Studies on the behavior of hantavirus envelope glycoprotein in adsorption and invasion to target cells.

汉坦病毒是啮齿动物传播的病毒性人畜共患病、HFRS和HPS的病原体。包膜糖蛋白G1和G2诱导中和抗体。具有中和活性的单克隆抗体显示融合抑制活性。这意味着汉坦病毒的疫苗靶表位是包膜蛋白中的一个融合负责区。我研究了包膜糖蛋白在细胞感染建立中的作用。首先，通过伪VSV筛选，我们发现了高敏感细胞系（A549、BHK和VeroE6细胞）和非允许细胞系（MDBK）。AlphaVbeta3整合素被报道为汉坦病毒进入的候选细胞受体。因此，我们尝试将alphaVbeta3诱导到MDBK中。我无法检测到细胞传染性的变化。然而，质粒结构对MDBK细胞的转染效率不足以评价分子的作用。另外，本研究中发现的抗- a3整合素抗体不能抑制汉坦病毒在允许细胞中的进入。所以这些分子被认为不是这些细胞中的主要受体分子。接下来，我从允许细胞系Vero E6细胞中筛选抗汉坦病毒糖蛋白的糖脂结合。在糖脂和包膜蛋白之间没有检测到任何显著的结合。这些结果提示汉坦病毒的细胞受体一定是除α vbeta3整合素外的蛋白质分子。最后，建立了MDBK细胞的克隆5，作为相对高DNA诱导和非允许细胞系。从允许细胞系A5449中获得cDNA文库。本研究为分析汉坦病毒包膜糖蛋白与细胞分子的相互作用奠定了基础。

Hantaviruses are causative agents for rodent-borne viral zoonosis, HFRS and HPS. Envelope glycoproteins G1 and G2 induce neutralizing antibodies. Monoclonal antibodies possessing neutralizing activities showed fusion inhibition activities. This means vaccine target epitope for hantavirus is a fusion responsible region in envelope protein. I studied the role of envelope glycoproteins in establishment of infection in cells. At the first, we found highly sensitive cell lines (A549, BHK and VeroE6 cells) and non-permissive cell lines (MDBK) by using pseudotype VSV screening. AlphaVbeta3 integrin was reported as candidate of the cellular receptor for hantavirus entry. Therefore, we tried to induce alphaVbeta3 into MDBK. I could not detect change of infectivity in that cells. However, transfection efficiency of plasmid constract to MDBK cells were not enough to evaluate the effect of molecules. Otherwise, anti-beta3 integrin antibodies were not inhibit cellular entry of hantavirus in permissive cells found in this study. So that this molecules was considered not to be major receptor molecules in these cells. Next, I screened bindings of glycolipids from permissive cell line Vero E6 cells against hantavirus glycoprotein. Any significant binding was not detected between glycolipids and envelope proteins. These results suggested that the cellular receptor of hantavirus must be protein molecules except alphaVbeta3 integrin. Finally, a clone 5 of MDBK cells was established as relative high DNA induction and non-permissive line. And cDNA libraly from permissive cell line A5449 was produced. This study established the basis for analysis of interaction between hantavirus envelope glycoproteins and cellular molecules.