Requirement of a functional ion channel for Sindbis virus glycoprotein transport, CPV-II formation, and efficient virus budding.
Requirement of a functional ion channel for Sindbis virus glycoprotein transport, CPV-II formation, and efficient virus budding.
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DOI：
10.1371/journal.ppat.1010892
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发表时间：
2022-10
期刊：
影响因子：
6.7
通讯作者：
中科院分区：
文献类型：
--
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许多病毒编码离子通道蛋白，其寡聚化以在病毒感染细胞的膜和一些包膜病毒的病毒膜中形成亲水性孔。甲病毒6 K、人类免疫缺陷病毒1型Vpu（HIV-Vpu）、甲型流感病毒M2（IAV-M2）和丙型肝炎病毒P7（HCV-P7）是跨膜离子通道蛋白，在病毒组装、出芽和进入中发挥重要作用。虽然M2和P7的离子通道活性的寡聚体结构和机制已得到充分确立，但6 K的这些结构和机制仍然未知。在这里，我们研究了离子通道活性的6 K在甲病毒组装的功能作用，通过利用一系列的辛德毕斯病毒（SINV）离子通道嵌合体表达的离子通道螺旋从Vpu或M2或取代整个6 K蛋白与全长P7，顺式。我们证明了Vpu螺旋有效地补充6 K，而M2和P7效率较低。我们的研究结果表明，虽然SINV主要是不敏感的M2离子通道抑制剂金刚烷胺，Vpu抑制剂5-N，N-六亚甲基阿米洛利（HMA），显着减少SINV的释放，这表明6 K的离子通道活性类似于Vpu，促进病毒出芽。使用活细胞成像的SINV与miniSOG标记的6 K和mCherry标记的E2，我们进一步证明，6 K和E2共定位与高尔基体的分泌途径。为了使6 K在高尔基体中的定位上下文中，我们使用透射电子显微镜分析了用SINV和SINV-离子通道嵌合体感染的细胞。我们的研究结果提供了证据，第一次为功能的作用，6 K在II型细胞病变空泡（CPV-II）的形成。我们证明，在没有6 K，CPV-II，这源于高尔基体，是没有检测到在感染的细胞，伴随着减少糖蛋白运输到质膜。取代功能性离子通道，M2或Vpu定位到高尔基体，恢复CPV-II生产，而P7，保留在ER中，是不足以诱导CPV-II的形成。总之，我们的研究结果表明，离子通道活性的6 K所需的形成CPV-II从高尔基体，促进糖蛋白穗运输到质膜和有效的病毒出芽。甲病毒引起急性和长期的疾病，范围从发热性疾病到感染个体的致命性脑炎。确定用于抗病毒开发的新药靶点对于控制和对抗甲病毒感染至关重要。病毒离子通道抑制剂，如流感M2、HIV Vpu和HCV P7已被证明是有效的抗病毒药物。甲病毒6 K是一种6 kDa的结构蛋白，在感染细胞的膜上形成离子通道。然而，没有结构信息可用于确定其在甲病毒生命周期中的功能作用。使用miniSOG标记的6 K SINV，我们首次在感染细胞中确定了6 K的空间和时间组织。我们的研究结果揭示了共定位的6 K与E2在高尔基体，其离子通道活性是必不可少的CPV-II的形成。本研究确定，6 K是所需的有效的糖蛋白运输到质膜，一个功能，可以部分补充顺式由其他病毒离子通道，交通到高尔基体。我们证明了通道阻断药物5-N，N-六亚甲基阿米洛利对6 K的抑制作用阻碍了甲病毒的出芽，为开发靶向6 K的抑制剂作为有吸引力的抗病毒策略奠定了基础。
Many viruses encode ion channel proteins that oligomerize to form hydrophilic pores in membranes of virus-infected cells and the viral membrane in some enveloped viruses. Alphavirus 6K, human immunodeficiency virus type 1 Vpu (HIV-Vpu), influenza A virus M2 (IAV-M2), and hepatitis C virus P7 (HCV-P7) are transmembrane ion channel proteins that play essential roles in virus assembly, budding, and entry. While the oligomeric structures and mechanisms of ion channel activity are well-established for M2 and P7, these remain unknown for 6K. Here we investigated the functional role of the ion channel activity of 6K in alphavirus assembly by utilizing a series of Sindbis virus (SINV) ion channel chimeras expressing the ion channel helix from Vpu or M2 or substituting the entire 6K protein with full-length P7, in cis. We demonstrate that the Vpu helix efficiently complements 6K, whereas M2 and P7 are less efficient. Our results indicate that while SINV is primarily insensitive to the M2 ion channel inhibitor amantadine, the Vpu inhibitor 5-N, N-Hexamethylene amiloride (HMA), significantly reduces SINV release, suggesting that the ion channel activity of 6K similar to Vpu, promotes virus budding. Using live-cell imaging of SINV with a miniSOG-tagged 6K and mCherry-tagged E2, we further demonstrate that 6K and E2 colocalize with the Golgi apparatus in the secretory pathway. To contextualize the localization of 6K in the Golgi, we analyzed cells infected with SINV and SINV-ion channel chimeras using transmission electron microscopy. Our results provide evidence for the first time for the functional role of 6K in type II cytopathic vacuoles (CPV-II) formation. We demonstrate that in the absence of 6K, CPV-II, which originates from the Golgi apparatus, is not detected in infected cells, with a concomitant reduction in the glycoprotein transport to the plasma membrane. Substituting a functional ion channel, M2 or Vpu localizing to Golgi, restores CPV-II production, whereas P7, retained in the ER, is inadequate to induce CPV-II formation. Altogether our results indicate that ion channel activity of 6K is required for the formation of CPV-II from the Golgi apparatus, promoting glycoprotein spike transport to the plasma membrane and efficient virus budding. Alphaviruses cause acute and long-term diseases ranging from febrile illness to fatal encephalitis in infected individuals. Identifying new drug targets for antiviral development is crucial to controlling and combating alphavirus infections. Inhibitors for viral ion channels such as Influenza M2, HIV Vpu, and HCV P7 have been proven to be effective antivirals. Alphavirus 6K is a 6 kDa structural protein that forms ion channels on membranes of infected cells. However, no structural information is available to establish its functional role in alphavirus life cycle. Using a miniSOG-tagged 6K SINV, we determined the spatial and temporal organization of 6K for the first time in infected cells. Our results reveal the colocalization of 6K with E2 in the Golgi, where its ion channel activity is essential for CPV-II formation. This study establishes that 6K is required for efficient glycoprotein transport to the plasma membrane, a function that can be partially complemented in cis by other viral ion channels that traffic to the Golgi. We demonstrate that the inhibition of 6K by a channel-blocking drug, 5-N, N-Hexamethylene amiloride impedes alphavirus budding, laying the foundation for developing inhibitors targeting 6K as an attractive antiviral strategy.
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