鉴定甲型流感病毒感染期间切割蛋白质的半胱天冬酶及其基序
Summary
甲型流感病毒（IAV）感染激活半胱天冬酶，切割宿主和病毒蛋白，而宿主和病毒蛋白又具有促病毒和抗病毒功能。通过使用抑制剂、RNA干扰、定点诱变以及蛋白质印迹和RT-qPCR技术，鉴定了受感染哺乳动物细胞中切割宿主皮质肌动蛋白和组蛋白脱乙酰酶的半胱天冬酶。
Abstract
半胱天冬酶是半胱氨酸蛋白酶家族，协调程序性细胞死亡以响应各种刺激，包括微生物感染。最初描述为由细胞凋亡发生，现在已知程序性细胞死亡包括三种相互关联的途径：焦亡，细胞凋亡和坏死性凋亡，共同创造为一个过程，即PANoptosis。影响 病毒（IAV）感染通过诱导不同半胱天冬酶的激活来诱导哺乳动物细胞中的PANoptosis，而半胱天冬酶又会切割各种宿主和病毒蛋白，从而导致宿主先天抗病毒反应的激活或拮抗宿主蛋白的降解等过程。在这方面，在动物和人类上皮细胞中发现了半胱天冬酶 3 介导的宿主皮质素、组蛋白脱乙酰酶 4 （HDAC4） 和组蛋白脱乙酰化酶 6 （HDAC6） 的切割，以响应 IAV 感染。为了证明这一点，采用了抑制剂、RNA干扰和定点诱变，随后通过蛋白质印迹测量了皮质素、HDAC4和HDAC6多肽的切割或抗性以及皮质素、HDAC4和HDAC6多肽的回收率。这些方法与RT-qPCR相结合，形成了一种简单而有效的策略，用于鉴定宿主以及在IAV或其他人类和动物病毒感染期间经历半胱天冬酶介导的切割的病毒蛋白。本议定书阐述了该战略的代表性结果，并讨论了使其更有效的方法。
Introduction
甲型流感病毒（IAV）是 正粘病毒科 的原型成员，已知会引起全球流行病和不可预测的大流行。IAV引起人类呼吸道疾病，流感，俗称“流感”。流感是一种急性疾病，可导致宿主促炎和抗炎先天免疫反应的诱导以及人类呼吸道中上皮细胞的死亡。这两个过程都受到一种称为程序性细胞死亡1的现象的支配。一旦各种病原体识别受体感知宿主细胞中传入的病毒颗粒，就会诱导程序性细胞死亡的信号传导。这导致通过三种相互关联的途径（称为焦亡，凋亡和坏死性凋亡）对感染细胞的死亡进行编程，并向邻近的健康细胞发出信号 – 最近被创造为一个过程，PANoptosis1。
全细胞凋亡涉及许多宿主和病毒蛋白从诱导到执行的蛋白水解加工。蛋白质的这种加工主要由称为半胱天冬酶1，2的半胱氨酸蛋白酶家族带头。已知多达18种半胱天冬酶（从半胱天冬酶1到半胱天冬酶18）3。大多数半胱天冬酶表达为半胱天冬酶原，并通过自催化或其他半胱天冬酶4进行自身的蛋白水解处理来激活，以响应病毒感染等刺激。IAV感染细胞的PANoptosis被认为是宿主防御机制，但IAV已经进化出逃避和利用它以促进其复制的方法1，2，5，6。其中之一是通过半胱天冬酶介导的裂解或降解来拮抗宿主因子，这些切割或降解要么具有固有的抗病毒作用，要么干扰IAV生命周期的一个步骤。为此，已发现宿主因子皮质素、HDAC4 和 HDAC6 在 IAV 感染的上皮细胞中经历半胱天冬酶介导的切割或降解7，8，9。HDAC4 和 HDAC6 是抗 IAV 因子8，10，皮质肌动蛋白在感染后期干扰 IAV 复制，可能在病毒组装和出芽期间干扰IAV 复制 11。
此外，各种半胱天冬酶也被激活，反过来，它们又切割多种蛋白质以激活IAV感染期间的宿主炎症反应1，2。此外，核蛋白（NP）、IAV 12，13，14的离子通道M2蛋白和其他病毒3，15，16的各种蛋白质在感染过程中也经历半胱天冬酶介导的切割，这会影响病毒发病机制。因此，需要持续研究半胱天冬酶介导的IAV和其他病毒感染过程中宿主和病毒蛋白的切割或降解，以了解病毒发病机制的分子基础。本文介绍了以下方法：（1）评估半胱天冬酶对这些蛋白质的切割或降解，（2）鉴定这些半胱天冬酶，以及（3）定位切割位点。
Protocol
Representative Results
Discussion
已经确定，病毒会根据宿主因素和途径进行调整。反过来，宿主细胞通过采用各种策略来抵抗它。其中一种策略是PANoptosis，宿主细胞将其用作对抗病毒感染的抗病毒策略。然而，像IAV这样的病毒已经进化出自己的策略来对抗PANoptosis并利用它来发挥自己的优势1，3，6。这种相互作用涉及半胱天冬酶切割各种宿主和病毒蛋白。鉴?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者感谢Jennifer Tipper，Bilan Li，Jesse vanWestrienen，Kevin Harrod，Da-Yuan Chen，Farjana Ahmed，Sonya Mros，Kenneth Yamada，Richard Webby，BEI Resources（NIAID），新西兰健康研究委员会，Maurice和Phyllis Paykel信托（新西兰），H.S.和J.C.安德森信托（但尼丁），微生物学和免疫学系以及生物医学科学学院（奥塔哥大学）。
Materials
| A549 cells | ATCC | CRM-CCL-185 | Human, epithelial, lung |
| Ammonium chloride | Sigma-Aldrich | A9434 | |
| Caspase 3 Inhibitor | Sigma-Aldrich | 264156-M | Also known as 'InSolution Caspase-3 Inhibitor II – Calbiochem' |
| cOmplete, Mini Protease Inhibitor Cocktail | Roche | 11836153001 | |
| Goat anti-NP antibody | Gift from Richard Webby (St Jude Children’s Research Hospital, Memphis, USA) to MH | ||
| Lipofectamine 2000 Transfection Reagent | ThermoFisher Scientific | 31985062 | |
| Lipofectamine RNAiMAX Transfection Reagent | ThermoFisher Scientific | 13778150 | |
| MDCK cells | ATCC | CCL-34 | Dog, epithelial, kidney |
| MG132 | Sigma-Aldrich | M7449 | |
| Minimum Essential Medium (MEM) | ThermoFisher Scientific | 11095080 | Add L-glutamine, antibiotics or other supplements as required |
| MISSION siRNA Universal Negative Control #1 | Sigma-Aldrich | SIC001 | |
| Odyssey Fc imager with Image Studio Lite software 5.2 | LI-COR | Odyssey Fc has been replaced with Odyssey XF and Image Studio Lite software has been replaced with Empiria Studio software. | |
| Pierce BCA Protein Assay Kit | ThermoFisher Scientific | 23225 | |
| Plasmid expressing human cortactin-GFP fusion | Addgene | 50728 | Gift from Kenneth Yamada to Addgene |
| Pre-designed small interferring RNA (siRNA) to caspase 3 | Sigma-Aldrich | NM_004346 | siRNA ID: SASI_Hs01_00139105 |
| Pre-designed small interferring RNA to caspase 6 | Sigma-Aldrich | NM_001226 | siRNA ID: SASI_Hs01_00019062 |
| Pre-designed small interferring RNA to caspase 7 | Sigma-Aldrich | NM_001227 | siRNA ID: SASI_Hs01_00128361 |
| Pre-designed SYBR Green RT-qPCR Primer pairs | Sigma-Aldrich | KSPQ12012 | Primer Pair IDs: H_CASP3_1; H_CASP6_1; H_CASP7_1 |
| Protran Premium nitrocellulose membrane | Cytiva (Fomerly GE Healthcare) | 10600003 | |
| Rabbit anti-actin antibody | Abcam | ab8227 | |
| Rabbit anti-cortactin antibody | Cell Signaling | 3502 | |
| Rabbit anti-GFP antibody | Takara | 632592 | |
| SeeBlue Pre-stained Protein Standard | ThermoFisher Scientific | LC5625 | |
| Transfection medium, Opti-MEM | ThermoFisher Scientific | 11668019 | |
| Tris-HCl, NaCl, SDS, Sodium Deoxycholate, Triton X-100 | Merck | ||
| Trypsin, TPCK-Treated | Sigma-Aldrich | 4370285 |
References
- Place, D. E., Lee, S., Kanneganti, T. -. D. PANoptosis in microbial infection. Current Opinion in Microbiology. 59, 42-49 (2021).
- Zheng, M., Kanneganti, T. -. D. The regulation of the ZBP1-NLRP3 inflammasome and its implications in pyroptosis, apoptosis, and necroptosis (PANoptosis). Immunological Reviews. 297 (1), 26-38 (2020).
- Connolly, P. F., Fearnhead, H. O. Viral hijacking of host caspases: An emerging category of pathogen-host interactions. Cell Death & Differentiation. 24 (8), 1401-1410 (2017).
- Julien, O., Wells, J. A. Caspases and their substrates. Cell Death & Differentiation. 24 (8), 1380-1389 (2017).
- Balachandran, S., Rall, G. F., Gack, M. U. Benefits and perils of necroptosis in influenza virus infection. Journal of Virology. 94 (9), 01101-01119 (2020).
- Ampomah, P. B., Lim, L. H. K. Influenza A virus-induced apoptosis and virus propagation. Apoptosis. 25 (1-2), 1-11 (2020).
- Chen, D. Y., Husain, M. Caspase-mediated degradation of host cortactin that promotes influenza A virus infection in epithelial cells. Virology. 497, 146-156 (2016).
- Galvin, H. D., Husain, M. Influenza A virus-induced host caspase and viral PA-X antagonize the antiviral host factor, histone deacetylase 4. Journal of Biological Chemistry. 294 (52), 20207-20221 (2019).
- Husain, M., Harrod, K. S. Influenza A virus-induced caspase-3 cleaves the histone deacetylase 6 in infected epithelial cells. FEBS Letters. 583 (15), 2517-2520 (2009).
- Husain, M., Cheung, C. Y. Histone deacetylase 6 inhibits influenza A virus release by downregulating the trafficking of viral components to the plasma membrane via its substrate, acetylated microtubules. Journal of Virology. 88 (19), 11229-11239 (2014).
- Chen, D. Y., Husain, M. Caspase-mediated cleavage of human cortactin during influenza A virus infection occurs in its actin-binding domains and is associated with released virus titres. Viruses. 12 (1), 87 (2020).
- Zhirnov, O. P., Syrtzev, V. V. Influenza virus pathogenicity is determined by caspase cleavage motifs located in the viral proteins. Journal of Molecular and Genetic Medicine. 3 (1), 124-132 (2009).
- Zhirnov, O. P., Klenk, H. -. D. Alterations in caspase cleavage motifs of NP and M2 proteins attenuate virulence of a highly pathogenic avian influenza virus. Virology. 394 (1), 57-63 (2009).
- Zhirnov, O. P., Konakova, T. E., Garten, W., Klenk, H. Caspase-dependent N-terminal cleavage of influenza virus nucleocapsid protein in infected cells. Journal of Virology. 73 (12), 10158-10163 (1999).
- Robinson, B. A., Van Winkle, J. A., McCune, B. T., Peters, A. M., Nice, T. J. Caspase-mediated cleavage of murine norovirus NS1/2 potentiates apoptosis and is required for persistent infection of intestinal epithelial cells. PLOS Pathogens. 15 (7), 1007940 (2019).
- Richard, A., Tulasne, D. Caspase cleavage of viral proteins, another way for viruses to make the best of apoptosis. Cell Death & Disease. 3 (3), 277 (2012).
- Brauer, R., Chen, P. Influenza virus propagation in embryonated chicken eggs. Journal of Visualized Experiments. (97), e52421 (2015).
- Lüthi, A. U., Martin, S. J. The CASBAH: A searchable database of caspase substrates. Cell Death & Differentiation. 14 (4), 641-650 (2007).
- Kumar, S., van Raam, B. J., Salvesen, G. S., Cieplak, P. Caspase cleavage sites in the human proteome: CaspDB, a database of predicted substrates. PLoS One. 9 (10), 110539 (2014).
- Igarashi, Y., et al. CutDB: A proteolytic event database. Nucleic Acids Research. 35 (Database issue). 35, 546-549 (2007).
- Crawford, E. D., et al. The DegraBase: A database of proteolysis in healthy and apoptotic human cells. Molecular & Cellular Proteomics. 12 (3), 813-824 (2013).
- Rawlings, N. D., Tolle, D. P., Barrett, A. J. MEROPS: The peptidase database. Nucleic Acids Research. 32, 160-164 (2004).
- Lange, P. F., Overall, C. M. TopFIND, a knowledgebase linking protein termini with function. Nature Methods. 8 (9), 703-704 (2011).
- Fortelny, N., Yang, S., Pavlidis, P., Lange, P. F., Overall, C. M. Proteome TopFIND 3.0 with TopFINDer and PathFINDer: Database and analysis tools for the association of protein termini to pre- and post-translational events. Nucleic Acids Research. 43, 290-297 (2015).
