Identification of potassium and calcium channel inhibitors as modulators of polyomavirus endosomal trafficking
Identification of potassium and calcium channel inhibitors as modulators of polyomavirus endosomal trafficking
复制标题
DOI：
10.1016/j.antiviral.2020.104819
复制
发表时间：
2020-07-01
影响因子：
7.6
通讯作者：
Whitehouse, Adrian
中科院分区：
文献类型：
--
作者：
Dobson, Samuel J.;Mankouri, Jamel;Whitehouse, Adrian
在病毒进入过程中，多瘤病毒科的成员在到达内质网（ER）的途中通过内溶酶体网络，降解的衣壳从内质网逃逸到细胞质中并进入细胞核。新出现的证据表明，病毒需要核内体酸化和核内体中K+和Ca 2+离子的正确离子平衡，以进行正确的病毒运输和基因组释放。在这里，使用两种多瘤病毒与不同的衣壳结构，即猿猴病毒40（SV 40）和默克尔细胞多瘤病毒（MCPyV），我们描述的方法，快速定量病毒感染IncuCyte ZOOM成像分析，并使用该系统调查的作用，K+和Ca 2+通道在病毒进入的早期阶段。使用广谱阻断剂的K+和Ca 2+通道特异性靶向宿主细胞离子通道的功能，我们表明，MCPyV，但不是SV 40可以抑制K+通道调节剂，而这两种病毒都受到限制的广谱钙通道抑制剂维拉帕米。使用一组更特异性的Ca 2+阻断剂，我们表明MCPyV和SV 40都依赖于双孔Ca 2+通道（TPC）的活性，因为TPC特异性阻断剂粉防己碱阻止了病毒进入所需的衣壳解体和核运输。因此，我们揭示了一个新的目标，以限制多瘤病毒的进入，这给了已知的作用，TPC在内溶酶体-ER融合，可能适用于其他病毒的过境这一途径。
During virus entry, members of the Polyomaviridae transit the endolysosomal network en route to the endoplasmic reticulum (ER), from which degraded capsids escape into the cytoplasm and enter the nucleus. Emerging evidence suggests that viruses require both endosomal acidification and the correct ionic balance of K+ and Ca2+ ions in endosomes for correct virus trafficking and genome release. Here, using two polyomaviruses with different capsid architectures, namely Simian virus 40 (SV40) and Merkel cell polyomavirus (MCPyV), we describe methods to rapidly quantify virus infection using IncuCyte ZOOM imaging analysis, and use this system to investigate the role of both K+ and Ca2+ channels during the early stages of virus entry. Using broad spectrum blockers of both K+ and Ca2+ channels to specifically target host cell ion channel functionality, we show that MCPyV, but not SV40 can be inhibited by K+ channel modulators, whilst both viruses are restricted by the broad spectrum Ca2+ channel inhibitor verapamil. Using a panel of more specific Ca2+ blockers, we show that both MCPyV and SV40 are dependent on the activity of two-pore Ca2+ channels (TPCs), as the TPC-specific blocker tetrandrine prevented capsid disassembly and nuclear transport required for virus entry. We therefore reveal a novel target to restrict the entry of polyomaviruses, which given the known role of TPCs during endolysosomal-ER fusion, is likely to be applicable to other viruses that transit this pathway.