MOLECULAR CLONING AND EXPRESSION OF MACAQUE MONKEY CD4 GENES ; FOR ESTABLISHMENT OF AIDS ANIMAL MODELS.

猕猴CD4基因的分子克隆和表达；

• 批准号: 05454120
• 负责人: TATSUMI Masashi
• 金额: $ 4.22万
• 依托单位: NATIONAL INSTITUTE OF HEALTH
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1993
• 资助国家: 日本
• 起止时间: 1993 至 1994
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05454120/
• 关键词: Macaque Monkeys; CD4; HIV-1; Vi Domain; CD4分子; V1ドメイン; SIV; HIV; 動物モデル

Although SIV/macaque monkey systems are known to provide useful animal models for the study of AIDS,SIV resembles HIV-2 in respect of its genetical elements more than HIV-1, of which prevalence is worldwide. Suitable animal models are clearly prerequisite for developements of effective vaccines and therapies against HIV-1, but are unfortunately few. So far SIV and HIV-1 have been extensively studied from the molecular aspects to elucidate the mechanisms determining its host range. In contrast, studies on the cellular receptor (s) of hosts, macaque monkeys are limited until now. We have reported that cynomolgus monkey CD4 could serve as a receptor for HIV-1 when expressed on a human cell line and that another cofactor (s) other than CD4, which exsits in a human cell but not in a monkey cell, might be required for efficient entry of HIV-1 into susceptible host cells. Therefore, it might substantially determine the narrow host range of HIV-1. To address this further, we make an attempt of … More molecular cloning of several macaque monkey CD4s to elucidate the relationship between their V1 domain structures and susceptibilties to HIV-1. We isolated monkey CD4 genes from the thymocyte or PBL libraries of several species of monkeys such as rhesus, pig-tailed, japanese, cynomolgus, african green and squirrel monkeys, which show high homology, depending upon their phylogenic proximity, to the human counterpart through the entire coding region but some critical differences exist in CDR regions of V1 domain, which is thought to constitute essential binding sites for gp120 of HIV-1. As these differences are suspected to reflect the unsusceptibility of monkeys to HIV-1, we constructed an array of mammalian expression vectors containing each monkey CD4 gene, transfected a human cell line HeLa under the seletion pressure of G418 and established several stable transformants expressing each monkey CD4 molecule. These transformants were then exposured with an infectious molecular clone HIV-1NL432 to study whether or not each monkey CD4 can serve as a receptor for the establishment of HIV-1 entry by IFA,syncythium assay and PCR detecting proviral genomic DNA.HeLa expressing monkey CD4 derived from japanese and african green monkeys could not only support the replication of HIV-1 but also form syncytia due to HIV-1. HeLa expressing monkey CD4 from cynomolgus and rhesus monkeys were also revealed to contain the integrated provirus of HIV-1 but not to form syncytia. In contrast HeLa expressing pig-tailed monkey CD4 showed no evidence of virus replication within their cytoplasm. Taken together, these findings indicated that there might exist some flexibility in the CD4 V1 domain structure for the binding with gp120 of HIV-1, and that syncytia formation might be attributed to mechanisms diffrent from the simple virus-cell fusion events. These experimental system might provide an analytical tool to shed some insight into the interaction between monkey CD4 and HIV-1. Less

虽然已知SIV/猕猴系统为艾滋病研究提供了有用的动物模型，但SIV在遗传成分方面更类似于HIV-2，而HIV-1在世界范围内流行。合适的动物模型显然是开发针对HIV-1的有效疫苗和疗法的先决条件，但不幸的是很少。迄今为止，人们从分子角度对SIV和HIV-1进行了广泛的研究，以阐明其宿主范围的决定机制。相比之下，对宿主猕猴的细胞受体的研究目前还很有限。我们已经报道了食蟹猴CD4在人类细胞系上表达时可以作为HIV-1的受体，并且除了CD4外，存在于人类细胞而不存在于猴子细胞中的另一个辅助因子可能需要HIV-1有效进入易感宿主细胞。因此，它可能从本质上决定了HIV-1的狭窄宿主范围。为了进一步解决这一问题，我们尝试对几个猕猴CD4s进行更多的分子克隆，以阐明其V1结构域结构与HIV-1易感性之间的关系。我们从恒河猴、猪尾猴、日本猴、食猴、非洲绿猴和松鼠猴等几种猴子的胸腺细胞或PBL文库中分离出猴子CD4基因，根据它们的系统发育接近性，它们在整个编码区显示出与人类对应的高度同源性，但在V1结构域的CDR区域存在一些关键差异，该区域被认为是构成HIV-1的gp120的必要结合位点。由于这些差异可能反映了猴子对HIV-1的不易感性，我们构建了包含每个猴子CD4基因的哺乳动物表达载体阵列，在G418的选择压力下转染人类细胞系HeLa，并建立了几个稳定的表达每个猴子CD4分子的转化子。然后将这些转化子暴露于传染性分子克隆HIV-1NL432中，通过IFA、合胞体测定和PCR检测前病毒基因组DNA，研究每个猴子CD4是否可以作为建立HIV-1进入的受体。来自日本和非洲绿猴的表达猴CD4的HeLa不仅支持HIV-1的复制，而且由于HIV-1而形成合胞体。从食蟹猴和恒河猴中表达猴CD4的HeLa也被发现含有HIV-1的整合前病毒，但不形成合胞体。相比之下，表达HeLa的猪尾猴CD4细胞在其细胞质中没有病毒复制的证据。综上所述，这些发现表明CD4 V1结构域与HIV-1的gp120结合可能存在一定的灵活性，合胞体的形成可能归因于不同于简单的病毒-细胞融合事件的机制。这些实验系统可能为揭示猴子CD4和HIV-1之间的相互作用提供一种分析工具。少

项目成果

巽 正志、武田 直和: "ウイルスレセプターとしてのCD抗原" 医学のあゆみ. 別冊. 68-73 (1995)

Masashi Tatsumi、Naokazu Takeda：“CD 抗原作为病毒受体”医学史 68-73（1995）。

TATSUMI,M.& TAKEDA,N.: "CD Antigens as Virus Receptors (in japanese)" IGAKUNO AYUMI. Supplements. 68-73 (1995)

辰巳，M.