Department of Pediatrics, Stanford University School of Medicine, Stanford, California, United States of America.
Department of Microbiology & Immunology, Stanford University School of Medicine, Stanford, California, United States of America.
PLoS Pathog. 2020 Nov 20;16(11):e1009022. doi: 10.1371/journal.ppat.1009022. eCollection 2020 Nov.
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.
细胞融合（简称细胞融合）是医学上重要病毒的特征病理学，包括水痘-带状疱疹病毒（VZV），即水痘和带状疱疹的病原体。细胞融合由 VZV 糖蛋白 gB 和 gH-gL 复合物介导，必须进行严格调节，才能根据 gB 和 gH 高融合性 VZV 突变体的研究在皮肤上发病。尽管已经研究了 gB 和 gH-gL 在调节细胞融合中的功能，但宿主因子是否直接参与该调节过程尚不清楚。在这里，我们通过对具有已知细胞靶标的生物活性化合物进行高通量筛选，发现了调节 VZV gB/gH-gL 介导的细胞融合的宿主因子。两种结构相关的非抗生素大环内酯类药物，他克莫司和吡美莫司，都显著增加了 VZV gB/gH-gL 介导的细胞融合。这些化合物与 FKBP1A 形成药物-蛋白复合物，与钙调磷酸酶结合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 syncytin-1 介导的细胞融合，表明钙调磷酸酶在调节这一过程中具有广泛的作用。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。FKBP1A 的 shRNA 敲低显著降低了吡美莫司诱导的增强的细胞融合，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，在 VZV 感染期间抑制钙调磷酸酶活性会导致合胞体形成过度，并抑制病毒复制，这与之前报道的 gB 和 gH 高融合性 VZV 突变体的表型一致。当钙调磷酸酶活性受到抑制时，有 7 种宿主细胞蛋白仍然被独特地磷酸化，被鉴定为参与融合调节的潜在下游因子。这些发现表明钙调磷酸酶是 VZV 诱导的细胞融合调节中的关键宿主细胞因子，对于 VZV 的发病机制至关重要。
