您好!欢迎访问洛阳富道生物科技有限公司官方网站!
富道生物耗材
专注高端生物耗材
助力全球生命科学发展
咨询电话
400-160-1996
您现在的位置: 首页 » 新闻动态 » 行业新闻 » 正文
SARS-CoV-2复制所需细胞因子的鉴定
发表日期:2021-12-20

Identification of Cellular Factors Required for SARS-CoV-2 Replication
SARS-CoV-2复制所需细胞因子的鉴定

  Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the recently emerged virus responsible for the COVID-19 pandemic. Clinical presentation can range from asymptomatic disease and mild respiratory tract infection to severe disease with lung injury, multiorgan failure, and death. SARS-CoV-2 is the third animal coronavirus to emerge in humans in the 21st century, and coronaviruses appear to possess a unique ability to cross borders between species and infect a wide range of organisms. This is somewhat surprising as, except for the requirement of host cell receptors, cellpathogen interactions are usually species-specific. Insights into these hostvirus interactions will provide a deeper understanding of the process of SARS-CoV-2 infection and provide a means for the design and development of antiviral agents. In this study, we describe a complex analysis of SARS-CoV-2 infection using a genome-wide CRISPR-Cas9 knock-out system in HeLa cells overexpressing entry receptor angiotensin-converting enzyme 2 (ACE2). This platform allows for the identification of factors required for viral replication. This study was designed to include a high number of replicates (48 replicates; 16 biological repeats with 3 technical replicates each) to prevent data instability, remove sources of bias, and allow multifactorial bioinformatic analyses in order to study the resulting interaction network. The results obtained provide an interesting insight into the replication mechanisms of SARS-CoV-2.

 严重急性呼吸系统综合症冠状病毒 2 (SARS-CoV-2) 是最近出现的导致 COVID-19 大流行的病毒。临床表现的范围可以从无症状疾病和轻度呼吸道感染到严重的肺损伤、多器官衰竭和死亡。SARS-CoV-2 21 世纪第三种在人类中出现的动物冠状病毒,冠状病毒似乎具有独特的跨越物种边界并感染广泛生物体的能力。这有点令人惊讶,因为除了需要宿主细胞受体外,细胞-病原体相互作用通常是物种特异性的。对这些宿主-病毒相互作用的深入了解将有助于更深入地了解 SARS-CoV-2 感染的过程,并为设计和开发抗病毒药物提供一种手段。在这项研究中,我们描述了在过度表达进入受体血管紧张素转换酶 2 (ACE2) HeLa 细胞中使用全基因组 CRISPR-Cas9 敲除系统对 SARS-CoV-2 感染进行的复杂分析。该平台允许识别病毒复制所需的因素。本研究旨在包括大量重复(48 个重复;16 个生物学重复,每个重复 3 个技术重复),以防止数据不稳定,消除偏差来源,并允许进行多因素生物信息学分析,以研究由此产生的相互作用网络。获得的结果提供了对 SARS-CoV-2 复制机制的有趣见解。该平台允许识别病毒复制所需的因素。本研究旨在包括大量重复(48 个重复;16 个生物学重复,每个重复 3 个技术重复),以防止数据不稳定,消除偏差来源,并允许进行多因素生物信息学分析,以研究由此产生的相互作用网络。获得的结果提供了对 SARS-CoV-2 复制机制的有趣见解。该平台允许识别病毒复制所需的因素。本研究旨在包括大量重复(48 个重复;16 个生物学重复,每个重复 3 个技术重复),以防止数据不稳定,消除偏差来源,并允许进行多因素生物信息学分析,以研究由此产生的相互作用网络。获得的结果提供了对 SARS-CoV-2 复制机制的有趣见解。

 SARS-CoV-2复制所需细胞因子的鉴定
高效摇瓶5L

Further knock-outs, including MACF1, PCDHGA1, GAGE1, and SPATA25, resulted in a decrease of viral RNA copies compared to non-targeting control (NTC). Microtubule-actin crosslinking factor 1 (MACF1), also widely known as actin crosslinking factor 7 (ACF7), plays a role in various cellular processes, including the regulation of cell polarization and motility through an interaction with microtubules and F-actin. In the context of coronavirus infection, microtubules and actin are critical in the transportation of internalized virus-containing vesicles. Interestingly, ACF7 is also involved in a wide range of cellular signaling networks, including Wnt/β-catenin signaling, the upregulation of which is associated with inflammation and cytokine storm in COVID-19 patients [46,47]. SPATA25 (spermatogenesis-associated protein 25) may play a role in spermatogenesis; however, this role is poorly understood. GAGE1 is a poorly defined protein, and its role in SARS-CoV-2 infection is currently unknown. Further examination of our top 178 list of targets and literature, suggested two further genes for validation: B4GALT7 and IL10RB.

B4GALT7 is a galactosyltransferase that functions in the heparan sulfate biosynthesis pathway and plays a role in DENV viral replication. B4GALT7 was identified in our screen and others [11,12]. The IL10 subunit IL10RB was similarly identified from our top 178 list of targets. Protein levels of IL10 may influence COVID-19 disease outcomes [48]. We, therefore, tested whether the depletion of these genes has an impact on SARS-CoV-2 replication, and using RT-qPCR analysis we found that both B4GALT7 and IL10RB KOs significantly reduced viral replication (Supplementary Figure S1), suggesting that they may play a role in SARS-CoV-2 infection. However, one may remember that using this methodology, only factors preventing productive infection would be identified.

SARS-CoV-2复制所需细胞因子的鉴定
细胞转瓶5L

We have prepared an extensive dataset using our genome-wide CRISPR/Cas9 knock-out screening approach, resulting in the identification of several cellular factors required for SARS-CoV-2 replication. This study was highly repetitive to remove false positives and stochastic noise, as the experiment design is very complex. We have identified and validated select factors using a broad range of analytic and experimental approaches. The data described provide an interesting insight into SARS-CoV-2 viral replication mechanisms and identify factors that may serve as potential therapeutic targets.

 包括MACF1PCDHGA1GAGE1SPATA25,与非靶向对照 (NTC) 相比,导致病毒 RNA 拷贝减少。微管-肌动蛋白交联因子 1 (MACF1),也广泛称为肌动蛋白交联因子 7 (ACF7),在各种细胞过程中发挥作用,包括通过与微管和 F-肌动蛋白的相互作用调节细胞极化和运动。在冠状病毒感染的背景下,微管和肌动蛋白对于内化含有病毒的囊泡的运输至关重要。有趣的是,ACF7 还涉及广泛的细胞信号网络,包括 Wnt/β-catenin 信号,其上调与 COVID-19 患者的炎症和细胞因子风暴有关 [ 46 , 47]SPATA25(精子发生相关蛋白25)可能在精子发生中起作用;然而,人们对这种作用知之甚少。GAGE1 是一种定义不清的蛋白质,目前尚不清楚其在 SARS-CoV-2 感染中的作用。进一步检查我们的前 178 个目标和文献列表,建议进一步验证两个基因:B4GALT7 IL10RB


10层细胞工厂

B4GALT7 是一种半乳糖基转移酶,在硫酸乙酰肝素生物合成途径中发挥作用,并在 DENV 病毒复制中发挥作用。B4GALT7 在我们的屏幕和其他人中被识别 [ 11 , 12 ]IL10 亚基 IL10RB 从我们的前 178 个目标列表中类似地确定。IL10 的蛋白质水平可能会影响 COVID-19 疾病的结果 [ 48 ]。因此,我们测试了这些基因的消耗是否对 SARS-CoV-2 复制有影响,并且使用 RT-qPCR 分析我们发现B4GALT7IL10RB KO 都显着减少了病毒复制(补充图 S1),表明它们可能在 SARS-CoV-2 感染中起作用。然而,人们可能还记得,使用这种方法,只能确定防止生产性感染的因素。

我们使用我们的全基因组 CRISPR/Cas9 敲除筛选方法准备了一个广泛的数据集,从而确定了 SARS-CoV-2 复制所需的几种细胞因子。由于实验设计非常复杂,因此该研究高度重复以消除误报和随机噪声。我们已经使用广泛的分析和实验方法确定并验证了选定的因素。所描述的数据提供了对 SARS-CoV-2 病毒复制机制的有趣见解,并确定了可能作为潜在治疗目标的因素。

 

 

 

关键词:

SARS-CoV-2,冠状病毒,CRISPR-Cas9,细胞因子,感染机制,病毒病机,SARS-CoV-2, coronavirus, CRISPR-Cas9, cellular factors, mechanisms of infection, viral pathogenesis,

推荐产品
网站首页 细胞培养耗材 细胞工厂 细胞摇瓶 SUS一次性耗材 关于富道 联系我们
首页 电话 联系