Name: identifying caspases and their motifs that cleave proteins during influenza a virus infection
Uploaded: 2022-07-21T15:00:00
Description: Watch this Scientific Journal Video about Identifying Caspases and their Motifs that Cleave Proteins During Influenza A Virus Infection at JoVE.com

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）。

奥塔哥大学机构生物安全委员会获得了监管部门的批准，与IAV和哺乳动物细胞合作。本研究使用Madin-Darby犬肾（MDCK）或人肺泡上皮A549细胞和IAV H1N1亚型。IAV是在鸡蛋中生长的，如其他地方所述17。无菌和无菌条件用于处理哺乳动物细胞，生物安全2级（或物理遏制2）设施和II类生物安全柜用于处理IAV亚型。
1. 评估半胱天冬酶对IAV感染细胞中蛋白质的切割?...

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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?...

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。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>A549 cells</td>
			<td>ATCC</td>
			<td>CRM-CCL-185</td>
			<td>Human, epithelial, lung</td>
		</tr>
		<tr>
			<td>Ammonium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>A9434</td>
			<td></td>
		</tr>
		<tr>
			<td>Caspase 3 Inhibitor</td>
			<td>Sigma-Aldrich</td>
			<td>264156-M</td>
			<td>Also known as &#39;InSolution Caspase-3 Inhibitor II - Calbiochem&#39;</td>
		</tr>
		<tr>
			<td>cOmplete, Mini Protease Inhibitor Cocktail</td>
			<td>Roche</td>
			<td>11836153001</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-NP antibody</td>
			<td></td>
			<td></td>
			<td>Gift from Richard Webby (St Jude Children&#8217;s Research Hospital, Memphis, USA) to MH</td>
		</tr>
		<tr>
			<td>Lipofectamine 2000 Transfection Reagent</td>
			<td>ThermoFisher Scientific</td>
			<td>31985062</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine RNAiMAX Transfection Reagent</td>
			<td>ThermoFisher Scientific</td>
			<td>13778150</td>
			<td></td>
		</tr>
		<tr>
			<td>MDCK cells</td>
			<td>ATCC</td>
			<td>CCL-34</td>
			<td>Dog, epithelial, kidney</td>
		</tr>
		<tr>
			<td>MG132</td>
			<td>Sigma-Aldrich</td>
			<td>M7449</td>
			<td></td>
		</tr>
		<tr>
			<td>Minimum Essential Medium (MEM)</td>
			<td>ThermoFisher Scientific</td>
			<td>11095080</td>
			<td>Add L-glutamine, antibiotics or other supplements as required</td>
		</tr>
		<tr>
			<td>MISSION siRNA Universal Negative Control #1</td>
			<td>Sigma-Aldrich</td>
			<td>SIC001</td>
			<td></td>
		</tr>
		<tr>
			<td>Odyssey Fc imager with Image Studio Lite software 5.2&#160;</td>
			<td>LI-COR</td>
			<td></td>
			<td>Odyssey Fc has been replaced with Odyssey XF and Image Studio Lite software has been replaced with Empiria Studio software.</td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay Kit</td>
			<td>ThermoFisher Scientific</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmid expressing human cortactin-GFP fusion&#160;</td>
			<td>Addgene</td>
			<td>50728</td>
			<td>Gift from Kenneth Yamada to Addgene</td>
		</tr>
		<tr>
			<td>Pre-designed small interferring RNA (siRNA) to caspase 3</td>
			<td>Sigma-Aldrich</td>
			<td>NM_004346</td>
			<td>siRNA ID: SASI_Hs01_00139105</td>
		</tr>
		<tr>
			<td>Pre-designed small interferring RNA to caspase 6</td>
			<td>Sigma-Aldrich</td>
			<td>NM_001226</td>
			<td>siRNA ID: SASI_Hs01_00019062</td>
		</tr>
		<tr>
			<td>Pre-designed small interferring RNA to caspase 7</td>
			<td>Sigma-Aldrich</td>
			<td>NM_001227</td>
			<td>siRNA ID: SASI_Hs01_00128361</td>
		</tr>
		<tr>
			<td>Pre-designed SYBR Green RT-qPCR Primer pairs</td>
			<td>Sigma-Aldrich</td>
			<td>KSPQ12012</td>
			<td>Primer Pair IDs: H_CASP3_1; H_CASP6_1; H_CASP7_1</td>
		</tr>
		<tr>
			<td>Protran Premium nitrocellulose membrane</td>
			<td>Cytiva (Fomerly GE Healthcare)</td>
			<td>10600003</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-actin antibody</td>
			<td>Abcam</td>
			<td>ab8227</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-cortactin antibody</td>
			<td>Cell Signaling</td>
			<td>3502</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-GFP antibody</td>
			<td>Takara</td>
			<td>632592</td>
			<td></td>
		</tr>
		<tr>
			<td>SeeBlue Pre-stained Protein Standard</td>
			<td>ThermoFisher Scientific</td>
			<td>LC5625</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfection medium, Opti-MEM</td>
			<td>ThermoFisher Scientific</td>
			<td>11668019</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl, NaCl, SDS, Sodium Deoxycholate, Triton X-100</td>
			<td>Merck</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin, TPCK-Treated</td>
			<td>Sigma-Aldrich</td>
			<td>4370285</td>
			<td></td>
		</tr>
	</tbody>
</table>

identifying caspases and their motifs that cleave proteins during influenza a virus infection

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。

用半胱天冬酶3抑制剂治疗 已经发现宿主皮质素，HDAC4和HDAC6多肽在犬（MDCK）和人（A549，NHBE）细胞中响应IAV感染而降解7，8，9。通过使用上述方法，发现IAV诱导的宿主半胱天冬酶，特别是半胱天冬酶3，导致其降解7，8，9。皮质肌动蛋白以感?...

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Identifying Caspases and their Motifs that Cleave Proteins During Influenza A Virus Infection

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。

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

Caspases, a family of cysteine proteases, orchestrate programmed cell death in response to various stimuli, including microbial infections. Initially ...

该方法将有助于确定和确认流感病毒感染细胞中的蛋白质是否经历半胱天冬酶的降解或切割，并鉴定半胱天冬酶切割位点。这种方法只需要基本的分子和细胞生物学实验室设置和研究技能，不需要任何专门的设备或软件培训。这种方法可以适应于实现类似的目标，包括感染其他动物病毒或微生物，以及暴露于外部刺激，如毒素和化学物质。首先，将MDCK或A549细胞接种在12孔细胞培养板中。将细胞在37摄氏度下在5%二氧化碳气氛下孵育过夜。第二天，从细胞中取出旧培养基，并用一毫升无血清MEM洗涤每个孔中的细胞两次。然后，通过加入400微升甲型流感病毒接种物来感染细胞，并将板放置孵育一小时。孵育后，取出病毒接种物并用MEM洗涤细胞。向每个孔中加入一毫升无血清MEM，并如上一步所示孵育细胞。24小时后，用一毫升注射器柱塞刮取细胞，并将其转移到1.5毫升管中。将试管在室温下以12， 000G离心两分钟，收集上清液。用250微升磷酸盐缓冲盐水和离心机洗涤细胞沉淀。除去上清液并加入100微升细胞裂解缓冲液。涡旋混合。将试管在 98 摄氏度下加热 10 分钟以完全裂解细胞。然后，通过标准SDS聚丙烯酰胺凝胶电泳分离未感染和感染样品中等量的蛋白质。电泳后，将蛋白质凝胶转移到硝酸纤维素或PVDF膜上进行蛋白质印迹。比较模拟处理和抑制剂处理的感染样品泳道中的蛋白质水平。在100微升适当的培养基（如Opti-MEM）中，稀释100纳摩尔的对照或半胱天冬酶siRNA，或适当体积的转染试剂。在室温下孵育五分钟。将 100 微升每种 siRNA 溶液与 100 微升转染试剂溶液混合，并在室温下孵育 20 至 45 分钟。在800微升的完整生长培养基中分裂并加入100，000 MDCK或A549细胞。加入 200 μL siRNA 转染试剂复合物，并将该悬浮液加入 12 孔培养板中。将细胞在37摄氏度的5%二氧化碳气氛下孵育72小时。用甲型流感病毒感染细胞，但没有抑制剂。进行蛋白质印迹，并比较如前所示的蛋白质水平。在多肽上找到半胱天冬酶切割基序XXXD和DXXD。通过定点诱变使 P1 天冬氨酸、两种谷氨酸或丙氨酸发生突变，并通过 DNA 测序进行确认。在100微升适当的培养基中，稀释两微克野生型或突变型血浆DNA或推荐体积的转染试剂，并在室温下孵育试管5分钟。将 100 微升血浆 DNA 溶液与 100 微升转染试剂溶液混合，并在室温下孵育 20 至 45 分钟。同时，分裂并将300，000个MDCK或A549细胞加入到800微升完全生长培养基中。加入200微升DNA转染试剂复合物，并将该悬浮液加入12孔培养板中。将板在37%二氧化碳气氛下在5%的气氛下孵育。48小时后，用不含抑制剂的甲型流感病毒感染细胞。进行蛋白质印迹，并比较如前所示的蛋白质水平。使用蛋白质印迹法，评估流感诱导的宿主半胱天冬酶对宿主皮质肌动蛋白的降解。用半胱天冬酶-3抑制剂治疗流感病毒感染细胞导致皮质肌动蛋白的挽救和病毒NP降解。皮质肌动蛋白条带强度的定量及其与相应肌动蛋白带的标准化表明，与未处理的感染细胞相比，半胱天冬酶-3抑制剂处理的感染细胞中的皮质肌动蛋白水平更高。在半胱天冬酶-6或半胱天冬酶-7耗尽的细胞中。没有观察到皮质肌动蛋白多肽水平的显着恢复，而在半胱天冬酶-3耗尽的细胞中，从流感病毒中恢复的皮质肌动蛋白诱导降解。多肽水平的定量和比较表明，半胱天冬酶-3表达耗尽的感染细胞中的皮质肌动蛋白多肽水平高于半胱天冬酶-3表达正常的细胞。位置116处的天冬氨酸突变为谷氨酸，使皮质肌动蛋白多肽对流感病毒诱导的降解具有抗性。多肽水平的定量和比较表明，感染细胞中突变型皮质肌动蛋白多肽的水平高于野生型皮质肌动蛋白多肽。需要产生数十个突变体并进行重复蛋白质印迹以鉴定多肽中的半胱天冬酶切割位点。可以开发一种含有纯化的半胱天冬酶和靶多肽的体外生化测定，以确认感兴趣的蛋白质是直接的基于气体的底物。该协议将有助于了解正在研究的系统中靶蛋白的重要性，降解或切割，例如病毒感染或疾病。

Research

JoVE Journal

Immunology and Infection

Generation of Recombinant Influenza Virus from Plasmid DNA

质粒DNA重组流感病毒的一代

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally ...

科研

免疫与感染

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

利用生物发光成像之间调查的协同作用肺炎链球菌 A型流感病毒的婴儿小鼠

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

High-throughput Detection Method for Influenza Virus

流感病毒的高通量检测方法

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

Peptide-based Identification of Functional Motifs and their Binding Partners

肽为基础的识别功能图案和具有约束力的合作伙伴

Specific short  peptides derived from motifs found in full-length proteins, in our case HIV-1  Nef, not only retain their biological function, but can ...

Influenza Virus Propagation in Embryonated Chicken Eggs

在鸡胚的流感病毒传播

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous ...

Co-immunoprecipitation of the Mouse Mx1 Protein with the Influenza A Virus Nucleoprotein

免疫共沉淀鼠标MX1蛋白与流感病毒核蛋白

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions ...

Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

鼻湿巾从猪流感病毒检测和​​隔离

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

中性粒细胞分离和分析，以确定他们的淋巴瘤细胞的敏感性对治疗药物的作用

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

在甲型流感病毒的衣壳的体外拆卸梯度离心

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus ...

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

荧光为基础的神经氨酸酶抑制测定流感病毒的易感性评估抗病毒药的神经氨酸酶抑制剂类

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...