PSEUDOTYPIC DEFECTIVE INTERFERING HIV PARTICLES AS AN ANTIVIRAL THERAPY FOR AIDS

假型缺陷干扰 HIV 颗粒作为艾滋病抗病毒疗法

• 批准号: 3782407
• 负责人: M SCHUBERT
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3782407
• 关键词: AIDS therapy; CD4 molecule; antiviral agents; chimeric proteins; computer simulation; defective virus; electron microscopy; helper virus; human immunodeficiency virus 1; immunofluorescence technique; interfering virus; intracellular parasitism; microorganism culture; recombinant DNA; transfection; transfection /expression vector; virus DNA; virus RNA; virus cytopathogenic effect; virus envelope; virus genetics; virus protein; virus replication

Extensive efforts in the development of drugs or therapeutic vaccines against HIV~1 have not been successful. Gene therapies, which involve the intracellular immunization of HIV~1 susceptible cells, face major challenges because of the diversity of HIV~1~ susceptible cells. The goal of this study is to develop an antiviral strategy against HIV~1 based on a defective interfering HIV~1 particle which interferes with the replication of wild type virus. If all elements of this strategy perform as hoped, an equilibrium of wild type and defective HIV~1 may be achieved which could potentially stabilize both virus load and T4 cell count and thereby delay the onset of AIDS. Several candidate defective interfering HIV~1 vector constructs (HD DNAs) were further evaluated: 1. The synthesis of Nef protein encoded by some of the HD DNAs was verified by immunofluorescent staining. Expression of the chimeric CD4~Env protein from two constructs which contain additional gag gene inserts was drastically reduced, suggesting that they may not be useful for the strategy. 2. HIV~1 released after cotransfection with HD DNAs appeared to be less infectious, suggesting a difference in the composition of released virus. 3. Several weeks of cocultivation of cells transfected with HIV~1 and HD DNA showed the presence of polyadenylated HD RNA in cell supernatants and the presence of excess HIV~1 RNA. 4. A recipient cell line for HD RNAs was selected. Cocultivation with HIV~1 and HD particle producing cells did not show a transfer of HD RNA at low sensitivity of the assay. 5. Packaging of an HIV~1 helper virus RNA into virus particles was detected despite the fact that the RNA lacks the "essential" cis~acting HIV~1 packaging signal. 6. Electron microscopy of HD virus and HIV~1 budding from cells showed expected morphological differences in the makeup of the two envelopes. The insertion of the CD4~Env protein into the envelope has not been confirmed until now. 7. Computer modeling of the potential use of HD viruses as antivirals seem to indicate that if all elements of the defective virus were functional, these particles may be effective in lymph nodes, the reservoir of the virus, where there is a higher density of persistently infected cells. The potential future use of HD vectors as antivirals will depend on the demonstration of HD RNA transfer to HIV~1 infected cells and the efficiency of packaging of HD RNAs relative to HIV~1 RNA.

大力开发药物或治疗性疫苗 对抗 HIV~1 尚未成功。 基因疗法，其中涉及 HIV~1易感细胞的细胞内免疫，面临重大挑战 由于 HIV~1~ 易感细胞的多样性而面临的挑战。 这 本研究的目标是制定针对 HIV~1 的抗病毒策略 基于有缺陷的干扰 HIV~1 粒子，该粒子会干扰 野生型病毒的复制。 如果该策略的所有要素都执行 正如所希望的，野生型和缺陷型 HIV~1 可以达到平衡 这有可能稳定病毒载量和 T4 细胞计数 从而延缓艾滋病的发病。几个候选人有缺陷干扰 进一步评估了 HIV~1 载体构建体（HD DNA）： 1. 由一些 HD DNA 编码的 Nef 蛋白的合成得到了验证 免疫荧光染色。 嵌合CD4~Env蛋白的表达 来自含有额外 gag 基因插入的两个构建体 急剧减少，表明它们可能对 战略。 2. HD DNA共转染后出现HIV~1释放 传染性较低，表明其成分存在差异 释放病毒。 3. 转染细胞共培养数周 与 HIV~1 和 HD DNA 一起显示多聚腺苷酸化 HD RNA 的存在 细胞上清液和过量 HIV~1 RNA 的存在。 4. 收件人 选择HD RNA的细胞系。 与 HIV~1 和 HD 共培养 颗粒产生细胞在低浓度下没有表现出 HD RNA 的转移 测定的灵敏度。 5. 将 HIV~1 辅助病毒 RNA 包装成 尽管RNA缺乏病毒颗粒，但仍检测到病毒颗粒 “必需的”顺式作用 HIV~1 包装信号。 6. 电子显微镜 HD病毒和HIV~1从细胞中出芽表现出预期的形态 两个信封的构成存在差异。 的插入 CD4~Env蛋白进入包膜至今尚未被证实。 7. HD 病毒作为抗病毒药物的潜在用途的计算机模型似乎 表明如果有缺陷的病毒的所有元素都具有功能， 这些颗粒可能对淋巴结（淋巴结的储存库）有效。 病毒，其中持续感染细胞的密度较高。 HD 载体作为抗病毒药物的潜在未来用途将取决于 HD RNA 转移至 HIV~1 感染细胞的演示 HD RNA 相对于 HIV~1 RNA 的包装效率。