human corneal stromal cells in such a manner that the competition between ACV and natural nucleosides for HSV-specific enzymes is shifted in favor of ACV. Our hypothesis is that this alteration in nucleoside metabolism is due to the selective repression of expression of HSV genes. We propose to determine the point in HSV replication inhibited by IFN treatment (i) by measurement of the activities of HSV-specific enzymes,(ii) by surveying the synthesis of HSV proteins using gel electrophoresis of radiolabeled infected-cell proteins,(iii) by quantitation of the production of viral mRNAs using slot blot hybridization with cloned HSV genomic DNA, and(iv) by mobility shift assays to detect the formation of HSV-cell protein/DNA complexes associated with immediate early gene transactivation. We propose that the altered nucleoside metabolism detected in IFN-treated, HSV-infected human corneal cells may reflect a modification of normal cellular nucleoside transporters. Using selective inhibitors of nucleoside transporters, we will determine the substrate specificity and number of nucleoside transporters on corneal cells and the effects of infection, IFN treatment, or combination of IFN and ACV on those transporter parameters. Using HSV as a model virus we will test our hypothesis and determine how combinations of IFN and ACV achieve synergistic antiherpes activity. We will then determine whether the synergistic anti-VZV activity we have demonstrated in corneal cells in culture is due to the same or different mechanism. Elucidation of the mechanism of those synergistic anti-herpes activity may allow therapy with combinations of nucleoside analog and IFN to be tailored to achieve maximum efficacy in the treatment of eye disease caused by human herpesviruses, including HSV, VZV, and CMV, that do not respond well to current monotherapy.

疱疹病毒引起的眼部感染是重要的临床问题。 由单纯疱疹病毒（HSV）引起的角膜炎是 美国的失明，有时需要角膜移植到 恢复视力。 用局部治疗HSV角膜感染 核苷类似物在治疗急性疾病方面有效，但 治疗其他形式的HSV诱导的角膜疾病和眼部疾病 由其他人类疱疹病毒引起 和巨细胞病毒（CMV）尚未证明非常成功。 处理 HSV角膜炎与核苷类似物（如cycycloguanosine）的组合 （ACV）和人干扰素-Alpha（IFN）已显示出更好的产生 治疗反应比任何一种抗病毒药物。 我们和其他人 已经表明，这种综合效应反映了这些效果 两种药物处于细胞水平。 协同抗病毒活性 已经证明了核苷类似物和IFN针对其他成员 疱疹病毒家族也是如此。 我们发现了IFN治疗的证据 改变HSV感染的人角膜基质中核苷的代谢 细胞以ACV与自然之间的竞争方式 HSV特异性酶的核苷转移，有利于ACV。 我们的 假设是核苷代谢的这种改变是由于 选择性抑制HSV基因的表达。 我们建议确定 IFN处理（i）通过测量抑制HSV复制的点 HSV特异性酶的活性，（ii）通过测量合成 使用放射性标记感染细胞的凝胶电泳的HSV蛋白 蛋白质，（iii）使用插槽定量生产病毒mRNA 与克隆的HSV基因组DNA和（IV）通过迁移率移位的杂交杂交 检测HSV细胞蛋白/DNA复合物相关的HSV细胞蛋白/DNA复合物的测定法 立即进行早期基因反式激活。 我们建议改变 在IFN处理的HSV感染的人角膜中检测到的核苷代谢 细胞可能反映正常细胞核苷的修饰 运输商。 使用核苷转运蛋白的选择性抑制剂，我们 将确定核苷的底物特异性和数量 角膜细胞上的转运蛋白以及感染的影响，IFN治疗， 或在这些转运蛋白参数上IFN和ACV的组合。 使用HSV 作为模型病毒，我们将检验我们的假设，并确定组合如何 IFN和ACV实现了协同的抗抗原活性。 然后我们会 确定我们是否在 培养中的角膜细胞是由于相同或不同的机制引起的。 阐明这些协同抗hearpes活性的机制可能 允许量身定制核苷类似物和IFN的疗法 为了在人类引起的眼科疾病治疗中获得最大的疗效 疱疹病毒，包括HSV，VZV和CMV，对 当前的单一疗法。