Rhinovirus C replication is associated with the endoplasmic reticulum and triggers cytopathic effects in an in vitro model of human airway epithelium.
Rhinovirus C replication is associated with the endoplasmic reticulum and triggers cytopathic effects in an in vitro model of human airway epithelium.
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DOI：
10.1371/journal.ppat.1010159
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发表时间：
2022-01
期刊：
影响因子：
6.7
通讯作者：
Scull MA
中科院分区：
文献类型：
--
作者：
Gagliardi TB;Goldstein ME;Song D;Gray KM;Jung JW;Ignacio MA;Stroka KM;Duncan GA;Scull MA
鼻病毒C（RV-C）的临床影响已得到充分记录;然而，病毒生命周期的定义仍然很差。因此，我们在单细胞水平上表征了RV-C15复制及其使用生理相关的体外模型对人气道上皮（HAE）的影响。RV-C15复制仅限于纤毛细胞，其中病毒RNA水平在感染后12小时（hpi）达到峰值，与24 hpi时顶端隔室中的滴度升高相关。值得注意的是，感染与RV-C受体（钙粘蛋白相关家族成员3）的极化表达丧失相关。在RV-C15复制期间双链RNA（dsRNA）的可视化揭示了细胞内两种不同的复制复合物排列，可能对应于感染中的不同时间点。为了进一步确定RV-C15复制位点，我们分析了巨蛋白、磷脂酰肌醇-4-磷酸和钙连接蛋白与dsRNA的表达和共定位。尽管在RV-C15感染期间通过免疫荧光观察到高尔基体片段化，如先前对其他RV所报道的，但高比率的钙连接蛋白-dsRNA共定位暗示内质网是HAE中RV-C15复制的主要位点。RV-C15感染还与干扰素基因刺激因子（STING）表达升高和诱导不完全自噬相关，不完全自噬是其他RV用于促进子代病毒体的非裂解性释放的机制。值得注意的是，HAE中STING的遗传消耗减弱了RV-C15和-A16（但不是-B14）复制，证实了先前提出的STING在一些RV感染中的前病毒作用。最后，RV-C15感染导致上皮屏障完整性的暂时丧失和紧密连接蛋白的易位，而粘膜纤毛清除率的降低表明对上皮功能的细胞病变作用。总之，我们的研究结果确定了RV-C复制与相关鼻病毒相比的共同和独特的特征，并确定了RV-C对上皮细胞组织和组织功能的影响-感染的各个方面可能有助于体内发病。鼻病毒C具有全球性分布和显著的临床影响，特别是在那些有潜在肺部疾病的患者中。尽管RV-C在遗传、结构和生物学上与RV-A和-B病毒不同，但我们对RV-C生命周期的理解主要是从这些和其他相关病毒推断的。在这里，我们进行了一个详细的分析RV-C15复制的生理相关模型的人气道上皮。我们的单细胞，显微镜为基础的方法显示，不像其他RV的内质网是RV-C15复制的主要网站。RV-C15复制也刺激STING表达，这是前病毒，并引发细胞组织的显着变化，包括改变病毒受体分布，破碎的高尔基体堆栈，和诱导不完全自噬。此外，我们观察到在RV-C15感染期间上皮屏障功能的丧失和粘膜纤毛清除的减少，粘膜纤毛清除是肺中的主要防御机制。总之，这些数据揭示了对RV-C15复制动力学的新见解以及在感染的主要靶细胞中产生的细胞病变效应，从而进一步加深了我们对RV-C发病机制的理解。我们的工作突出了与相关病原体相比RV-C15复制的相似和独特方面，这将有助于指导未来对RV-C感染分子机制的研究。
The clinical impact of rhinovirus C (RV-C) is well-documented; yet, the viral life cycle remains poorly defined. Thus, we characterized RV-C15 replication at the single-cell level and its impact on the human airway epithelium (HAE) using a physiologically-relevant in vitro model. RV-C15 replication was restricted to ciliated cells where viral RNA levels peaked at 12 hours post-infection (hpi), correlating with elevated titers in the apical compartment at 24hpi. Notably, infection was associated with a loss of polarized expression of the RV-C receptor, cadherin-related family member 3. Visualization of double-stranded RNA (dsRNA) during RV-C15 replication revealed two distinct replication complex arrangements within the cell, likely corresponding to different time points in infection. To further define RV-C15 replication sites, we analyzed the expression and colocalization of giantin, phosphatidylinositol-4-phosphate, and calnexin with dsRNA. Despite observing Golgi fragmentation by immunofluorescence during RV-C15 infection as previously reported for other RVs, a high ratio of calnexin-dsRNA colocalization implicated the endoplasmic reticulum as the primary site for RV-C15 replication in HAE. RV-C15 infection was also associated with elevated stimulator of interferon genes (STING) expression and the induction of incomplete autophagy, a mechanism used by other RVs to facilitate non-lytic release of progeny virions. Notably, genetic depletion of STING in HAE attenuated RV-C15 and -A16 (but not -B14) replication, corroborating a previously proposed proviral role for STING in some RV infections. Finally, RV-C15 infection resulted in a temporary loss in epithelial barrier integrity and the translocation of tight junction proteins while a reduction in mucociliary clearance indicated cytopathic effects on epithelial function. Together, our findings identify both shared and unique features of RV-C replication compared to related rhinoviruses and define the impact of RV-C on both epithelial cell organization and tissue functionality–aspects of infection that may contribute to pathogenesis in vivo. Rhinovirus C has a global distribution and significant clinical impact–especially in those with underlying lung disease. Although RV-C is genetically, structurally, and biologically distinct from RV-A and -B viruses, our understanding of the RV-C life cycle has been largely inferred from these and other related viruses. Here, we performed a detailed analysis of RV-C15 replication in a physiologically-relevant model of human airway epithelium. Our single-cell, microscopy-based approach revealed that–unlike other RVs–the endoplasmic reticulum is the primary site for RV-C15 replication. RV-C15 replication also stimulated STING expression, which was proviral, and triggered dramatic changes in cellular organization, including altered virus receptor distribution, fragmented Golgi stacks, and the induction of incomplete autophagy. Additionally, we observed a loss of epithelial barrier function and a decrease in mucociliary clearance, a major defense mechanism in the lung, during RV-C15 infection. Together, these data reveal novel insight into RV-C15 replication dynamics and resulting cytopathic effects in the primary target cells for infection, thereby furthering our understanding of the pathogenesis of RV-C. Our work highlights similar, as well as unique, aspects of RV-C15 replication compared to related pathogens, which will help guide future studies on the molecular mechanisms of RV-C infection.
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DOI：
10.1183/09031936.00092410
发表时间：
2011-05
期刊：
The European respiratory journal
影响因子：
--
作者：
Bizzintino J;Lee WM;Laing IA;Vang F;Pappas T;Zhang G;Martin AC;Khoo SK;Cox DW;Geelhoed GC;McMinn PC;Goldblatt J;Gern JE;Le Souëf PN
通讯作者：
Le Souëf PN
影响因子：
5.5
作者：
Colegio, OR;Van Itallie, C;Anderson, JM
通讯作者：
Anderson, JM
DOI：
10.1152/ajplung.00182.2003
发表时间：
2003-11-01
影响因子：
4.9
作者：
Coyne, CB;Gambling, TM;Johnson, LG
通讯作者：
Johnson, LG
DOI：
10.1128/aem.16.10.1445-1450.1968
发表时间：
1968-01-01
期刊：
APPLIED MICROBIOLOGY
影响因子：
--
作者：
FIALA, M
通讯作者：
FIALA, M
影响因子：
30.8
作者：
Bonnelykke, Klaus;Sleiman, Patrick;Bisgaard, Hans
通讯作者：
Bisgaard, Hans