Name: confocal imaging of double-stranded rna and pattern recognition receptors in negative-sense rna virus infection
Uploaded: 2019-01-26T15:00:00
Description: Watch this Scientific Journal Video about Confocal Imaging of Double-Stranded RNA and Pattern Recognition Receptors in Negative-Sense RNA Virus Infection at JoVE.com

我们要感谢 utmb 成像核心设施和 maxim ivnikov 的显微镜协助。
这项工作得到了公共卫生服务机构向 sp、给方案的 utmb 承诺基金 p84373 和 t32 ai007526 至 em 的 RO1AI093445 和 ro1aia129198 赠款的支持。

1. a549 细胞的制备和 junv 感染
<ol><li>种子 2 x 10 5 人肺上皮 a549 细胞到多 d-赖氨酸 (pdl) 涂层玻璃覆盖物在12孔板在感染前24小时。</li>
	<li>在 dulbecco 的修饰鹰培养基 (dmem) 培养基中稀释的每细胞1.0 斑块形成单位的感染 (moi) 中, 以2% 的胎牛血清 (fbs) 和1% 的青霉素和链霉素 (p/s) 为补充, 制备150毫升的 junm13 等价物).</li>
	<li>从细胞培养中删除介质。在每口含有盖板的井上加入病...

<ol>
	<li>Jensen, S., Thomsen, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensing+of+RNA+viruses:+a+review+of+innate+immune+receptors+involved+in+recognizing+RNA+virus+invasion.">Sensing of RNA viruses: a review of innate immune receptors involved in recognizing RNA virus invasion.</a> Journal of Virology. 86, 2900-2910 (2012).</li><li>Weber, F., Wagner, V., Rasmussen, S. B., Hartmann, R., Paludan, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double-stranded+RNA+is+produced+by+positive-strand+RNA+viruses+and+DNA+viruses+but+not+in+detectable+amounts+by+negative-strand+RNA+viruses.">Double-stranded RNA is produced by positive-strand RNA viruses and DNA viruses but not in detectable amounts by negative-strand RNA viruses.</a> Journal of Virology. 80, 6 (2006).</li><li>Triantafilou, K., Vakakis, E., Kar, S., Richer, E., Evans, G. L., Triantafilou, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualisation+of+direct+interaction+of+MDA5+and+the+dsRNA+replicative+intermediate+form+of+positive+strand+RNA+viruses.">Visualisation of direct interaction of MDA5 and the dsRNA replicative intermediate form of positive strand RNA viruses.</a> Journal of Cell Science. 125, 4761-4769 (2012).</li><li>Onomoto, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+role+of+an+antiviral+stress+granule+containing+RIG-I+and+PKR+in+viral+detection+and+innate+immunity.">Critical role of an antiviral stress granule containing RIG-I and PKR in viral detection and innate immunity.</a> PLoS One. 7, e43031 (2012).</li><li>Son, K. N., Liang, Z., Lipton, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double-stranded+RNA+is+detected+by+immunofluorescence+analysis+in+RNA+and+DNA+virus+infections,+including+those+by+negative-sense+RNA+viruses.">Double-stranded RNA is detected by immunofluorescence analysis in RNA and DNA virus infections, including those by negative-sense RNA viruses.</a> Journal of Virology. 89, 9383-9392 (2015).</li><li>Mateer, E. J., Paessler, S., Huang, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+double-stranded+RNA+colocalizing+with+pattern+recognition+receptors+in+arenavirus+infected+cells.">Visualization of double-stranded RNA colocalizing with pattern recognition receptors in arenavirus infected cells.</a> Frontiers Cellular and Infection Microbiology. 8, 251 (2018).</li><li>Buchmeier, M. J., de la Torre, J. C., Peters, C. J., Knipe, D. M., Howley, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arenavirdae:+The+viruses+and+their+replication.">Arenavirdae: The viruses and their replication.</a> Fields Virology. 2, (2006).</li><li>Levis, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endogenous+interferon+in+Argentine+hemorrhagic+fever.">Endogenous interferon in Argentine hemorrhagic fever.</a> The Journal of Infectious Diseases. 149, 428-433 (1984).</li><li>Levis, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+between+endogenous+interferon+and+the+clinical+evolution+of+patients+with+Argentine+hemorrhagic+fever.">Correlation between endogenous interferon and the clinical evolution of patients with Argentine hemorrhagic fever.</a> Journal of Interferon Research. 5, 383-389 (1985).</li><li>Huang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+pathogenic+new+world+and+old+world+human+arenaviruses+induce+distinct+interferon+response+in+human+cells.">Highly pathogenic new world and old world human arenaviruses induce distinct interferon response in human cells.</a> Journal of Virology. 89, 7079-7088 (2015).</li><li>Huang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Junin+virus+infection+activates+the+type+I+interferon+pathway+in+a+RIG-I-dependent+manner.">Junin virus infection activates the type I interferon pathway in a RIG-I-dependent manner.</a> PLoS Neglected Tropical Diseases. 6, e1659 (2012).</li><li>Huang, C., Kolokoltsova, O. A., Mateer, E. J., Koma, T., Paessler, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+pathogenic+new+world+arenavirus+infection+activates+the+pattern+recognition+receptor+protein+kinase+R+without+attenuating+virus+replication+in+human+cells.">Highly pathogenic new world arenavirus infection activates the pattern recognition receptor protein kinase R without attenuating virus replication in human cells.</a> Journal of Virology. 91, 20 (2017).</li><li>Emonet, S. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rescue+from+cloned+cDNAs+and+in+vivo+characterization+of+recombinant+pathogenic+Romero+and+live-attenuated+Candid#1+strains+of+Junin+virus,+the+causative+agent+of+Argentine+hemorrhagic+fever+disease.">Rescue from cloned cDNAs and in vivo characterization of recombinant pathogenic Romero and live-attenuated Candid#1 strains of Junin virus, the causative agent of Argentine hemorrhagic fever disease.</a> Journal of Virology. 85 (4), 1473-1483 (2011).</li><li>Child, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antagonism+of+the+protein+kinase+R+pathway+in+human+cells+by+Rhesus+Cytomegalovirus.">Antagonism of the protein kinase R pathway in human cells by Rhesus Cytomegalovirus.</a> Journal of Virology. 92, 6 (2018).</li><li>Hobro, A. J., Smith, N. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+evaluation+of+fixation+methods:+Spatial+and+compositional+cellular+changes+observed+by+Raman+imaging.">An evaluation of fixation methods: Spatial and compositional cellular changes observed by Raman imaging.</a> Vibrational Spectroscopy. 91, 31-45 (2017).</li></ol>

对于正感 rna 病毒和 dsdna 病毒, dsrna 很容易被广泛使用的 j2 抗 dsrna 抗体检测。然而, 负感 rna 病毒被认为在低或低于检测水平产生 dsrna 使用相同的抗体2。因此, 病毒 rna 和 prr 相互作用的许多方面在很大程度上是不清楚的负感 rna 病毒。我们试图使用 j2 抗体染色牙病毒感染中的 dsrna, 但与模拟感染相比, 荧光信号是不可微的。最初用于泛肠病毒检测的 mab 9d5 被发现是特定于 dsrna 的,...

诱导先天免疫反应的第一步是通过模式识别受体 (prr) (如维甲酸 (rigi) 样受体 (rlr) r-i 和黑色素瘤差异相关的双链 rna 的宿主识别蛋白 5 (mda-5)1。对于正感 rna 病毒, 通常可以使用 j2 或 k1 抗 dsrna 单克隆抗体 (mab)2很容易检测到 dsrna。利用共聚焦显微镜3对阳性链 rna 病毒 (如 picornas-病毒) 中 dsrna 和 prr 之间的相互作用进行了表征。然而, 对于负感 rna 病毒, prr 和 dsrna 相互作用的可视化和表征由于缺乏对 dsrna 的敏感抗体而受到阻碍。rna 荧光原位杂交 (fish) 已被应用于病毒 rna 和 prrs4 的可视化。然而, fish 方法要求了解目标 rna 序列, 并且可能与 prr 共染色不兼容。最近, 9d5 mab, 最初是为诊断泛肠病毒感染而开发的, 被发现比 j2 mab 更敏感, 并且可以很容易地检测到 dsrna 在负感 rna 病毒感染5,6。因此, mab 9d5 是研究负感 rna 病毒的病毒复制以及 prr 与病毒 rna 相互作用的一种新的有用工具。
竞技场病毒是单链、负感 rna 病毒的家族, 包括几种人类病原体, 如拉萨病毒 (lasv)、junín 病毒 (junv) 和 machupo 病毒 (macv), 这些病毒在人类中引起严重的出血热疾病7。新世界腺病毒引起的阿根廷严重和致命出血热病例的临床数据显示出异常高的血清 ifn-α08,9。我们已经证明, 致病性的 nw 芳烃 (junv 和 macv), 而不是致病性的旧大陆腺病毒, lasv, 诱导 i 型干扰素 (ifn) 反应在人类单核细胞衍生树突状细胞10。此外, rigi 是在 junv 感染细胞11中介导 i 型 ifn 响应的传感器之一。我们还发现, 蛋白激酶 r (pkr) 受体, 这是传统上已知的 dsrna 识别, 被激活的致病性 nw 腺病毒感染12。为了进一步了解腺病毒感染过程中病毒特异性 ifn 反应的机制, 我们的目标是开发一个协议, 以可视化病毒 dsrna 与细胞质 prr 之间的相互作用。
对致病性 junv、macv 和 lasv 的感染实验必须在生物安全第4级 (bsl-4) 设施中进行。因此, 除了在芳烃病毒感染中形成的 dsrna 水平可能较低之外, 在对这些高致病性病毒进行成像研究时, 满足生物安全要求是另一个技术挑战。本报告利用 junv 的9d5 抗体和 candid1# 疫苗株, 描述了一种基于共聚焦显微镜的协议, 该协议已成功地用于在感染芳烃病毒的细胞中同时显示 dsrna、病毒蛋白和 prr。bsl2 实验室。该方案也适用于 bsl4 设施致病性腺病毒感染过程中 dsrna 和 prr 的细胞内分布的可视化。

<table>
	<tbody>
		<tr>
			<td>APEX Alexa Fluor 647 antibody labeling kit</td>
			<td>Invitrogen</td>
			<td>A10475</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma Aldrich</td>
			<td>A4503</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Cell Signaling</td>
			<td>4083</td>
			<td>1:1,000 dilution</td>
		</tr>
		<tr>
			<td>Donkey-anti rabbit Alexa Fluor 594</td>
			<td>Invitrogen</td>
			<td>A-11058</td>
			<td>1:2,000 dilution; Lot #: 1454437</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified Eagle&#39;s medium</td>
			<td>Corning</td>
			<td>10-013-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Atlanta Bio</td>
			<td>S11150</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass microscope slides</td>
			<td>Fisher</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat-anti mouse Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>A-11029</td>
			<td>1:2000 dilution; Lot #: 1874804</td>
		</tr>
		<tr>
			<td>Human lung epithelial A549 cells</td>
			<td>ATCC</td>
			<td>CCL-185</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher</td>
			<td>A412</td>
			<td>Stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Mouse MAb anti-JUNV NP</td>
			<td>BEI</td>
			<td>NA05-AG12</td>
			<td>Conjugated to Alexa-647 at 1:1,000 dilution</td>
		</tr>
		<tr>
			<td>Mouse MAb pan-Enterovirus 9D5 Reagent</td>
			<td>Millipore Sigma</td>
			<td>3361</td>
			<td>ready for use; diluted to 1:2; Lot #: 3067445</td>
		</tr>
		<tr>
			<td>PBS supplemented with Ca and Mg</td>
			<td>Corning</td>
			<td>21-030-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>PDL Coated coverslips</td>
			<td>Neuvitro</td>
			<td>H-12-1.5-pdl</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold antifade</td>
			<td>Invitrogen</td>
			<td>P10144</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit MAb MDA-5</td>
			<td>Abcam</td>
			<td>ab126630</td>
			<td>1:250 dilution; Lot #: GR97758-7</td>
		</tr>
		<tr>
			<td>recombiant Candid#1 strain of JUNV</td>
			<td>Lab generated</td>
			<td>Lab generated</td>
			<td>As previously described in reference 13.</td>
		</tr>
		<tr>
			<td>RIG-I mouse MAb conjugated to Alexa-594</td>
			<td>Santa Cruz</td>
			<td>sc-376845</td>
			<td>1:1000 dilution; Lot #: AO218</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma Aldrich</td>
			<td>T8787&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

confocal imaging of double-stranded rna and pattern recognition receptors in negative-sense rna virus infection

双链 rna 是在 rna 病毒感染过程中作为复制中间体产生的。通过宿主模式识别受体 (prr) (如维甲酸 (MDA-5) 样受体 (rlr) 和黑色素瘤差异相关蛋白 5 (mda-5)) 识别 dsrna, 从而诱导先天免疫反应。在正感 rna 病毒感染中, dsrna 的形成和细胞内分布已通过显微镜观察。许多负面的 rna 病毒, 包括一些芳烃病毒, 在感染过程中触发先天免疫反应。然而, 负感 rna 病毒被认为产生低水平的 dsrna, 这阻碍了对病毒 dsrna 的 prr 识别的成像研究。此外, 高致病性芳烃的感染实验必须在高遏制生物安全设施 (bsl-4) 中进行。病毒 rna 和高致病性 rna 病毒的 prr 之间的相互作用在很大程度上是未知的, 因为研究人员需要在 bsl-4 设施中面临额外的技术挑战。最近, 一种最初用于泛肠病毒检测的单克隆抗体 (mab) (克隆 9d5) 被发现能够以比传统的 j2 或 k1 抗 dsrna 抗体更高的灵敏度专门检测 dsrna。在此, 通过使用9d5 抗体, 我们描述了一种共聚焦显微镜协议, 该协议已成功地用于在感染芳烃病毒的单个细胞中同时显示 dsrna、病毒蛋白和 prr。该方案也适用于 bsl4 设施中致病性腺病毒感染细胞的 dsrna 和 prr 分布的成像研究。

该方案用于研究 rlr (rigi 和 mda-5) 与 dsrna 在 junv 感染细胞中的分布和共化。如图 1和图 2所示, 随着病毒感染的进展, dsrna 的积累会随着时间的推移而增加。浓度 mda-5 (图 1) 和 rigi (图 2) 信号与 np 和 dsrna 的点状结构同相化。
<img alt="Figure 1" c...

Watch this Scientific Journal Video about Confocal Imaging of Double-Stranded RNA and Pattern Recognition Receptors in Negative-Sense RNA Virus Infection at JoVE.com

Confocal Imaging of Double-Stranded RNA and Pattern Recognition Receptors in Negative-Sense RNA Virus Infection

在 rna 病毒复制过程中产生的双链 rna 可以通过模式识别受体来识别, 从而诱导先天免疫反应。对于负感 rna 病毒, 低水平 dsrna 和 prr 之间的相互作用仍不清楚。我们开发了一种共聚焦显微镜方法, 用于在单个细胞中显示腺病毒 dsrna 和 prr。

微链 rna 的共聚焦成像和模式识别受体在负感 rna 病毒感染中的应用

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors ...

传统的双链RNA检测检测通常不够灵敏，无法检测在负感RNA病毒感染中形成的双链RNA。因此，双链RNA和宿主模式识别受体之间的相互作用对于这些病毒来说基本上仍然难以捉摸。利用该协议，我们能够可视化和描述病毒核蛋白、双链RNA和在单细胞水平上宿主的PRR对负感RNA病毒的相互作用。这是一种多路复用检测，允许在单个细胞中同时染色双链RNA和两个病毒或宿主蛋白靶点。与RNA-FISH测定不同，该技术与RNA序列无关，通常与蛋白质靶点抗体检测相容。在感染前24小时开始，将20万个人类肺上皮 A549细胞播种到12孔板中的聚-D-Lysine涂层玻璃盖上，并在37孔生长

Research

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Immunology and Infection

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RNA干扰在蜱

Ticks are obligate hematophagous ectoparasites of wild and domestic animals and humans, and are considered to be second worldwide to mosquitoes as vectors ...

科研

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Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency

富达的RNA病毒和病毒的突变频率的表征的变种的分离

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the ...

Development of Cell-type specific anti-HIV gp120 aptamers for siRNA delivery

发展的细胞类型特异性的抗艾滋病毒gp120基因的siRNA传递适配

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering ...

Separation of Single-stranded DNA, Double-stranded DNA and RNA from an Environmental Viral Community Using Hydroxyapatite Chromatography

分离单链DNA，双链DNA和RNA病毒使用羟基磷灰石色谱从环境社区

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2.  Viruses modulate host cell abundance and diversity, ...

Monitoring Activation of the Antiviral Pattern Recognition Receptors RIG-I And PKR By Limited Protease Digestion and Native PAGE

在抗病毒模式识别受体RIG-I和PKR通过有限蛋白酶消化和Native PAGE监测激活

Host defenses to virus infection are dependent on a rapid detection by pattern recognition receptors (PRRs) of the innate immune system. In the cytoplasm, ...

Nucleocapsid Annealing-Mediated Electrophoresis (NAME) Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

Nucleocapsid Annealing-Mediated Electrophoresis NAME Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

核衣壳退火介导电泳（NAME）分析允许HIV-1核衣壳抑制剂的快速鉴定

RNA or DNA folded in stable tridimensional folding are interesting targets in the development of antitumor or antiviral drugs. In the case of HIV-1, viral ...

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 ...

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

该<em&gt;离体</em&gt;文化与模式识别小鼠肠道组织体受体刺激

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the ...

Combining Analysis of DNA in a Crude Virion Extraction with the Analysis of RNA from Infected Leaves to Discover New Virus Genomes

粗病毒提取中 DNA 的组合分析及对感染叶 RNA 的分析发现新的病毒基因组

This metagenome approach is used to identify plant viruses with circular DNA genomes and their transcripts. Often plant DNA viruses that occur in low ...

Imaging Cell Interaction in Tracheal Mucosa During Influenza Virus Infection Using Two-photon Intravital Microscopy

双光子活体显微镜感病毒感染时气管黏膜的影像细胞相互作用

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. ...