Calcineurin phosphatase activity regulates Varicella-Zoster Virus induced cell-cell fusion.
Calcineurin phosphatase activity regulates Varicella-Zoster Virus induced cell-cell fusion.
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DOI：
10.1371/journal.ppat.1009022
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发表时间：
2020-11
期刊：
影响因子：
6.7
通讯作者：
Oliver SL
中科院分区：
文献类型：
--
作者：
Zhou M;Kamarshi V;Arvin AM;Oliver SL
细胞-细胞融合（缩写为细胞融合）是医学上重要病毒的一种特征性病理学，包括水痘-带状疱疹病毒（VZV）、水痘和带状疱疹的病原体。细胞融合由VZV糖蛋白gB和gH-gL的复合物介导，并且必须严格调节以使基于gB和gH超致流VZV突变体的研究的皮肤发病机制成为可能。虽然gB和gH-gL在细胞融合调节中的功能已被探索，但宿主因子是否直接参与这一调节过程尚不清楚。在这里，我们发现宿主因子，调制VZV gB/gH-gL介导的细胞融合，通过高通量筛选具有已知细胞靶点的生物活性化合物。两种结构上相关的非抗生素大环内酯类药物，他克莫司和吡美莫司，均显著增加VZV gB/gH-gL介导的细胞融合。这些化合物与FKBP 1A形成药物-蛋白质复合物，FKBP 1A结合钙调磷酸酶并特异性抑制钙调磷酸酶活性。钙调磷酸酶活性的抑制也增强了单纯疱疹病毒-1融合复合物和合胞素-1介导的细胞融合，表明钙调磷酸酶在调节这一过程中的广泛作用。为了表征钙调磷酸酶活性在VZV gB/gH-gL介导的融合中的作用，进行了一系列生化、生物学和感染性测定。吡美莫司诱导的增强的细胞融合被FKBP 1A的shRNA敲低显著降低，进一步支持钙调磷酸酶活性在融合调节中的作用。重要的是，VZV感染过程中钙调磷酸酶活性的抑制导致夸大的合胞体形成和抑制病毒繁殖，这与先前报道的gB和gH超流性VZV突变体的表型一致。七个宿主细胞蛋白，仍然独特的磷酸化时，钙调磷酸酶活性被抑制被确定为潜在的下游因子参与融合调节。这些结果表明，钙调神经磷酸酶是一个关键的宿主细胞因子在调节VZV诱导的细胞融合，这是VZV的发病机制是必不可少的。VZV是一种医学上重要的人类病毒，可引起水痘（水痘）并重新激活为带状疱疹（带状疱疹）。带状疱疹后神经痛（PHN）是带状疱疹的常见后遗症，带状疱疹后持续数月至数年的持续性神经疼痛。PHN对抗病毒药物和疼痛治疗无效，显著影响生活质量。尽管VZV减毒活疫苗在健康人群中取得了成功，但免疫功能低下的个体仍然容易发生严重的发病率。最近批准的亚单位带状疱疹疫苗是有效的，但它是否对水痘有效尚不清楚。因此，更好地了解VZV发病机制的分子基础将为替代治疗提供重大进展。VZV发病机制的一个标志是在感染的皮肤和神经节中观察到合胞体形成。VZV诱导的细胞融合由包含糖蛋白gB、gH和gL的病毒融合复合物介导。这种融合过程的失调导致皮肤中VZV感染受损。在这里，我们报告说，抑制细胞磷酸酶，钙调神经磷酸酶，显着增加gB/gH-gL介导的细胞融合，并加强合胞体形成VZV复制过程中，但抑制病毒传播。我们的研究表明，钙调神经磷酸酶活性调节VZV诱导的细胞融合，为靶向宿主因子的潜在抗病毒策略提供了新的视角。
Cell-cell fusion (abbreviated as cell fusion) is a characteristic pathology of medically important viruses, including varicella-zoster virus (VZV), the causative agent of chickenpox and shingles. Cell fusion is mediated by a complex of VZV glycoproteins, gB and gH-gL, and must be tightly regulated to enable skin pathogenesis based on studies with gB and gH hyperfusogenic VZV mutants. Although the function of gB and gH-gL in the regulation of cell fusion has been explored, whether host factors are directly involved in this regulation process is unknown. Here, we discovered host factors that modulated VZV gB/gH-gL mediated cell fusion via high-throughput screening of bioactive compounds with known cellular targets. Two structurally related non-antibiotic macrolides, tacrolimus and pimecrolimus, both significantly increased VZV gB/gH-gL mediated cell fusion. These compounds form a drug-protein complex with FKBP1A, which binds to calcineurin and specifically inhibits calcineurin phosphatase activity. Inhibition of calcineurin phosphatase activity also enhanced both herpes simplex virus-1 fusion complex and syncytin-1 mediated cell fusion, indicating a broad role of calcineurin in modulating this process. To characterize the role of calcineurin phosphatase activity in VZV gB/gH-gL mediated fusion, a series of biochemical, biological and infectivity assays was performed. Pimecrolimus-induced, enhanced cell fusion was significantly reduced by shRNA knockdown of FKBP1A, further supporting the role of calcineurin phosphatase activity in fusion regulation. Importantly, inhibition of calcineurin phosphatase activity during VZV infection caused exaggerated syncytia formation and suppressed virus propagation, which was consistent with the previously reported phenotypes of gB and gH hyperfusogenic VZV mutants. Seven host cell proteins that remained uniquely phosphorylated when calcineurin phosphatase activity was inhibited were identified as potential downstream factors involved in fusion regulation. These findings demonstrate that calcineurin is a critical host cell factor pivotal in the regulation of VZV induced cell fusion, which is essential for VZV pathogenesis. VZV is a medically important human virus that causes varicella (chicken pox) and reactivates as zoster (shingles). Postherpetic neuralgia (PHN) is a common sequela of zoster, a persistent nerve pain that can last from months to years after zoster. Refractory to antiviral drugs and pain treatment, PHN significantly impacts quality of life. Despite the success of VZV live-attenuated vaccines in the healthy population, immunocompromised individuals are still vulnerable to significant morbidity. The recently approved subunit zoster vaccine is effective but whether it works against varicella is not known. Therefore, a better understanding of the molecular basis of VZV pathogenesis would provide significant advances towards alternative treatments. A hallmark of VZV pathogenesis is syncytia formation observed in infected skin and nerve ganglia. VZV-induced cell fusion is mediated by the viral fusion complex comprised of glycoproteins gB, gH, and gL. Dysregulation of this fusion process leads to impaired VZV infection in skin. Here, we report that inhibition of a cellular phosphatase, calcineurin, significantly increased gB/gH-gL mediated cell fusion, and intensified syncytia formation during VZV replication, but suppressed virus spread. Our study demonstrated that calcineurin phosphatase activity regulates VZV-induced cell fusion, providing a new perspective for potential antiviral strategies that target host factors.
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