Cardona Sandra M, Dunphy Jaclyn M, Das Alvin S, Lynch Connor R, Lynch William P
Department of Integrative Medical Sciences, Northeast Ohio Medical University, Rootstown, OH, United States.
Program in Cellular and Molecular Biology, School of Biomedical Sciences, Kent State University, Kent, OH, United States.
Front Neurosci. 2019 Oct 29;13:1166. doi: 10.3389/fnins.2019.01166. eCollection 2019.
The ability of retroviruses (RVs) to cause neurodegeneration is critically dependent upon two activities of the envelope protein (Env). First, Env facilitates viral genome delivery to CNS target cells through receptor binding and membrane fusion. Second, Env expression within one or more targets indirectly alters the physiology of certain neurons. Although the major Env expressing CNS cell types have been identified for many neurovirulent RVs, it remains unresolved, which targets play a causal role in neuropathogenesis. Moreover, this issue is complicated by the potential for post-infection virus suppression. To address these questions we explored herein, whether and how cryptic neurotropism differences between ecotropic and amphotropic murine leukemia viruses (MLVs) impacted neurovirulence. Neurotropism was first explored using (1) acute primary glial cell cultures and (2) neural progenitor cell (NPC)- neural stem cell (NSC) neural sphere (NPH) chimeras. These experiments indicated that primary astrocytes and NPCs acutely restrict amphotropic but not ecotropic virus entry. CNS tropism was investigated using NSC transplant-based Cre-vector pseudotyping wherein mTmG transgenic fluorescent protein reporter mice revealed both productive and suppressed infection. Cre-pseudotyping with FrCasE, a prototypic neurovirulent ecotropic virus, identified glia and endothelia, but not neurons, as targets. Almost two-thirds (62%) of mGFP+ cells failed to show Env expression, suggesting widespread virus suppression. To circumvent RV superinfection interference confounds, targets were also identified using ecotropic packaging NSCs. These experiments identified known ecotropic targets: microglia, oligodendrocyte progenitor cells (OPCs) and endothelia. Additionally, one third of mGFP+ cells were identified as protoplasmic astrocytes, cells that rarely express virus . A CNS targeting comparison between isogenic ecotropic (FrCasE) and amphotropic (FrAmE) viruses showed a fourfold higher astrocyte targeting by FrCasE. Since ecotropic Env pseudotyping of amphotropic virus in the CNS dramatically exacerbates neurodegeneration, these results strongly suggest that astrocyte infection is a major disease requirement. Moreover, since viral Env protein expression is largely subdetectable in astrocytes, minimal viral protein expression appears sufficient for affecting neuronal physiology. More broadly, these findings raise the specter that subdetectable astrocyte expression of exogenous or endogenous RVs could play a major role in human and animal neurodegenerative diseases.
逆转录病毒（RVs）引起神经退行性变的能力严重依赖于包膜蛋白（Env）的两种活性。首先，Env通过受体结合和膜融合促进病毒基因组传递至中枢神经系统（CNS）靶细胞。其次，一种或多种靶细胞内的Env表达间接改变某些神经元的生理功能。尽管对于许多神经毒性RVs，主要的表达Env的CNS细胞类型已被确定，但哪些靶细胞在神经病理发生中起因果作用仍未解决。此外，感染后病毒抑制的可能性使这个问题变得复杂。为了解决这些问题，我们在此探讨亲嗜性和兼嗜性鼠白血病病毒（MLVs）之间潜在的隐匿性嗜神经性差异是否以及如何影响神经毒性。首先使用（1）急性原代神经胶质细胞培养物和（2）神经祖细胞（NPC）-神经干细胞（NSC）神经球（NPH）嵌合体来研究嗜神经性。这些实验表明，原代星形胶质细胞和NPC急性限制兼嗜性病毒而非亲嗜性病毒的进入。使用基于NSC移植的Cre载体假型化来研究CNS嗜性，其中mTmG转基因荧光蛋白报告小鼠揭示了 productive感染和抑制性感染。用原型神经毒性亲嗜性病毒FrCasE进行Cre假型化，确定神经胶质细胞和内皮细胞而非神经元为靶细胞。几乎三分之二（62%）的mGFP+细胞未显示Env表达，表明存在广泛的病毒抑制。为了规避RV重复感染干扰的混淆因素，还使用亲嗜性包装的NSC来确定靶细胞。这些实验确定了已知的亲嗜性靶细胞：小胶质细胞、少突胶质前体细胞（OPCs）和内皮细胞。此外，三分之一的mGFP+细胞被确定为原浆性星形胶质细胞，这种细胞很少表达病毒。同基因亲嗜性（FrCasE）和兼嗜性（FrAmE）病毒之间的CNS靶向比较显示，FrCasE对星形胶质细胞的靶向率高四倍。由于在CNS中亲嗜性Env对兼嗜性病毒进行假型化会显著加剧神经退行性变，这些结果强烈表明星形胶质细胞感染是疾病的主要必要条件。此外，由于病毒Env蛋白表达在星形胶质细胞中大多难以检测到，最小限度的病毒蛋白表达似乎足以影响神经元生理功能。更广泛地说，这些发现引发了一种担忧，即外源性或内源性RVs在星形胶质细胞中难以检测到的表达可能在人类和动物神经退行性疾病中起主要作用。
