Respiratory Syncytial Virus Sequesters NF-κB Subunit p65 to Cytoplasmic Inclusion Bodies To Inhibit Innate Immune Signaling.
Respiratory Syncytial Virus Sequesters NF-κB Subunit p65 to Cytoplasmic Inclusion Bodies To Inhibit Innate Immune Signaling.
复制标题
呼吸道合胞病毒将 NF-κB 亚基 p65 隔离到细胞质包涵体中，以抑制先天免疫信号传导。
DOI：
10.1128/jvi.01380-20
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发表时间：
2020-10-27
影响因子：
5.4
通讯作者：
Bailey D
中科院分区：
文献类型：
--
作者：
Jobe F;Simpson J;Hawes P;Guzman E;Bailey D
许多病毒几乎完全在感染细胞的细胞质中复制;然而，这些病原体如何能够划分其生命周期以提供有利的复制条件并避免细胞质中的一连串抗病毒检测机制仍然相对不确定。在这份手稿中，我们表明，牛呼吸道合胞病毒（bRSV），感染牛，这样做是通过在感染细胞的细胞质中产生包涵体。我们证实bRSV和人RSV病毒RNA复制都发生在这些包涵体中，这可能意味着这些细胞器是这组病毒（正肺病毒）功能保守的特征。重要的是，我们还发现这些细胞器能够捕获重要的先天免疫转录因子（在这种情况下是NF-κ B），阻断告诉细胞核细胞被感染的正常信号传导过程，这可能有助于我们了解这些病毒如何引起疾病。病毒通常采用策略来阻止受感染细胞中先天免疫信号的激活。呼吸道合胞病毒（RSV）也不例外，因为它编码两种辅助蛋白（NS 1和NS 2），这两种蛋白已被公认可阻断干扰素信号传导。然而，RSV编码的抑制NF-κB信号传导的机制还不太清楚。在这项研究中，我们确定了RSV介导的这种途径的拮抗作用，独立于NS 1和NS 2蛋白，确实不同于其他已知的NF-κB抑制的病毒机制。在人和牛RSV感染的细胞中，我们证明NF-κB的p65亚基被重新路由到细胞质中的核周点，这与病毒包涵体（IB）同义，是病毒RNA复制的位点。捕获的p65在肿瘤坏死因子α（TNF-α）刺激后不能易位到细胞核或反式激活NF-κB报告基因，证实了这种隔离的免疫拮抗性质。随后，我们使用相关的光电子显微镜（CLEM）共定位的RSV N蛋白和p65内牛RSV（bRSV）IBs，这是颗粒状，无膜区域的细胞质与液体细胞器样的属性。bRSV IB的其他表征表明，尽管它们可能通过液-液相分离（LLPS）形成，但它们对低渗休克的敏感性与其大小成比例。总之，这些数据确定了一种新的先天免疫信号转导的病毒拮抗机制，该机制依赖于NF-κB亚基p65与生物分子缩合物的隔离-这是一种在正肺病毒属中保守的机制，而不是宿主细胞特异性的。更一般地说，它们提供了额外的证据，证明RNA病毒IB是感染细胞内重要的免疫调节复合物。重要性许多病毒几乎完全在感染细胞的细胞质中复制;然而，这些病原体如何能够划分其生命周期，为复制提供有利条件，并避免细胞质中的抗病毒检测机制仍然相对不确定。在这份手稿中，我们表明，牛呼吸道合胞病毒（bRSV），感染牛，这样做是通过在感染细胞的细胞质中产生包涵体。我们证实bRSV和人RSV病毒RNA复制都发生在这些包涵体中，这可能意味着这些细胞器是这组病毒（正肺病毒）功能保守的特征。重要的是，我们还发现这些细胞器能够捕获重要的先天免疫转录因子（在这种情况下是NF-κ B），阻断告诉细胞核细胞被感染的正常信号传导过程，这可能有助于我们了解这些病毒如何引起疾病。
Many viruses replicate almost entirely in the cytoplasm of infected cells; however, how these pathogens are able to compartmentalize their life cycle to provide favorable conditions for replication and to avoid the litany of antiviral detection mechanisms in the cytoplasm remains relatively uncharacterized. In this manuscript, we show that bovine respiratory syncytial virus (bRSV), which infects cattle, does this by generating inclusion bodies in the cytoplasm of infected cells. We confirm that both bRSV and human RSV viral RNA replication takes place in these inclusion bodies, likely meaning these organelles are a functionally conserved feature of this group of viruses (the orthopneumoviruses). Importantly, we also showed that these organelles are able to capture important innate immune transcription factors (in this case NF-KB), blocking the normal signaling processes that tell the nucleus the cell is infected, which may help us to understand how these viruses cause disease. Viruses routinely employ strategies to prevent the activation of innate immune signaling in infected cells. Respiratory syncytial virus (RSV) is no exception, as it encodes two accessory proteins (NS1 and NS2) which are well established to block interferon signaling. However, RSV-encoded mechanisms for inhibiting NF-κB signaling are less well characterized. In this study, we identified RSV-mediated antagonism of this pathway, independent of the NS1 and NS2 proteins and indeed distinct from other known viral mechanisms of NF-κB inhibition. In both human and bovine RSV-infected cells, we demonstrated that the p65 subunit of NF-κB is rerouted to perinuclear puncta in the cytoplasm, which are synonymous with viral inclusion bodies (IBs), the site for viral RNA replication. Captured p65 was unable to translocate to the nucleus or transactivate a NF-κB reporter following tumor necrosis factor alpha (TNF-α) stimulation, confirming the immune-antagonistic nature of this sequestration. Subsequently, we used correlative light electron microscopy (CLEM) to colocalize the RSV N protein and p65 within bovine RSV (bRSV) IBs, which are granular, membraneless regions of cytoplasm with liquid organelle-like properties. Additional characterization of bRSV IBs indicated that although they are likely formed by liquid-liquid phase separation (LLPS), they have a differential sensitivity to hypotonic shock proportional to their size. Together, these data identify a novel mechanism for viral antagonism of innate immune signaling which relies on sequestration of the NF-κB subunit p65 to a biomolecular condensate—a mechanism conserved across the Orthopneumovirus genus and not host-cell specific. More generally, they provide additional evidence that RNA virus IBs are important immunomodulatory complexes within infected cells. IMPORTANCE Many viruses replicate almost entirely in the cytoplasm of infected cells; however, how these pathogens are able to compartmentalize their life cycle to provide favorable conditions for replication and to avoid the litany of antiviral detection mechanisms in the cytoplasm remains relatively uncharacterized. In this manuscript, we show that bovine respiratory syncytial virus (bRSV), which infects cattle, does this by generating inclusion bodies in the cytoplasm of infected cells. We confirm that both bRSV and human RSV viral RNA replication takes place in these inclusion bodies, likely meaning these organelles are a functionally conserved feature of this group of viruses (the orthopneumoviruses). Importantly, we also showed that these organelles are able to capture important innate immune transcription factors (in this case NF-KB), blocking the normal signaling processes that tell the nucleus the cell is infected, which may help us to understand how these viruses cause disease.