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

到目前为止，人们从分子水平对SIV和HIV-1进行了广泛的研究，以阐明决定其宿主范围的机制。相比之下，对猕猴宿主细胞受体(S)的研究到目前为止还很有限。我们已经报道了几个猕猴CD4在人类细胞系上表达时可以作为HIV-1的受体，而不是在猕猴细胞上表达，并且这些猕猴CD4基因在整个编码区有很高的同源性，尽管在HIV-1诱导的合胞体形成方面在这些猕猴CD4表达转化子中发现了关键的差异。关于除V1以外的其他结构域在合体形成中的作用仍存在争议，在V1中定义了与gp120的结合部位。由于猕猴…在编码区具有很高的同源性，但在合体形成方面的表型不同，可能为进一步解决这一问题提供了一个合适的实验系统，我们试图表达CD4更多的S嵌合的CD_4来源于几个猕猴的CD_4，它们形成合胞体的能力不同，以阐明结构域结构与HIV-1诱导的合胞体形成敏感性之间的关系。我们选择日本猴(JpT4)的CD4cDNAs作为合胞体阳性表型，食蟹猴(CyT4)的CD4cDNAs作为合胞体阴性表型。我们利用这两个猕猴CD4之间保守的两个酶切位点，构建了一组哺乳动物表达载体，其中含有多个嵌合的CD4cDNAs，在G418的选择压力下，转染了人细胞系HeLa，建立了几个稳定表达嵌合CD4分子的转化子。然后将这些转化子与具有感染性的分子克隆HIV-1NL432接触，以研究每个嵌合的CD4是否能够支持合胞体的形成。合胞体仅在由阳性表型衍生的V2和V3结构域的转化子表达的情况下发生。比较JpT4和CyT4的氨基酸序列，在第144位氨基酸上只有一个差异：Leu(CyT4)和Ile(JpT4)。接下来，我们通过定点突变的方法构建了在第144位含有Ile的CyT4突变体，并建立了表达CyT4突变体的转化子，发现该突变体在暴露于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的直接结合部位外，另一个涉及144个氨基酸的V2结构域可能在合体形成中发挥重要作用，作为gp120的第二结合部位，或有效地使gp120与含有疏水融合结构域的gp41非共价结合。这些实验系统可能提供一种分析工具，以深入了解HIV-1进入HIV-1过程中CD4和HIV-1之间的相互作用。较少

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