Robert Koch Institute, Berlin, Germany.
PLoS One. 2018 Jul 12;13(7):e0200570. doi: 10.1371/journal.pone.0200570. eCollection 2018.
Immunosuppression by retroviruses including the human immunodeficiency virus-1 (HIV-1) is well known, however the mechanisms how retroviruses induce this immunosuppression is not fully investigated. It was shown that non-infectious retroviral particles as well as retroviral or recombinant retroviral transmembrane envelope (TM) proteins demonstrated immunosuppressive properties. The same was shown for peptides corresponding to a highly conserved domain in the TM protein. This domain is called immunosuppressive (ISU) domain and it induces modulation of the cytokine release of peripheral blood mononuclear cells (PBMCs) from healthy donors. In addition, it changes the gene expression of these cells. Common indications for the immunosuppressive activity were tumour growth in vivo and interleukin-10 (IL-10) release from human PBMCs in vitro. Single mutations in the ISU domain abrogated the immunosuppressive activity. In order to develop a new model system for the expression of the ISU domain and presentation to PBMCs which is not prone to possible endotoxin contaminations, two expression systems were developed. In the first system, designated pOUT, retroviral proteins containing the ISU domain were expressed and released into the cell culture medium, and in the second system, tANCHOR, the ISU domain was presented by a tetraspanin-anchored sequence on the cell surface of human cells. Both systems were exploited to express the wild-type (wt) ISU domains of HIV-1, of the porcine endogenous retrovirus (PERV) and of the murine leukaemia virus (MuLV) as well as to express mutants (mut) of these ISU domains. PERV is of special interest in the context of virus safety of xenotransplantation using pig organs. Expression of the TM proteins was demonstrated by confocal laser scanning microscopy, ELISA and Western blot analyses using specific antibodies. However, when cells expressing and releasing the ISU were co-incubated with human PBMCs, no increased production of IL-10 was observed when compared with the mutants. Similar results were obtained when the released TM proteins were concentrated by immunoprecipitation and added to PBMCs. We suggest that the absence of IL-10 induction can be explained by a low amount of protein, by the lack of a biologically active conformation or the absence of additional factors.
逆转录病毒（包括人类免疫缺陷病毒 1（HIV-1））的免疫抑制作用是众所周知的，然而，逆转录病毒如何诱导这种免疫抑制作用还没有得到充分的研究。已经表明，非感染性逆转录病毒颗粒以及逆转录病毒或重组逆转录病毒跨膜包膜（TM）蛋白具有免疫抑制特性。与 TM 蛋白中高度保守结构域相对应的肽也具有相同的特性。这个结构域被称为免疫抑制（ISU）结构域，它诱导来自健康供体的外周血单个核细胞（PBMCs）的细胞因子释放的调制。此外，它还改变这些细胞的基因表达。免疫抑制活性的常见迹象是体内肿瘤生长和体外人 PBMCs 中白细胞介素 10（IL-10）的释放。ISU 结构域中的单个突变消除了免疫抑制活性。为了开发一种新的表达 ISU 结构域并呈递给 PBMCs 的模型系统，该系统不易受到内毒素污染的影响，开发了两种表达系统。在第一个系统中，指定为 pOUT，含有 ISU 结构域的逆转录病毒蛋白被表达并释放到细胞培养液中，在第二个系统中，tANCHOR，ISU 结构域通过人细胞表面的四跨膜蛋白锚定序列呈现。这两个系统都被用来表达 HIV-1、猪内源性逆转录病毒（PERV）和鼠白血病病毒（MuLV）的野生型（wt）ISU 结构域，以及表达这些 ISU 结构域的突变体（mut）。在使用猪器官进行异种移植的病毒安全性方面，PERV 特别令人关注。通过使用特异性抗体的共聚焦激光扫描显微镜、ELISA 和 Western blot 分析，证明了 TM 蛋白的表达。然而，当表达和释放 ISU 的细胞与人类 PBMCs 共孵育时，与突变体相比，IL-10 的产生没有增加。当用免疫沉淀浓缩释放的 TM 蛋白并添加到 PBMCs 中时，也获得了类似的结果。我们认为，IL-10 诱导的缺失可以通过以下原因来解释：蛋白量低、缺乏生物活性构象或缺乏其他因素。
