Name: using click chemistry to measure the effect of viral infection on host-cell rna synthesis
Uploaded: 2013-08-09T13:00:00
Description: Watch this Scientific Journal Video about Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis Video at JoVE.com

我们感谢RB TESH的提供鼠标RVFV多克隆抗血清和M.格里芬UTMB流式细胞仪核心基金流式细胞仪的帮助。 5 U54 AI057156支持通过这项工作是支持的詹姆斯·麦克劳克林奖学金基金在UTMB.OL被支持西部区域的卓越中心，美国国立卫生研究院授予R01 AI08764301和资金从西利疫苗研发中心UTMB.BK一个莫里斯·希勒曼早期阶段职业研究者奖。

通过荧光显微镜转录分析 1。感染细胞与MP-12和标签新生RNA与欧盟<ol><li>放置12毫米的圆形盖玻片，盖玻片，每孔到12孔组织培养板。 </li></ol> 注意：如果细胞有麻烦秉 ​​承盖玻片，放置在井前大衣与聚-L-赖氨酸（MW≥70,000）。为了这个目标，淹没在无菌的0.1毫克/毫升的溶液5分钟，在H 2 O的聚-L-赖氨酸的盖玻片。冲洗盖玻片，用无菌H ...

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A., 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=Characterization+of+Rift+Valley+Fever+Virus+MP-12+Strain+Encoding+NSs+of+Punta+Toro+Virus+or+Sandfly+Fever+Sicilian+Virus.">Characterization of Rift Valley Fever Virus MP-12 Strain Encoding NSs of Punta Toro Virus or Sandfly Fever Sicilian Virus.</a> PLoS Negl Trop Dis. 7, e2181 (2013).</li><li>Kalveram, B., Ikegami, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toscana+Virus+NSs+Protein+Promotes+Degradation+of+Double-Stranded+RNA-Dependent+Protein+Kinase.">Toscana Virus NSs Protein Promotes Degradation of Double-Stranded RNA-Dependent Protein Kinase.</a> J. Virol. , (2013).</li><li>Schmaljohn, C., Nichol, S., Knipe, D. M., 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=.">.</a> In Fields Virology. , 1741-1789 (2007).</li><li>Le May, N., 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=TFIIH+transcription+factor,+a+target+for+the+Rift+Valley+hemorrhagic+fever+virus.">TFIIH transcription factor, a target for the Rift Valley hemorrhagic fever virus.</a> Cell. 116, 541-550 (2004).</li><li>Caplen, H., Peters, C. J., Bishop, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutagen-directed+attenuation+of+Rift+Valley+fever+virus+as+a+method+for+vaccine+development.">Mutagen-directed attenuation of Rift Valley fever virus as a method for vaccine development.</a> J. Gen. Virol. 66, 2271-2277 (1985).</li><li>Ikegami, T., Won, S., Peters, C. J., Makino, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rescue+of+infectious+Rift+Valley+fever+virus+entirely+from+cDNA,+analysis+of+virus+lacking+the+NSs+gene,+and+expression+of+a+foreign+gene.">Rescue of infectious Rift Valley fever virus entirely from cDNA, analysis of virus lacking the NSs gene, and expression of a foreign gene.</a> J. Virol. 80, 2933-2940 (2006).</li><li>Vyboh, K., Ajamian, L., Mouland, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+viral+RNA+by+fluorescence+in+situ+hybridization+(FISH).">Detection of viral RNA by fluorescence in situ hybridization (FISH).</a> J. Vis. Exp. , e4002 (2012).</li><li>Reich, E., Goldberg, I. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actinomycin+and+nucleic+acid+function.">Actinomycin and nucleic acid function.</a> Prog. Nucleic Acid Res. Mol. Biol. 3, 183-234 (1964).</li><li>Holmes, K. L., Lantz, L. M., Russ, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conjugation+of+fluorochromes+to+monoclonal+antibodies.">Conjugation of fluorochromes to monoclonal antibodies.</a> Curr. Protoc. Cytom. Chapter 4 (Unit 4 2), (2001).</li></ol>

所提供的协议描述了一种方法来衡量效果的的尿苷模拟欧盟通过掺入到新生RNA病毒感染宿主细胞的转录。这种方法比以前的方法有以下几个优点：它是快速，灵敏，它不依赖于使用放射性同位素。此外，该方法可适应通过荧光显微镜或流式细胞仪定量数据通过使用本质上是相同的试剂，得到的定性数据。当结合病毒抗原的免疫荧光染色，这种方法也使得研究者能够区分感染未受感染的细胞。 <p...

<html>传统上，转录活性已通过设立或放射性（3 H-尿嘧啶）1测量或卤化（BRU）2核苷新生RNA。这些尿嘧啶核苷类似物被细胞，通过核苷的补救途径转换成尿苷三磷酸（UTP），并随后并入新合成的RNA，3 H-尿嘧啶核苷，可以通过放射自显影，这可能是耗时的，或闪烁检测计数，需要专门的设备。此外，使用放射性同位素可能不实用，在一些实验室设置，包括高遏制生物安全实验室。 BRU可以被检测到，通过与抗-BRU抗体，这可能需要苛刻的通透性，以确保进入到细胞核或部分变性的RNA，RNA二级结构可能是一个空间位阻的抗体结合的抗体的免疫染色。 _content“&gt;这里描述的协议利用最近开发的点击化学3来衡量RVFV MP-12菌株感染的细胞中的转录活性。点击化学依赖于一个具有高度选择性和高效的铜（I）催化炔和环加成反应叠氮基部分4,5，在该协议中，在感染的细胞的新生RNA首先通过掺入标记的尿苷模拟欧盟注册成立的欧盟在第二步骤中，通过用荧光叠氮化物反应检测，该方法的主要优点是（1），它不要求使用放射性同位素和（2），它并没有要求苛刻的通透性或变性的RNA不妨碍随着分子量小于50沓，获得的叠氮化物荧光不足透化或RNA二级结构。此外，研究人员正在并不限于在他们所选择的初级抗体结合的RNA的检测时，用类的iCal免疫病毒蛋白。 在这个协议中描述的两种不同的方法：一个用于可视化通过荧光显微镜（步骤1-4），并通过流式细胞仪（步骤5-8）用于定量转录的转录。这两种方法结合标记的新生RNA与细胞内的病毒蛋白的免疫染色，允许转录活性之间的相关性和病毒感染的细胞通过细胞的基础上。 作者已经成功地使用这里描述的协议，以确定RVFV MP-12菌株6，与不同的MP-12的突变体7,8，9和细胞感染托斯卡纳病毒（TOSV）的10感染的细胞感染的细胞中的转录活性。这里所描述的协议，可以很容易地适应用于不同的病毒，并有可能进行修改，以包括特定的病毒或细胞蛋白的免疫荧光染色。

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>5-ethynyl-uridine</td>
			<td>Berry &amp; Associates</td>
			<td>PY 7563</td>
			<td>dissolve in DMSO for 100 mM stock</td>
		</tr>
		<tr>
			<td>12-mm round coverslips</td>
			<td>Fisherbrand</td>
			<td>12-545-82</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>5 ml polystyrene tubes</td>
			<td>BD Biosciences</td>
			<td>352058</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Actinomycin D (ActD)</td>
			<td>Sigma</td>
			<td>9415</td>
			<td>dissolve in DMSO for 10 mM stock</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 goat anti-mouse IgG</td>
			<td>Invitrogen</td>
			<td>A-11029</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Santa Cruz</td>
			<td>sc-2323</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>CuSO4</td>
			<td>Sigma</td>
			<td>C-8027</td>
			<td>dissolve in H2O for 100 mM stock</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma</td>
			<td>D-9542</td>
			<td>dissolve in H2O for 5 mg/ml stock</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Invitrogen</td>
			<td>11965092</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Invitrogen</td>
			<td>16000044</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>fluorescent azide (Alexa Fluor 594-coupled)</td>
			<td>Invitrogen</td>
			<td>A10270</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>fluorescent azide (Alexa Fluor 657-coupled)</td>
			<td>Invitrogen</td>
			<td>A10277</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fluoromount-G</td>
			<td>Southern Biotech</td>
			<td>0100-01</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>L-ascorbic acid</td>
			<td>Sigma</td>
			<td>A-5960</td>
			<td>dissolve in H2O for 500 mM</td>
		</tr>
		<tr>
			<td>paper filter</td>
			<td>VWRbrand</td>
			<td>28333-087</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>paraformaldehyde</td>
			<td>Sigma</td>
			<td>158127</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Invitrogen</td>
			<td>15140122</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>poly-L-lysine (MW &ge; 70000)</td>
			<td>Sigma</td>
			<td>P1274</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>T-8787</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Trizma base</td>
			<td>Sigma</td>
			<td>T-1503</td>
			<td>dissolve in H2O for 1.5 M stock, pH 8.5</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Invitrogen</td>
			<td>25200056</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fluorescence Microscope</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>e.g. Olympus IX71</td>
		</tr>
		<tr>
			<td>Flow Cytometer</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>e.g. BD Biosciences LSRII Fortessa</td>
		</tr>
	</tbody>
</table>

using click chemistry to measure the effect of viral infection on host-cell rna synthesis

许多RNA病毒已经进化作为一种​​手段来规避细胞防御能力，抑制宿主细胞的转录。如果在这些病毒的研究中，因此，它是重要的是要有一种快速和可靠的方式测量在感染的细胞中的转录活性。传统上，转录测量可以通过掺入放射性核苷如3 H-尿嘧啶核苷其次是检测通过放射自显影或液闪计数或卤代尿嘧啶核苷类似物，如5 -溴尿苷（BRU），其次是通过免疫染色检测掺入。但是，使用放射性同位素，需要专门的设备，在一些实验室的设置是不可行的，，而BRU检测可能会很麻烦，并可能遭受低灵敏度。 最近开发的点击化学反应，其中涉及铜催化的叠氮化物和炔三唑形成，现在提供了一个快速，highlÝ敏感替代这两种方法。点击化学反应是一个两步骤的过程中，首先通过掺入标记的5 - 乙炔基尿嘧啶核苷模拟（欧盟），然后检测荧光叠氮化物中的标签新生RNA。这些叠氮化物可作为几个不同的荧光团，允许用于可视化的选择，适用范围广。 本协议描述的方法来衡量细胞中的转录抑制感染裂谷热病毒（RVFV）MP-12菌株利用点击化学。同时，在这些细胞中的病毒蛋白表达由古典细胞免疫测定。步骤1到步骤4详细的方法，通过荧光显微镜可视化抑制转录，而步骤5至8详细的方法通过流式细胞仪量化转录抑制。此协议是用于与其他病毒很容易适应。

奈米RVFV蛋白（家庭布尼亚病毒科 ，属Phlebovirus）11抑制宿主细胞的转录，通过两种不同的机制：（i）在螯合P44亚单位的基础转录因子TFIIH 11及（ii）通过促 ​​进p62的蛋白酶体降解亚基的TFIIH 6。由于MP-12菌株可在生物安全2级实验室处理，被排除在美国，选择代理规则适用于其他野生RVFV MP-12疫苗株，在这个协议中描述的实验已用于13键入RVFV株。 MP-12菌...

Watch this Scientific Journal Video about Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis Video at JoVE.com

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis Video (Video) | JoVE

这个方法描述了使用的点击化学感染裂谷热病毒（RVFV）MP-12菌株后，在宿主细胞的转录变化来衡量。结果可以可视化，通过荧光显微镜定性或定量地获得通过流式细胞仪。此方法是适应性强，用于与其他病毒。

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Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

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Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro Video (Video) | JoVE

建立的液体覆盖的文化偏光人类呼吸道上皮的CALU-3细胞研究呼吸系统病原宿主细胞的反应<em在体外</em

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

Using the Overlay Assay to Qualitatively Measure Bacterial Production of and Sensitivity to Pneumococcal Bacteriocins

Using the Overlay Assay to Qualitatively Measure Bacterial Production of and Sensitivity to Pneumococcal Bacteriocins Video (Video) | JoVE

使用复分析定性测量细菌生产和敏感性，以肺炎球菌细菌素

Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms.  We have ...

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans Video (Video) | JoVE

巨噬细胞胆固醇消耗及其对的吞噬作用<em&gt;新型隐球菌</em

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are ...

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii Video (Video) | JoVE

冻干环介导等温扩增试剂的检测中的发展<em&gt;贝氏柯克斯体</em

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. ...