Geis Franziska K, Galla Melanie, Hoffmann Dirk, Kuehle Johannes, Zychlinski Daniela, Maetzig Tobias, Schott Juliane W, Schwarzer Adrian, Goffinet Christine, Goff Stephen P, Schambach Axel
Institute of Experimental Hematology, Hannover Medical School, Carl-Neuberg-Str. 1, Hannover, Germany.
REBIRTH Cluster of Excellence, Hannover Medical School, Hannover, Germany.
Retrovirology. 2017 May 31;14(1):34. doi: 10.1186/s12977-017-0358-1.
Retroviral vectors are derived from wild-type retroviruses, can be used to study retrovirus-host interactions and are effective tools in gene and cell therapy. However, numerous cell types are resistant or less permissive to retrovirus infection due to the presence of active defense mechanisms, or the absence of important cellular host co-factors. In contrast to multipotent stem cells, pluripotent stem cells (PSC) have potential to differentiate into all three germ layers. Much remains to be elucidated in the field of anti-viral immunity in stem cells, especially in PSC.
In this study, we report that transduction with HIV-1-based, lentiviral vectors (LV) is impaired in murine PSC. Analyses of early retroviral events in induced pluripotent stem cells (iPSC) revealed that the restriction is independent of envelope choice and does not affect reverse transcription, but perturbs nuclear entry and proviral integration. Proteasomal inhibition by MG132 could not circumvent the restriction. However, prevention of cyclophilin A (CypA) binding to the HIV-1 capsid via use of either a CypA inhibitor (cyclosporine A) or CypA-independent capsid mutants improved transduction. In addition, application of higher vector doses also increased transduction. Our data revealed a CypA mediated restriction in iPSC, which was acquired during reprogramming, associated with pluripotency and relieved upon subsequent differentiation.
We showed that murine PSC and iPSC are less susceptible to LV. The block observed in iPSC was CypA-dependent and resulted in reduced nuclear entry of viral DNA and proviral integration. Our study helps to improve transduction of murine pluripotent cells with HIV-1-based vectors and contributes to our understanding of retrovirus-host interactions in PSC.
逆转录病毒载体源自野生型逆转录病毒，可用于研究逆转录病毒与宿主的相互作用，是基因治疗和细胞治疗的有效工具。然而，由于存在主动防御机制或缺乏重要的细胞宿主辅助因子，许多细胞类型对逆转录病毒感染具有抗性或较低的易感性。与多能干细胞不同，多能干细胞（PSC）有分化为所有三个胚层的潜力。干细胞抗病毒免疫领域，尤其是PSC中的抗病毒免疫领域，仍有许多有待阐明的问题。
在本研究中，我们报告基于HIV-1的慢病毒载体（LV）转导在小鼠PSC中受损。对诱导多能干细胞（iPSC）中早期逆转录病毒事件的分析表明，这种限制与包膜选择无关，不影响逆转录，但会干扰核进入和前病毒整合。MG132对蛋白酶体的抑制不能规避这种限制。然而，通过使用环孢素A（一种环孢菌素A）或不依赖环孢素A的衣壳突变体来防止亲环素A（CypA）与HIV-1衣壳结合，可改善转导。此外，应用更高的载体剂量也可增加转导。我们的数据揭示了iPSC中一种由CypA介导的限制，这种限制在重编程过程中获得，与多能性相关，并在随后的分化过程中得到缓解。
我们表明小鼠PSC和iPSC对LV的敏感性较低。在iPSC中观察到的阻断是CypA依赖性的，导致病毒DNA的核进入和前病毒整合减少。我们的研究有助于改善基于HIV-1的载体对小鼠多能细胞的转导，并有助于我们理解PSC中逆转录病毒与宿主的相互作用。
