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

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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