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

尽管SIV/猕猴系统已知为研究AIDS提供有用的动物模型，但SIV在其遗传元件方面比HIV-1更类似于HIV-2，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，SIV和HIV-1已被广泛地从分子水平上研究，以阐明其宿主范围的决定机制。相比之下，迄今为止对宿主猕猴细胞受体的研究还很有限。我们已经报道了食蟹猴CD 4在人细胞系上表达时可以作为HIV-1的受体，并且HIV-1有效进入易感宿主细胞可能需要存在于人细胞中而不存在于猴细胞中的除CD 4之外的另一种辅因子。因此，它可能在很大程度上决定了HIV-1的狭窄宿主范围。为了进一步解决这个问题，我们尝试 ...更多信息 对几种猕猴CD 4进行分子克隆，以阐明其V1结构域结构与HIV-1易感性之间的关系。我们从几种猴如恒河猴、猪尾猴、日本猴、食蟹猴、非洲绿色猴和松鼠猴的胸腺细胞或PBL文库中分离了猴CD 4基因，根据它们的基因同源性，它们通过整个编码区与人类对应物显示出高度同源性，但在V1结构域的CDR区存在一些关键差异，其被认为构成HIV-1的gp 120的必需结合位点。由于这些差异被怀疑反映了猴对HIV-1的不敏感性，我们构建了含有每个猴CD 4基因的哺乳动物表达载体阵列，在G418的选择压力下转染人细胞系HeLa，并建立了几个表达每个猴CD 4分子的稳定转化子。用感染性分子克隆HIV-1 NL 432对这些转化子进行转染，通过IFA、合胞体试验和PCR检测前病毒基因组DNA，研究猴CD 4是否可以作为HIV-1进入细胞的受体。来自食蟹猴和恒河猴的表达猴CD 4的HeLa也显示含有HIV-1的整合前病毒，但不形成合胞体。相反，表达HeLa的猪尾猴CD 4细胞在其细胞质内没有病毒复制的证据。综上所述，这些发现表明，可能存在一些灵活的CD 4 V1结构域与HIV-1的gp 120的结合，合胞体的形成可能归因于不同的机制，从简单的病毒-细胞融合事件。这些实验系统可能提供一个分析工具，以摆脱一些洞察猴CD 4和HIV-1之间的相互作用。少

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