The Golgi Calcium ATPase Pump Plays an Essential Role in Adeno-associated Virus Trafficking and Transduction.
The Golgi Calcium ATPase Pump Plays an Essential Role in Adeno-associated Virus Trafficking and Transduction.
复制标题
高尔基体钙ATP酶泵在腺相关病毒运输和转导中起重要作用。
DOI：
10.1128/jvi.01604-20
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发表时间：
2020-10-14
影响因子：
5.4
通讯作者：
Asokan A
中科院分区：
文献类型：
--
作者：
Madigan VJ;Berry GE;Tyson TO;Nardone-White D;Ark J;Elmore ZC;Murlidharan G;Vincent HA;Asokan A
腺相关病毒（AAV）已被证明是有效的基因转移载体。然而，我们对宿主细胞环境如何影响 AAV 转导的理解仍在不断发展。在本研究中，我们研究了 ATP2C1 的作用，ATP2C1 编码膜钙转运泵 SPCA1，对于维持 AAV 转导中的细胞钙稳态至关重要。我们的结果表明，细胞钙对于 AAV 衣壳中有效的细胞内运输和构象变化至关重要，而 AAV 衣壳支持有效的基因组转录。此外，我们还表明，细胞钙水平的药理学调节可潜在地应用于提高 AAV 基因转移效率。腺相关病毒 (AAV) 是依赖细小病毒，已被证明可用于治疗性基因转移；然而，我们对影响 AAV 贩运和转导的宿主因素的理解仍在不断发展。在这里，我们研究了细胞钙在 AAV 感染途径中的作用。首先，我们证明了由 ATP2C1 基因编码的宿主高尔基体 ATP 驱动的钙泵（分泌途径钙 ATP 酶 1 [SPCA1]）在 AAV 感染中的关键作用。基于 CRISPR 的 ATP2C1 敲除 (KO) 减少了不同 AAV 血清型的转导。 ATP2C1 KO 似乎不会抑制 AAV 结合、细胞摄取或核进入；然而，ATP2C1 KO 细胞内的衣壳表现出分散和点状的运输，与正常细胞中观察到的核周、跨高尔基体模式不同。此外，我们观察到 AAV 衣壳在 ATP2C1 KO 细胞中进行构象变化和支持有效载体基因组转录的能力存在缺陷。钙螯合剂 BAPTA-AM 可降低胞质钙，在体外挽救有缺陷的 ATP2C1 KO 表型和 AAV 转导。相反，钙离子载体离子霉素会破坏钙梯度，从而阻断 AAV 转导。此外，我们证明使用 BAPTA-AM 调节小鼠大脑中的钙可以增强体内 AAV 基因的表达。综合这些数据，我们假设钙 ATP 酶维持细胞内钙梯度和高尔基体内部的处理对于启动衣壳以支持有效的 AAV 基因组转录至关重要。重要性 腺相关病毒 (AAV) 已被证明是有效的基因转移载体。然而，我们对宿主细胞环境如何影响 AAV 转导的理解仍在不断发展。在本研究中，我们研究了 ATP2C1 的作用，ATP2C1 编码膜钙转运泵 SPCA1，对于维持 AAV 转导中的细胞钙稳态至关重要。我们的结果表明，细胞钙对于 AAV 衣壳中有效的细胞内运输和构象变化至关重要，而 AAV 衣壳支持有效的基因组转录。此外，我们还表明，细胞钙水平的药理学调节可潜在地应用于提高 AAV 基因转移效率。
Adeno-associated viruses (AAVs) have proven to be effective gene transfer vectors. However, our understanding of how the host cell environment influences AAV transduction is still evolving. In the present study, we investigated the role of ATP2C1, which encodes a membrane calcium transport pump, SPCA1, essential for maintaining cellular calcium homeostasis on AAV transduction. Our results indicate that cellular calcium is essential for efficient intracellular trafficking and conformational changes in the AAV capsid that support efficient genome transcription. Further, we show that pharmacological modulation of cellular calcium levels can potentially be applied to improve the AAV gene transfer efficiency. Adeno-associated viruses (AAVs) are dependoparvoviruses that have proven useful for therapeutic gene transfer; however, our understanding of host factors that influence AAV trafficking and transduction is still evolving. Here, we investigated the role of cellular calcium in the AAV infectious pathway. First, we demonstrated a critical role for the host Golgi compartment-resident ATP-powered calcium pump (secretory pathway calcium ATPase 1 [SPCA1]) encoded by the ATP2C1 gene in AAV infection. CRISPR-based knockout (KO) of ATP2C1 decreases transduction by different AAV serotypes. ATP2C1 KO does not appear to inhibit AAV binding, cellular uptake, or nuclear entry; however, capsids within ATP2C1 KO cells demonstrate dispersed and punctate trafficking distinct from the perinuclear, trans-Golgi pattern observed in normal cells. In addition, we observed a defect in the ability of AAV capsids to undergo conformational changes and support efficient vector genome transcription in ATP2C1 KO cells. The calcium chelator BAPTA-AM, which reduces cytosolic calcium, rescues the defective ATP2C1 KO phenotype and AAV transduction in vitro. Conversely, the calcium ionophore ionomycin, which disrupts calcium gradients, blocks AAV transduction. Further, we demonstrated that modulating calcium in the murine brain using BAPTA-AM augments AAV gene expression in vivo. Taking these data together, we postulate that the maintenance of an intracellular calcium gradient by the calcium ATPase and processing within the Golgi compartment are essential for priming the capsid to support efficient AAV genome transcription. IMPORTANCE Adeno-associated viruses (AAVs) have proven to be effective gene transfer vectors. However, our understanding of how the host cell environment influences AAV transduction is still evolving. In the present study, we investigated the role of ATP2C1, which encodes a membrane calcium transport pump, SPCA1, essential for maintaining cellular calcium homeostasis on AAV transduction. Our results indicate that cellular calcium is essential for efficient intracellular trafficking and conformational changes in the AAV capsid that support efficient genome transcription. Further, we show that pharmacological modulation of cellular calcium levels can potentially be applied to improve the AAV gene transfer efficiency.