STUDY ON THE MECHANISM OF HIV/SIV-INDUCED SYNCYTIUM FORMATION USING TRANSFORMANTS EXPRESSING CHIMERA CD4 AMONG SEVERAL SPECIES OF MACAQUES.

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。我们已经报道了几种猕猴CD4在人类细胞系而不是猴子细胞上表达时可以作为HIV-1的受体，并且这些猕猴CD4基因在整个编码区具有高度的同源性，尽管在这些表达CD4的猕猴转化子中检测到HIV-1诱导的合胞体形成的关键差异。对于CD4分子的V1以外的其他结构域在合胞体形成中的作用，目前仍有争议，其中gp120的结合位点是明确的。由于猕猴CD4s在编码区具有高度的同源性，但在合胞体形成方面存在不同的表型，可能为进一步解决这一问题提供了合适的实验系统，因此我们尝试表达来自几种合胞体形成能力不同的猕猴CD4s的嵌合CD4，以阐明域结构与HIV-1诱导合胞体形成易感性之间的关系。我们选择合胞体阳性表型的猴CD4 cdna来自日本猴（JpT4），合胞体阴性表型的猴CD4 cdna来自食蟹猴（CyT4）。我们利用这两种猕猴CD4s之间保守的两个限制性内切位点，构建了包含这些猕猴CD4s中多个嵌合CD4 cdna的哺乳动物表达载体阵列，并在G418的选择压力下转染人细胞系HeLa，建立了多个稳定表达嵌合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，以研究每个嵌合CD4是否可以支持合胞体的形成。仅在来自阳性表型的V2和V3结构域的转化子表达的情况下发生合胞体。在这些区域的氨基酸序列比较中，JpT4与CyT4仅在第144个氨基酸处有1个差异；Leu （CyT4）和Ile （JpT4）。接下来，我们通过位点定向诱变构建了含有Ile的第144位CyT4突变体，并建立了表达CyT4突变体的转化体，该突变体暴露于HIV-1后形成合胞体。综上所述，这些发现表明，除了V1结构域作为gp120的直接结合位点外，另一个包含144个氨基酸的V2结构域可能在合胞体形成过程中发挥重要作用，作为gp120的第二个结合位点，或者gp120有效脱离与gp41的非共价结合，gp41含有疏水融合结构域。这些实验系统可能为深入了解CD4和HIV-1在HIV-1进入时的相互作用提供一种分析工具。少

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 already reported that several macaque monkey CD4s could serve as a receptor for HIV-1 when expressed on a human cell line but not a monkey cells, and that there is high homology in the entire coding region among these macaque monkey CD4 genes although critical differences were detected in syncytium formation induced by HIV-1 among these macaque CD4 expressing transformants. There are still controversies on the role in syncytium formation of other domains of CD4 molecule than V1, in which the binding site to gp120 is defined. Since macaque monkey CD4s showing high homology in the coding region but different phenotypes in term of syncytium formation might provide a suitable experimental system to address this issue further, we make an attempt to expres … More s chimeric CD4 derived from several macaque monkey CD4s, which differ in the ability of syncytium formation to elucidate the relationship between domain strucrures and susceptibilities to syncytium formation induced by HIV-1. We chose monkey CD4 cDNAs from japanese monkey (JpT4) as a syncytium positive phenotype and from cynomolgus monkey (CyT4) as a syncytium negative phenotype. We construct an array of mammalian expression vectors which contain several chimeric CD4 cDNAs among these macaque CD4s by using the two restriction sites conserved between these two macaque CD4s, transfected a human cell line HeLa under the selection pressure of G418 and established several stable transformants expressing chimeric CD4 molecule. These transformants were then exposured with an infectious molecular clone HIV-1NL432 to study whether or not each chimeric CD4 can support the syncytium formation. Syncytia occurred only in the cases of transformants expression of V2 and V3 domains derived from the positive phenotype. On the comparison of amino acid sequence in such regions between JpT4 and CyT4, there was only one difference at the 144th amino acid ; Leu (CyT4) and Ile (JpT4). Next we construct the CyT4 Mutant which contained Ile at 144th position by site-directed mutagenesis and established a transformant expressing CyT4 mutant, which was revealed to form syncytia after exposure to HIV-1. Taken together, these findings indicated that aside from the V1 domain as the direct binding site for gp120, the other V2 domain involving 144th amino acid might play an important role in the syncytium formation as the second binding site for gp120 or for the effective shedding of gp120 from noncovalent association with gp41, which contains the hydrophobic fusion domain. These experimental system might provide an analytical tool to shed some insight into the interaction between CD4 and HIV-1 on the HIV-1 entry. Less