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 2 O和风干至少2小时。 <ol start="2"><li>种子293细胞到盖玻片上，在1毫升生长培养基（含有10％胎牛血清，100 U / ml的青霉素和100毫克/毫升链霉素的DMEM培养液），每孔中加入5×10 4细胞。在培养箱孵育在37℃和5％CO 2 O / N </li><li> 3感染复数（MOI）感染细胞与MP-12。至少包括两个<html>未受感染的井，两口井将作为未感染控制，其他的将被视为与放线菌素D（ACTD）作为转录抑制控制在1.5步。移除生长培养基中，稀释的病毒原液，加入到各孔的总体积是200毫升。增湿培养箱中，孵育1小时，在37℃和5％CO 2。 </li></ol> 注:感染细胞，细胞也可以被转染的质粒DNA或体外合成的RNA编码一个特定的病毒蛋白质。为了这个目标，转染细胞根据制造商的说明，用适当的化学转染试剂，在转染后16小时，进行到步骤1.5。 RNA标记和固定之前，明智的做法是优化转染条件和培养时间。 <ol start="4"><li>删除接种，并添加1毫升新鲜的生长培养基，每孔。在37°C孵育15小时。 </li></ol>ove_content&gt; 备注:如果此协议用 ​​于与其他病毒适应，最好是执行一个时间过程实验，以确定最佳的时间点，RNA标记和固定。设置此时间过程使感染时间是交错排列的，和所有样品可标记的，固定的，和在同一时间染色。 <ol start="5"><li>在15小时后感染（HPI），更换生长培养基中，培养基中含有1毫米欧盟。模拟感染井之一，也加入5微克/毫升ACTD。这将作为控制转录抑制，，ACTD抑制依赖于DNA的RNA合成。返回细胞的孵化器。 </li><li> 16 HPI，除去介质和洗涤盖玻片一次，用1毫升的PBS（氯化钾0.20克/升，KH 2 PO 4 0.20克/升，氯化钠8.00克/升的Na 2 HPO 4 1.15克/升，pH值7.4）每口井。修复细胞，每孔中加入1毫升4％多聚甲醛（PFA），在RT下孵育30分钟。 </li></ol>注意:要准备4％PFA固定的解决方案，溶解10克的多聚甲醛在150毫升H 2 O加热至60°C在加热磁力搅拌器。加入500μl1M的NaOH，继续搅拌，直到溶液变得清晰。筛选PFA溶液通过纸过滤器，以去除微粒，并添加62.5毫升磷酸盐缓冲液（77毫摩尔的NaH 2 PO 4，287毫米的Na 2 HPO 4，pH 7.4）中。加入H 2 O至总体积为250毫升。冻结在-20°C的单用途等分。 <ol start="7"><li>每口井用1毫升的PBS洗一次。没有孵化需要时间，这和所有随后的洗涤步骤。立即进行第2步。 </li></ol> 注:重要的是立即着手进行点击反应，RNA退化较长时间保持在这个阶段，如果，RNA荧光信号的损失已经可以观察到细胞保持1小时4°C 。同样，如果每形成一个时间过程实验，一定要标签，修复，并在同一时间，所有的样品染色。 2。点击反应检测标记的RNA <ol><li>通透细胞加入1 ml 0.2％的Triton X-100的PBS中。在RT下孵育10分钟。 </li></ol> 注:所有步骤中使用的解决方案，需要2.1至2.3核酸自由。 <ol start="2"><li>用1ml PBS中，每孔洗涤三次。 </li><li>删除盖玻片的12孔板并转入湿热试验箱。涵盖50 - 100μL点击染色溶液（100毫米的Tris pH值8.5，1毫米硫酸铜 ，20μM荧光叠氮化[最大激发/发射:590/617纳米，100毫米抗坏血酸），每个每个盖玻片。孵育1小时RT在黑暗中。 </li></ol> 注意:要组装湿热试验箱，线的细胞培养或培养皿底部，一个直径10厘米的塑料石蜡膜。线的内侧边缘的二SH与湿纸巾。放置到塑料盖玻片石蜡膜。 注意:要小心，不要让干燥的盖玻片，在此期间或在协议中的任何其他步骤。最好是添加染色液每个盖玻片后直接被放置在潮湿的腔。 注意:，准备点击染色液新鲜紧接到这一步。 1.5 M Tris pH值8.5，100mM的CuSO 4的，和500mM的抗坏血酸储备液可被存储在4℃下然而，放弃任何抗坏血酸原液已经变成黄色。 注意:一定要选择相匹配的过滤器上的荧光显微镜的荧光。 <ol start="4"><li>取出盖玻片湿热试验箱和地方到一个新的12孔板，每孔1毫升PBS洗三次。 </li></ol> 注意:如果没有去model的病毒蛋白进行需要的话，可被安装盖玻片，在此步骤之后（进行步骤3.3）成像。 3。免疫荧光检测病毒蛋白的表达<ol><li>向每孔加入1 ml的封闭缓冲液（3％（重量/体积）牛血清白蛋白（BSA）的PBS中），并孵育30分钟，在RT下在黑暗中。 </li><li>删除盖玻片12孔板，转移到湿热试验箱，覆盖50 - 100微升的主要抗体（抗-RVFV，一种礼品从RB TESH博士，UTMB）的阻断缓冲液（1:500）稀释。孵育1小时RT在黑暗中。 </li></ol> 注意:当使用不同的病毒适应这个协议，确定您的主要抗体最佳稀释。 注:另外，这一步可以在4°C O / N在黑暗中。 <ol start="3"><li>到12孔板取出盖玻片湿热试验箱和地方洗三次，每孔用1ml PBS。 </li><li>从12孔的地方，放置到潮湿的腔室和盖50 - 100μl的第二抗体（抗小鼠IgG共轭的荧光染料[激发/发射最大:五百一十九分之四百九十五nm]的在封闭缓冲液（1）稀释清除盖玻片: 500）。孵育1小时RT在黑暗中。 </li></ol> 注意:一定要选择二次抗体，可以识别你的主要抗体和筛选器匹配的荧光显微镜。 注意:如果需要的话，200 ng / ml的DAPI可以加入第二抗体稀释至可视化的细胞核。 <ol start="5"><li>删除从潮湿的腔室盖玻片和地点到12孔板中，每孔1毫升PBS洗三次。 </li><li>安装1滴（ 约 15微升）Fluoromount-G放置到载玻片盖玻片。盖玻片浸入到H 2 O，通过触摸届，去除多余的H 2 OE侧对纸巾，单元侧到下拉的Fluoromount-G。空气干燥5分钟。 </li></ol> 注意:如果倒置显微镜成像，允许Fluoromount-G，以巩固在4°CO / N或加盖盖玻片，在显微镜幻灯片，通过密封的边缘用透明的指甲油。 注:幻灯片可以立即成像或储存在4℃下在黑暗中长达一个星期。 4。收购数据<ol><li>用荧光显微镜的图像。 </li></ol> 注意:一定要选择适当的荧光基团，可以可视化使用显微镜。仅供参考，以下是荧光用于收购本出版物中的图像和过滤器集。 DAPI: 激发滤光片:BP 330 - 385 双色镜:400马克 排放过滤器:LP 420 激发滤光片:BP 460 - 490 双色镜:DM 505 排放过滤器:LP 510 荧光最大激发/发射六百十七分之五百九十: 激发滤光片:BP 545 - 580 双色镜:DM 600 排放过滤器:LP 610 通过流式细胞仪转录分析 5。感染细胞与MP-12和标签新生RNA与欧盟<ol><li>种子293细胞到6孔组织培养板中生长培养基（含有10％胎牛血清，100 U / ml的青霉素，100μg/ ml的链霉素的DMEM培养基）中。增湿培养箱中，孵育在37℃，5％CO 2，直到细胞已经达到了80 - 100％汇合。 </li><li>感染细胞与MP-12在MOI 3。包括至少两个未感染的井两口井将作为未受感染的控制，其他的将被视为与ACTD步骤5.4。移除生长培养基中，稀释的病毒原液，使总体积，每孔加入400微升。增湿培养箱中，孵育1小时，在37℃和5％CO 2。 </li><li>删除接种，每孔加入2毫升新鲜的生长培养基。在37°C孵育12小时。 </li></ol> 注意:如果此协议用 ​​于与其他病毒适应，最好是执行一个时间过程实验，以确定最佳的时间点，用于标识和收获。设置此时间过程，使感染时间是交错的，所有的样品都可以被标记，收获，并在同一时间染色。 <ol start="4"><li>在12 HPI，更换生长培养基中，培养基中含有0.5毫欧。模拟感染井之一，也加入5微克/毫升ACTD。这将作为控制转录抑制，，ACTD抑制依赖于DNA的RNA合成。返回细胞的孵化器。 </li><li>在13 HPI，洗细胞一次机智ħPBS和收获胰酶消化细胞。三次洗涤收获的细胞用含有1mM EDTA的溶液（PBS-EDTA）。 </li></ol> 注:在这和随后的步骤中，不要离心细胞的速度比500 - 1,000 XG防止细胞断裂。离心细胞，1 - 2分钟，去滓。 注意:如果表面免疫计划后，点击反应，收获用橡皮警察或一次性细胞刮刀代替。 <ol start="6"><li>修复细胞重新悬浮在1ml的4％多聚甲醛，并培育30分钟，在RT。参见步骤说明如何准备4％PFA 1.6。 </li><li>与1毫升PBS-EDTA洗一次，每口井。立即着手第6步。 </li></ol> 注:重要的是立即着手进行点击反应，RNA退化，如果较长时间保持在这个阶段。同样，如果执行时间c未尝不是实验，同时所有样品的标签，固定和染色。 6。点击反应检测标记的RNA <ol><li>通透细胞重新悬浮于0.5ml的0.2％的Triton X-100的PBS中。在RT下孵育10分钟。 </li></ol> 注意:所有步骤中使用的解决方案，需要6.1至6.3无核酸酶的。 注意:如果细胞不正确沉积物在此步骤中，增加离心时间为5分钟。沉淀的行为将提高细胞一旦被暂停在FACS缓冲液（6.4）。 <ol start="2"><li>用1毫升PBS洗一次。 </li></ol> 注意:不要使用EDTA在这一步，因为这将螯合铜离子的点击反应的必要。 <ol start="3"><li>悬浮细胞在200微升点击染色溶液（100毫米的Tris pH值8.5，1毫米硫酸铜 ，200nm的叠氮标记用荧光染料[EXC最大itation /发射:六百六十五分之六百五十〇纳米，100毫米抗坏血酸）。孵育1小时RT在黑暗中。 </li></ol> 注意:，准备点击染色液新鲜紧接到这一步。 1.5 M Tris pH值8.5，100mM的CuSO 4的，和500mM的抗坏血酸储备液可被存储在4℃下然而，放弃任何抗坏血酸原液已经变成黄色。 注意:如果重悬细胞聚集在一起，在此步骤中，通过上下吸几次。 注意:一定要选择流式细胞仪，可以检测到荧光。 注意:还准备点击染色过程不会受到感染的细胞的一个样品，这些将用于流式细胞仪的校准。要么使用一半的受感染的细胞，包括一个额外的步骤5.2感染。这些CON在步骤7.1中，控制细胞将被免疫染色。 <ol start="4"><li>用FACS缓冲液（含1mM EDTA和0.5％（重量/体积）牛血清白蛋白的PBS）洗两次。 </li></ol> 注:如果没有病毒蛋白的免疫细胞可以立即分析（继续执行步骤8）。 7。免疫荧光检测病毒蛋白的表达<ol><li>悬浮细胞在200微升的主要抗体（抗-RVFV）稀释FACS缓冲液（1:10000）孵育1小时即时在黑暗中。 </li></ol> 注:另外，这一步可以在4°C O / N在黑暗中。 注意:还准备进行点击染色过程，但不会被免疫染色的未感染的细胞的一个样品，这些将用于流式细胞仪的校准。要么使用模拟感染细胞的一半，或包括一个额外的未感染以及步骤5.2。 注意:当使用不同的病毒适应这个协议，确定您的主要抗体最佳稀释。 <ol start="2"><li>洗涤细胞两次在流式细胞仪缓冲。 </li><li> 200微升的二次抗体（抗鼠IgG共轭荧光染料[最大激发/发射:519分之495纳米）的悬浮细胞在FACS缓冲液（1:1,000）和稀释孵育1小时即时在黑暗中。 </li><li>一次洗涤细胞在流式细胞仪缓冲。 </li></ol> 注:此时，细胞可以储存在4℃的O / N在黑暗中。 8。收购数据<ol><li>于500μlFACS缓冲液，转移到5毫升聚苯乙烯管中重悬细胞，并用流式细胞仪分析。使用MP-12感染的细胞（没有点击反应，抗RVFV染色）和模拟感染细胞（点击反应）对仪器进行校准。 </li></ol> 不E:一定要选择合适的荧光基团，可以检测到您的仪器。仅供参考，以下是激光线和过滤器集，用于收购本出版物中显示的数据。 荧光与激发/发射光谱五百十九分之四百九十五: 激光:488纳米 过滤器:530/30 荧光与激发/发射光谱六百六十五分之六百五: 激光:640纳米 过滤器:670/20

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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>

没有利益冲突的声明。

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 c...

<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 at JoVE.com

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

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。

使用点击化学测量的影响，病毒感染宿主细胞RNA合成

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

using-click-chemistry-to-measure-effect-viral-infection-on-host-cell

Research

JoVE Journal

Immunology and Infection

11.6K 次观看.  University of Texas Medical Branch. 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。

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

Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence of novel viral pathogens, such as SARS coronavirus and 2009 pandemic H1N1 influenza virus, that are not detectable by traditional methods. To address these challenges, we have previously developed and validated a pan-viral microarray platform called the Virochip with the capacity to detect all known viruses as well as novel variants on the basis of conserved sequence homology1. Using the Virochip, we have identified the full spectrum of viruses associated with respiratory infections, including cases of unexplained critical illness in hospitalized patients, with a sensitivity equivalent to or superior to conventional clinical testing2-5. The Virochip has also been used to identify novel viruses, including the SARS coronavirus6,7, a novel rhinovirus clade5, XMRV (a retrovirus linked to prostate cancer)8, avian bornavirus (the cause of a wasting disease in parrots)9, and a novel cardiovirus in children with respiratory and diarrheal illness10. The current version of the Virochip has been ported to an Agilent microarray platform and consists of ~36,000 probes derived from over ~1,500 viruses in GenBank as of December of 2009. Here we demonstrate the steps involved in processing a Virochip assay from start to finish (~24 hour turnaround time), including sample nucleic acid extraction, PCR amplification using random primers, fluorescent dye incorporation, and microarray hybridization, scanning, and analysis.

Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

The Virochip is a pan-viral microarray designed to simultaneously detect all known viruses as well as novel viruses on the basis of conserved sequence homology. Here we demonstrate how to run a Virochip assay to analyze clinical samples for the presence of both known and unknown viruses.

使用泛病毒基因芯片检测（Virochip）屏幕病毒病原体的临床标本

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence ...

科研

免疫与感染

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

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 generation of extreme population diversity that facilitates virus adaptation and evolution. Increasing evidence shows that the intrinsic error rates, and the resulting mutation frequencies, of RNA viruses can be modulated by subtle amino acid changes to the viral polymerase. Although biochemical assays exist for some viral RNA polymerases that permit quantitative measure of incorporation fidelity, here we describe a simple method of measuring mutation frequencies of RNA viruses that has proven to be as accurate as biochemical approaches in identifying fidelity altering mutations. The approach uses conventional virological and sequencing techniques that can be performed in most biology laboratories. Based on our experience with a number of different viruses, we have identified the key steps that must be optimized to increase the likelihood of isolating fidelity variants and generating data of statistical significance. The isolation and characterization of fidelity altering mutations can provide new insights into polymerase structure and function1-3. Furthermore, these fidelity variants can be useful tools in characterizing mechanisms of virus adaptation and evolution4-7.

The present article describes the steps required to isolate and characterize RNA polymerase fidelity variants of RNA viruses and how to use mutation frequency data to confirm fidelity changes in tissue culture.

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 ...

Amplifying and Quantifying HIV-1 RNA in HIV Infected Individuals with Viral Loads Below the Limit of Detection by Standard Clinical Assays

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below 50-75 copies/ml) is necessary to gain insight to viral dynamics and virus host interactions in patients who naturally control the infection and those who are on combination antiretroviral therapy (cART). 
Here we describe how to amplify viral genomes by single genome sequencing (the SGS protocol) 13, 19 and how to accurately quantify HIV-1 RNA in patients with low viral loads (the single-copy assay (SCA) protocol) 12, 20. 
The single-copy assay is a real-time PCR assay with sensitivity depending on the volume of plasma being assayed. If a single virus genome is detected in 7 ml of plasma, then the RNA copy number is reported to be 0.3 copies/ml. The assay has an internal control testing for the efficiency of RNA extraction, and controls for possible amplification from DNA or contamination. Patient samples are measured in triplicate.
The single-genome sequencing assay (SGS), now widely used and considered to be non-labor intensive 3, 7, 12, 14, 15,is a limiting dilution assay, in which endpoint diluted cDNA product is spread over a 96-well plate. According to a Poisson distribution, when less than 1/3 of the wells give product, there is an 80% chance of the PCR product being resultant of amplification from a single cDNA molecule. SGS has the advantage over cloning of not being subjected to resampling and not being biased by PCR-introduced recombination 19. However, the amplification success of SCA and SGS depend on primer design. Both assays were developed for HIV-1 subtype B, but can be adapted for other subtypes and other regions of the genome by changing primers, probes, and standards.

Quantifying levels of HIV-1 RNA in plasma and sequencing single HIV-1 genomes from individuals with viral loads below the limit of detection (50-75 copies/ml) is difficult. Here we describe how to extract and quantify plasma viral RNA using a real time PCR assay that reliably measures HIV-1 RNA down to 0.3 copies/ml and how to amplify viral genomes by single genome sequencing, from samples with very low viral loads.

在艾滋病毒感染者病毒载量低于检测限制标准的临床实验放大和量化HIV - 1 RNA

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below ...

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

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2. Viruses modulate host cell abundance and diversity, contribute to the cycling of nutrients, alter host cell phenotype, and influence the evolution of both host cell and viral communities through the lateral transfer of genes 3. Numerous studies have highlighted the staggering genetic diversity of viruses and their functional potential in a variety of natural environments. 
Metagenomic techniques have been used to study the taxonomic diversity and functional potential of complex viral assemblages whose members contain single-stranded DNA (ssDNA), double-stranded DNA (dsDNA) and RNA genotypes 4-9. Current library construction protocols used to study environmental DNA-containing or RNA-containing viruses require an initial nuclease treatment in order to remove nontargeted templates 10. However, a comprehensive understanding of the collective gene complement of the virus community and virus diversity requires knowledge of all members regardless of genome composition. Fractionation of purified nucleic acid subtypes provides an effective mechanism by which to study viral assemblages without sacrificing a subset of the community&#x2019;s genetic signature. 
Hydroxyapatite, a crystalline form of calcium phosphate, has been employed in the separation of nucleic acids, as well as proteins and microbes, since the 1960s11. By exploiting the charge interaction between the positively-charged Ca2+ ions of the hydroxyapatite and the negatively charged phosphate backbone of the nucleic acid subtypes, it is possible to preferentially elute each nucleic acid subtype independent of the others. We recently employed this strategy to independently fractionate the genomes of ssDNA, dsDNA and RNA-containing viruses in preparation of DNA sequencing 12. Here, we present a method for the fractionation and recovery of ssDNA, dsDNA and RNA viral nucleic acids from mixed viral assemblages using hydroxyapatite chromotography.

We describe an efficient method to separate single-stranded DNA, double-stranded DNA and RNA molecules from environmental viral communities. Nucleic acids are fractionated using hydroxyapatite chromatography with increasing concentrations of phosphate-containing buffers. This method permits the isolation of all viral nucleic acid types from environmental samples.

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, ...

Chemoselective Modification of Viral Surfaces via Bioorthogonal Click Chemistry

The modification of virus particles has received a significant amount of attention for its tremendous potential for impacting gene therapy, oncolytic applications and vaccine development.1,2,3 Current approaches to modifying viral surfaces, which are mostly genetics-based, often suffer from attenuation of virus production, infectivity and cellular transduction.4,5 Using chemoselective click chemistry, we have developed a straightforward alternative approach which sidesteps these issues while remaining both highly flexible and accessible.1,2
The goal of this protocol is to demonstrate the effectiveness of using bioorthogonal click chemistry to modify the surface of adenovirus type 5 particles. This two-step process can be used both therapeutically1 or analytically,2,6 as it allows for chemoselective ligation of targeting molecules, dyes or other molecules of interest onto proteins pre-labeled with azide tags. The three major advantages of this method are that (1) metabolic labeling demonstrates little to no impact on viral fitness,1,7 (2) a wide array of effector ligands can be utilized, and (3) it is remarkably fast, reliable and easy to access.1,2,7
In the first step of this procedure, adenovirus particles are produced bearing either azidohomoalanine (Aha, a methionine surrogate) or the unnatural sugar O-linked N-azidoacetylglucosamine (O-GlcNAz), both of which contain the azide (-N3) functional group. After purification of the azide-modified virus particles, an alkyne probe containing the fluorescent TAMRA moiety is ligated in a chemoselective manner to the pre-labeled proteins or glycoproteins. Finally, an SDS-PAGE analysis is performed to demonstrate the successful ligation of the probe onto the viral capsid proteins. Aha incorporation is shown to label all viral capsid proteins (Hexon, Penton and Fiber), while O-GlcNAz incorporation results in labeling of Fiber only.
In this evolving field, multiple methods for azide-alkyne ligation have been successfully developed; however only the two we have found to be most convenient are demonstrated herein &#x2013; strain-promoted azide-alkyne cycloaddition (SPAAC) and copper-catalyzed azide-alkyne cycloaddition (CuAAC) under deoxygenated atmosphere.

Adenovirus particles are engineered to contain either the unnatural amino acid analogue azidohomoalanine or the azido sugar O-GlcNAz. The azide group of each is chemoselectively ligated via "click" chemistry reactions as a means of viral surface modification.

通过Bioorthogonal点击化学化学选择性病毒表面改性

The modification of virus particles has received a significant amount of attention for its tremendous potential for impacting gene therapy, oncolytic ...

The Synergistic Effect of Visible Light and Gentamycin on Pseudomona aeruginosa Microorganisms

Recently there were several publications on the bactericidal effect of visible light, most of them claiming that blue part of the spectrum (400 nm-500 nm) is responsible for killing various pathogens1-5. The phototoxic effect of blue light was suggested to be a result of light-induced reactive oxygen species (ROS) formation by endogenous bacterial photosensitizers which mostly absorb light in the blue region4,6,7. There are also reports of biocidal effect of red and near infra red8 as well as green light9.
In the present study, we developed a method that allowed us to characterize the effect of high power green (wavelength of 532 nm) continuous (CW) and pulsed Q-switched (Q-S) light on Pseudomonas aeruginosa. Using this method we also studied the effect of green light combined with antibiotic treatment (gentamycin) on the bacteria viability. P. aeruginosa is a common noscomial opportunistic pathogen causing various diseases. The strain is fairly resistant to various antibiotics and contains many predicted AcrB/Mex-type RND multidrug efflux systems10.
The method utilized free-living stationary phase Gram-negative bacteria (P. aeruginosa strain PAO1), grown in Luria Broth (LB) medium exposed to Q-switched and/or CW lasers with and without the addition of the antibiotic gentamycin. Cell viability was determined at different time points. The obtained results showed that laser treatment alone did not reduce cell viability compared to untreated control and that gentamycin treatment alone only resulted in a 0.5 log reduction in the viable count for P. aeruginosa. The combined laser and gentamycin treatment, however, resulted in a synergistic effect and the viability of P. aeruginosa was reduced by 8 log's.
The proposed method can further be implemented via the development of catheter like device capable of injecting an antibiotic solution into the infected organ while simultaneously illuminating the area with light.

The Synergistic Effect of Visible Light and Gentamycin on Pseudomona aeruginosa Microorganisms

We show that a developed biomedical device involving continuous or pulsed visible laser based treatment that is combined with antibiotic treatment (gentamycin), results in a statistically significant synergistic effect leading to a reduction in the viability of P. aeruginosa PAO1, by 8 log's compared to antibiotic treatment alone.

可见光和庆大霉素的协同作用<em&gt;假单胞菌假单胞菌</em&gt;微生物

Recently there were several publications on the  bactericidal effect of visible light, most of them claiming that blue part of  the spectrum (400 nm-500 ...

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

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

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

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

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 RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

利用RNA干扰调查的先天免疫反应的小鼠巨噬细胞

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

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

Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms. We have shown that bacterially produced antimicrobial peptides (bacteriocins) dictate the outcome of these competitive interactions. All fully-sequenced pneumococcal strains harbor a bacteriocin-like peptide (blp) locus. The blp locus encodes for a range of diverse bacteriocins and all of the highly conserved components needed for their regulation, processing, and secretion. The diversity of the bacteriocins found in the bacteriocin immunity region (BIR) of the locus is a major contributor of pneumococcal competition. Along with the bacteriocins, immunity genes are found in the BIR and are needed to protect the producer cell from the effects of its own bacteriocin. The overlay assay is a quick method for examining a large number of strains for competitive interactions mediated by bacteriocins. The overlay assay also allows for the characterization of bacteriocin-specific immunity, and detection of secreted quorum sensing peptides. The assay is performed by pre-inoculating an agar plate with a strain to be tested for bacteriocin production followed by application of a soft agar overlay containing a strain to be tested for bacteriocin sensitivity. A zone of clearance surrounding the stab indicates that the overlay strain is sensitive to the bacteriocins produced by the pre-inoculated strain. If no zone of clearance is observed, either the overlay strain is immune to the bacteriocins being produced or the pre-inoculated strain does not produce bacteriocins. To determine if the blp locus is functional in a given strain, the overlay assay can be adapted to evaluate for peptide pheromone secretion by the pre-inoculated strain. In this case, a series of four lacZ-reporter strains with different pheromone specificity are used in the overlay.

Competition in Streptococcus pneumoniae is mediated by bacteriocins, small antimicrobial peptides with inhibitory activity towards pneumococcus and other related species.&#160; Here we describe an optimized bacterial overlay assay that allows for the characterization of bacteriocin activity and inhibitory spectrum, bacteriocin-specific immunity, and detection of secreted quorum sensing peptides.

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 ...

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

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 proteins involved in the regulation of the virus activity recognize several nucleic acids. The nucleocapsid protein NCp7 (NC) is a key protein regulating several processes during virus replication. NC is in fact a chaperone destabilizing the secondary structures of RNA and DNA and facilitating their annealing. The inactivation of NC is a new approach and an interesting target for anti-HIV therapy. The Nucleocapsid Annealing-Mediated Electrophoresis (NAME) assay was developed to identify molecules able to inhibit the melting and annealing of RNA and DNA folded in thermodynamically stable tridimensional conformations, such as hairpin structures of TAR and cTAR elements of HIV, by the nucleocapsid protein of HIV-1. The new assay employs either the recombinant or the synthetic protein, and oligonucleotides without the need of their previous labeling. The analysis of the results is achieved by standard polyacrylamide gel electrophoresis (PAGE) followed by conventional nucleic acid staining. The protocol reported in this work describes how to perform the NAME assay with the full-length protein or its truncated version lacking the basic N-terminal domain, both competent as nucleic acids chaperones, and how to assess the inhibition of NC chaperone activity by a threading intercalator. Moreover, NAME can be performed in two different modes, useful to obtain indications on the putative mechanism of action of the identified NC inhibitors.

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

This protocol describes NAME, an assay that allows the rapid identification of molecules able to inhibit in vitro the chaperone activities of HIV-1 nucleocapsid protein.

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 ...

使用点击化学测量的影响，病毒感染宿主细胞RNA合成

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