Mouse hepatitis coronavirus RNA replication depends on GBF1-mediated ARF1 activation.
Mouse hepatitis coronavirus RNA replication depends on GBF1-mediated ARF1 activation.
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DOI：
10.1371/journal.ppat.1000088
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发表时间：
2008-06-13
期刊：
影响因子：
6.7
通讯作者：
de Haan, Cornelis A. M.
中科院分区：
文献类型：
--
作者：
Verheije, Monique H.;Raaben, Matthijs;Mari, Muriel;Lintelo, Eddie G. te;Reggiori, Fulvio;van Kuppeveld, Frank J. M.;Rottier, Peter J. M.;de Haan, Cornelis A. M.
冠状病毒在受感染的细胞中诱导形成双膜囊泡，这是 RNA 复制的位点。尽管最近的观察表明内质网在小鼠肝炎冠状病毒（MHV）复制复合物（RC）的形成中发挥着重要作用，但人们对这些囊泡的形成知之甚少。我们现在证明 MHV 复制对布雷菲德菌素 A (BFA) 敏感。一致的是，ARF1 的显性失活突变体的表达（已知可模仿该药物的作用）可显着抑制 MHV 感染。免疫荧光分析和定量电子显微镜表明，BFA 本身并没有阻止 RC 的形成，而是减少了 RC 的数量。 MDCK 细胞中的 MHV RNA 复制对 BFA 不敏感，而 MDCK 细胞表达抗 BFA 的鸟嘌呤核苷酸交换因子 GBF1。因此，通过转染小干扰RNA (siRNA)对BFA的高尔基体驻留靶标进行个体敲低表明，GBF1而非BIG1或BIG2在MHV RNA复制中发挥着重要作用。 ARF1（GBF1 的细胞效应子）似乎也参与了 MHV 复制，因为针对这种小 GTP 酶的 siRNA 显着抑制了 MHV 感染。总的来说，我们的结果表明 GBF1 介导的 ARF1 激活是 MHV RNA 有效复制所必需的，并揭示早期分泌途径和 MHV 复制复合物的形成密切相关。冠状病毒是人类和动物许多呼吸道和肠道感染的病原体。与所有病毒一样，实际上其感染周期的所有步骤都取决于宿主细胞因素。作为进入细胞后的第一步也是最关键的一步，冠状病毒组装其复制复合物（RC），并与特征性的、新诱导的膜结构相关联。这些正链RNA病毒劫持来创建这些“工厂”的细胞途径尚未阐明。在这里，我们通过使用药物布雷菲德菌素 A (BFA) 研究分泌途径在小鼠肝炎冠状病毒 (MHV) 复制中的参与，已知该药物通过抑制 ADP-核糖基化因子 (ARF) 小 GTP 酶的激活来干扰 ER-高尔基体膜运输。我们的观察表明 MHV RNA 复制对 BFA 敏感。与此一致，我们通过使用各种技术证明，BFA 敏感的胍核苷酸交换因子 GBF1 及其下游效应子 ARF1 对于冠状病毒复制至关重要。根据我们的结果，我们得出结论，MHV RNA 复制依赖于 GBF1 介导的 ARF1 激活。我们的研究为 MHV 复制与早期分泌途径之间的密切联系提供了新的见解。
Coronaviruses induce in infected cells the formation of double membrane vesicles, which are the sites of RNA replication. Not much is known about the formation of these vesicles, although recent observations indicate an important role for the endoplasmic reticulum in the formation of the mouse hepatitis coronavirus (MHV) replication complexes (RCs). We now show that MHV replication is sensitive to brefeldin A (BFA). Consistently, expression of a dominant-negative mutant of ARF1, known to mimic the action of the drug, inhibited MHV infection profoundly. Immunofluorescence analysis and quantitative electron microscopy demonstrated that BFA did not block the formation of RCs per se, but rather reduced their number. MHV RNA replication was not sensitive to BFA in MDCK cells, which are known to express the BFA-resistant guanine nucleotide exchange factor GBF1. Accordingly, individual knockdown of the Golgi-resident targets of BFA by transfection of small interfering RNAs (siRNAs) showed that GBF1, but not BIG1 or BIG2, was critically involved in MHV RNA replication. ARF1, the cellular effector of GBF1, also appeared to be involved in MHV replication, as siRNAs targeting this small GTPase inhibited MHV infection significantly. Collectively, our results demonstrate that GBF1-mediated ARF1 activation is required for efficient MHV RNA replication and reveal that the early secretory pathway and MHV replication complex formation are closely connected. Coronaviruses are the causative agents of many respiratory and enteric infections in humans and animals. As with all viruses, virtually all of the steps of their infection cycle depend on host cellular factors. As the first and most crucial step after their entry into cells, coronaviruses assemble their replication complexes (RCs) in association with characteristic, newly induced membranous structures. The cellular pathways hijacked by these plus-strand RNA viruses to create these “factories” have not been elucidated. Here, we study the involvement of the secretory pathway in mouse hepatitis coronavirus (MHV) replication by using the drug brefeldin A (BFA), which is known to interfere with ER–Golgi membrane traffic by inhibiting the activation of ADP-ribosylation factor (ARF) small GTPases. Our observations show that MHV RNA replication is sensitive to BFA. In agreement herewith we demonstrate, by using various techniques, that the BFA-sensitive guanidine nucleotide exchange factor GBF1 and its downstream effector ARF1 are of critical importance for coronavirus replication. From our results we conclude that MHV RNA replication depends on GBF1-mediated ARF1 activation. Our study provides new insights into the close connection between MHV replication and the early secretory pathway.
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