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

利用在几种猕猴中表达嵌合 CD4 的转化子研究 HIV/SIV 诱导的合胞体形成机制。

• 批准号: 08456164
• 负责人: TATSUMI Masashi
• 金额: $ 4.29万
• 依托单位: NATIONAL INSTITUTE OF INFECTIOUS DISEASES
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08456164/
• 关键词: Macaque; CD4; Domain 2; HIV-1; Entry; Syncytium; Entry

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

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。相反，对宿主猕猴细胞受体的研究至今还很有限。我们已经报道了当在人细胞系而不是猴细胞上表达时，几种猕猴CD 4可以作为HIV-1的受体，并且在这些猕猴CD 4基因之间的整个编码区中具有高度同源性，尽管在这些猕猴CD 4表达转化体中由HIV-1诱导的合胞体形成中检测到关键差异。除了V1以外，CD 4分子的其他结构域在合胞体形成中的作用仍存在争议，其中确定了与gp 120的结合位点。由于猕猴CD 4s在编码区具有高度同源性，但在合胞体形成方面表现出不同的表型，这可能为进一步解决这一问题提供了一个合适的实验系统，我们尝试表达CD 4s， ...更多信息 的嵌合CD 4来自几个猕猴CD 4，不同的合胞体形成的能力，阐明域结构和亲和性之间的关系，以合胞体形成由HIV-1诱导。我们选择来自日本猴（JpT 4）的猴CD 4 cDNA作为合胞体阳性表型，并且选择来自食蟹猴（CyT 4）的猴CD 4 cDNA作为合胞体阴性表型。我们利用猕猴CD 4分子中的两个保守位点，构建了一系列含有这两种猕猴CD 4分子中的嵌合CD 4分子的哺乳动物表达载体，并在G418筛选压力下转染人HeLa细胞，建立了稳定表达嵌合CD 4分子的转化子。然后用感染性分子克隆HIV-1 NL 432对这些转化体进行转染，以研究每个嵌合CD 4是否能够支持合胞体形成。合胞体仅发生在V2和V3结构域的转化体表达的情况下，来自阳性表型。在JpT 4和CyT 4之间的这些区域中的氨基酸序列的比较中，仅在第144个氨基酸处存在一个差异：Leu（CyT 4）和Ile（JpT 4）。接下来，我们通过定点突变构建了第144位含有Ile的CyT 4突变体，并建立了表达CyT 4突变体的转化体，发现该突变体在接触HIV-1后形成合胞体。这些结果表明，除了V1结构域作为gp 120的直接结合位点外，第144位氨基酸的V2结构域可能作为gp 120的第二结合位点或作为gp 120与gp 41的非共价结合的有效脱落在合胞体形成中起重要作用，其中包含疏水融合结构域。这些实验系统可能提供一个分析工具，以揭示一些相互作用的CD 4和HIV-1的HIV-1进入。少

项目成果

Yabe,M.,Matsuura,Y.and Tatsumi,M.: "Molecular cloning and expression of cynomolgus monkey IL-2 cDNA by the baculovirus system." Int.Arches.Immunol.Allergy. (in press). (1997)

Yabe,M.、Matsuura,Y. 和 Tatsumi,M.：“通过杆状病毒系统进行食蟹猴 IL-2 cDNA 的分子克隆和表达。”

Li, T-C,Yamakawa, Y., Suzuki, k., Tatsumi, M., Razak, M.A., Uchida, T., Takeda, N., and Miyamura, T.: "Expression and self-assembly of empty virus-like particles of hepatitis E virus." J.Virol.71. 7207-7213 (1997)

Li, T-C、Yamakawa, Y.、Suzuki, k.、Tatsumi, M.、Razak, M.A.、Uchida, T.、Takeda, N. 和 Miyamura, T.：“空病毒样的表达和自组装

Li,T-Cet al:"Expression and self-assembly of empty virus-like particles of hepatitis E virus." J.Virol.71. 7207-7213 (1997)

Li，T-Cet al：“戊型肝炎病毒空病毒样颗粒的表达和自组装。”

Takakura, H., Mori, Y., and Tatsumi, M.: "Molecular cloning and expression of caprine IL-6 cDNA in insect cells." Int.Archiev Allergy Immunol.113. 409-416 (1997)

Takakura, H.、Mori, Y. 和 Tatsumi, M.：“山羊 IL-6 cDNA 在昆虫细胞中的分子克隆和表达。”

Totsuka, K., Takakura, H., Hashimoto, O.and Tatsumi, M.: "Molecular cloning and Expression of cynomolgus monkey Interleukin-1β." Int.Archiev Allergy Immunol.115(in press).

Totsuka, K.、Takakura, H.、Hashimoto, O. 和 Tatsumi, M.：“食蟹猴 Interleukin-1β 的分子克隆和表达。Int.Archiev Allergy Immunol.115（出版中）。”