Name: vaccinia reporter viruses for quantifying viral function at all stages of gene expression
Uploaded: 2014-05-15T13:45:00
Description: Watch this Scientific Journal Video about Vaccinia Reporter Viruses for Quantifying Viral Function at All Stages of Gene Expression at JoVE.com

我们感谢SIGA技术（俄勒冈州Corvallis）提供ST-246。 DKR是由美国国立卫生研究院的练金在免疫学，以波士顿大学（5T32AI 7309）的支持。这项工作是由P41 086180部分支持，美国国立卫生研究院RO1AI1096159-01，和RO3（以JHC）。

1，电池板<ol><li>从板分离HeLa细胞在生长培养基（DMEM，2mM的L-过剩，10％FBS）中稀释至约2.0×10 5个细胞/ ml。分配100μl/孔在黑色壁，透明平底96孔板（20000个细胞/孔）。 </li><li>孵育细胞24小时，直至汇合在37℃培养箱+，5％CO 2。 </li></ol> 2，感染细胞<ol><li>稀释病毒和感染细胞。 <ol><li>解冻TRPV（三人间病毒;早期金星，中级的mCherry，晚TagBFP）和PLV（启动子的维纳斯?...

<ol>
	<li>Yen, J., Golan, R., Rubins, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vaccinia+virus+infection+&amp;+temporal+analysis+of+virus+gene+expression:+part+1.">Vaccinia virus infection &amp; temporal analysis of virus gene expression: part 1.</a> J Vis Exp. 26 (26), (2009).</li><li>Hansen, S. G., Cope, T. A., Hruby, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BiZyme:+a+novel+fusion+protein-mediating+selection+of+vaccinia+virus+recombinants+by+fluorescence+and+antibiotic+resistance.">BiZyme: a novel fusion protein-mediating selection of vaccinia virus recombinants by fluorescence and antibiotic resistance.</a> BioTechniques. 32 (5), 1182-1187 (2002).</li><li>Popov, S., Mirshahidi, S., Essono, S., Song, R., Wang, X., Ruprecht, R. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poxvirus+dilemmas--monkeypox,+smallpox,+and+biologic+terrorism.+The+New+England.">Poxvirus dilemmas--monkeypox, smallpox, and biologic terrorism. The New England.</a> Journal of Medicine. 339 (8), 556-559 (1998).</li><li>Baker, R. O., Bray, M., Huggins, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+antiviral+therapeutics+for+smallpox,+monkeypox+and+other+orthopoxvirus+infections.">Potential antiviral therapeutics for smallpox, monkeypox and other orthopoxvirus infections.</a> Antiviral Research. 57, 1-2 (2003).</li><li>Rimoin, A. W., 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=Major+increase+in+human+monkeypox+incidence+30+years+after+smallpox+vaccination+campaigns+cease+in+the+Democratic+Republic+of+Congo.">Major increase in human monkeypox incidence 30 years after smallpox vaccination campaigns cease in the Democratic Republic of Congo.</a> Proceedings of the National Academy of Sciences of the U S A. 107 (37), 16262-167 (2010).</li><li>Sodeik, B., Griffiths, G., Ericsson, M., Moss, B., Doms, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+of+vaccinia+virus:+effects+of+rifampin+on+the+intracellular+distribution+of+viral+protein+p65.">Assembly of vaccinia virus: effects of rifampin on the intracellular distribution of viral protein p65.</a> Journal of Virology. 68 (2), 1103-1114 (1994).</li><li>Grosenbach, D. W., Jordan, R., Hruby, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+small-molecule+antiviral+ST-246+as+a+smallpox+therapeutic.">Development of the small-molecule antiviral ST-246 as a smallpox therapeutic.</a> Future Virology. 6 (5), 653-671 (2011).</li><li>Broyles, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vaccinia+virus+transcription.">Vaccinia virus transcription.</a> Journal of General Virology. 84 (9), 2293-2303 (2003).</li><li>Condit, R. C., Niles, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+viral+transcription+elongation+and+termination+during+vaccinia+virus+infection.">Regulation of viral transcription elongation and termination during vaccinia virus infection.</a> Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression. 1577 (2), 325-336 (2002).</li><li>D&#237;az-Guerra, M., Rivas, C., Esteban, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducible+expression+of+the+2-5A+synthetase/RNase+L+system+results+in+inhibition+of+vaccinia+virus+replication.">Inducible expression of the 2-5A synthetase/RNase L system results in inhibition of vaccinia virus replication.</a> Virology. 227 (1), 220-228 (1997).</li><li>Cohrs, R. J., Condit, R. C., Pacha, R. F., Thompson, C. L., Sharma, O. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+ppp(A2&#8217;p)nA-dependent+RNase+by+a+temperature-sensitive+mutant+of+vaccinia+virus.">Modulation of ppp(A2&#8217;p)nA-dependent RNase by a temperature-sensitive mutant of vaccinia virus.</a> Journal of Virology. 63 (2), 948-951 (1989).</li><li>S&#225;nchez-Puig, J. M., S&#225;nchez, L., Roy, G., Blasco, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Susceptibility+of+different+leukocyte+cell+types+to+Vaccinia+virus+infection.">Susceptibility of different leukocyte cell types to Vaccinia virus infection.</a> Virology Journal. 1 (1), (2004).</li><li>Bengali, Z., Satheshkumar, P. S., Yang, Z., Weisberg, A. S., Paran, N., Moss, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+S2+cells+are+non-permissive+for+vaccinia+virus+DNA+replication+following+entry+via+low+pH-dependent+endocytosis+and+early+transcription.">Drosophila S2 cells are non-permissive for vaccinia virus DNA replication following entry via low pH-dependent endocytosis and early transcription.</a> PLoS One. 6 (2), (2011).</li></ol>

这里，我们描述了一种多级牛痘记者病毒（TRPV），它提供病毒复制的重现性，实时反馈测量的实际使用情况。通过使用良好定义的痘病毒的抑制剂，我们能够显示，TRPV与动作的每个抑制剂的理解机制相一致的方式进行响应。而TRPV病毒提供了有关病毒阶段级数最全面的信息，两阶段（IREV，LREV）和单级（EV，IV LV）的病毒，也可以类似地使用，同时考虑最优的蛋白质折叠的优点，稳定和优异的信号?...

传统上，病毒表达是利用分子生物学技术（ 例如，RNA印迹法，蛋白质印迹法，微阵列杂交， 等等 ）1研究。虽然这些方法能够提供详细的信息相对于个别分类的mRNA或蛋白质的表达的变化，它们通常不适合于实时性和高通量处理。采用基于荧光的记者替代方法与痘病毒工作时，先前已被应用;然而，他们的开发和使用已经出于不同的目的。一些这样的方法被设计为选择的重组病毒2,3。这些技术表达绿色荧光蛋白在适当的成立和从重组痘苗病毒克隆外源DNA的表达。类似地，一些牛痘株稳定表达可溶性EGFP或下一个本机牛痘启动子表达GFP标记的蛋白已被广泛使用。这些都是典型售货机，ically用于定量病毒进入及复制过程中的基于抗体的中和测定法，化学抑制，或抗病毒效力4-6比较。尽管这些病毒已经被证明非常有用，他们在向因单一暧昧早/晚病毒启动子及其用法提供了有关抑制的点的详细信息的能力是有限的。以前的方法也取得了利用EGFP蛋白的次优的信号 - 噪声特性。 由于痘病毒复制和缺乏可用以测定在病毒基因表达的实时变动对现有工具的复杂性，我们开发了一套单级和多级记者病毒7,8。如在以前的出版物中描述的，这些病毒表达一种，两种，或从天然痘苗病毒启动子3在光谱上不同的荧光蛋白在早期，在感染中间，或最后阶段。这些病毒可能是我们编为病毒复制进度使用荧光显微镜的指标和它们同样适合于高通量的基于平板和阅读器测定。这些病毒很容易在地方的野生型病毒的使用，具有类似的动力学成长，达到相当于滴度7。在策划和创作的这些病毒，很在意拍摄于选择具有高信背景特征具有超强的折叠效率的荧光基团，以方便可靠的定量分析与快速反馈的变化，表达（金星的mCherry和TagBFP）。此外，病毒启动子的组合被选择能产生高保真的，明确的阶段特异性表达，提供有关的全方位早期（C11R启动子）的信息，中间体（G8R启动子）和晚期（F17R启动子）的基因表达，而相比之下，暧昧早/晚推动者。 这些病毒可以作为用于英伟的工具stigating原型痘病毒，牛痘病毒的生命周期。现在还是有很多不知道的宿主 - 病毒相互作用，尽管其悠久的研究历史。牛痘是复杂的，生产超过200独特的蛋白质，其中有许多是免疫调节和主机对立。一旦感染病毒，牛痘病毒立即开始早期mRNA的转录。这是通过被在病毒粒子的包装上的病毒基因组中加载并在暂停状态保持到随后的感染RNA聚合酶和转录因子的促进。这种早期的表现主要生产所需的抑制宿主的免疫系统（mRNA的脱帽，dsRNA的封存和诱饵受体蛋白以及细胞凋亡的抑制剂，应激反应和收费，白细胞介素，和NF-κB信号）和基因组复制的蛋白质。早期表达也产生必要的中间表达的转录因子。中间表达式包括后期转录因子的表达。这表达级联导致生产结构和酶的病毒蛋白质在感染的晚期阶段，这是必要的成熟牛痘病毒体的完整组件。 我们组的荧光记者病毒可以快速进展痘病毒生物学的理解作出。一个在病毒学领域中最常见的和费时的方法是在生长测定。这通常涉及到感染细胞，通过裂解感染的细胞颁布一系列的处理，收获病毒，并通过噬菌斑测定法量化所得到的病毒滴度。使用此处描述的记者病毒允许病毒生长的实时测量，可以很容易检测和并行执行大量的处理之间进行比较。我们预计这组试剂将用于各种协议确定响应药物治疗，RNA干扰K的病毒基因表达的改变nockdown，或宿主范围的限制。 此方法也允许的规模比以前提供更大的高内涵分析，允许高通量的抗病毒的药物屏幕对于感兴趣专门定义的目标的阶段。虽然打击痘病毒感染许多潜在的治疗方法已经确定，唯一被FDA批准的治疗有效治疗痘病毒感染是无环核苷膦酸酯，西多福韦和牛痘免疫球蛋白9,10治疗。尽管消灭天花，1977年11，痘病毒仍然是一个显著威胁人类健康12。对天花病毒的广泛接种疫苗的终止，导致易感性增加其他痘病毒13。例如，中非以前接种疫苗保护的地区在猴痘病毒感染14激增。显著的关注也一直提出了关于潜在的易感性天花病毒的有意释放。由于目前可用的有限的疗法，目前迫切需要对新疗法的发展。这些记者病毒允许快速和高通量小分子抑制剂筛选抑制病毒复制的特定阶段。针对病毒表达阶段抑制目前不是目标抑制剂的鉴定将有利于联合疗法的发展与提高的效力。 要通过观察变化，每个阶段的基因表达进行搜集了关于牛痘细胞生物学。衰减通过化学或感染的宿主细胞的遗传操作通常以降低病毒滴度表示。然而，通过比较改变的病毒表达级联的每一级，可以得到怎样的病毒适应度的影响更完整的理解由一个特定的治疗主编。这些数据已经显示出与传统的病毒滴度输出良好的相关性，但提供更详细的机理信息，以及高吞吐量能力7。

<table border="0" cellpadding="0" cellspacing="0" width="1249">
	<tbody>
		<tr height="24">
			<td height="24" style="height:24px;width:747px;">Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td style="width:231px;">Gibco</td>
			<td style="width:104px;">11995-065</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Fetal Bovine Serum - Optima, Heat Inactivated (FBS)</td>
			<td>Atlanta Biologicals</td>
			<td>S12450H</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">200 mM L-Glutamine, (L-glut)</td>
			<td>Gibco</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">96 Well Flat Clear Bottom Black Polystyrene TC-Treated Microplates</td>
			<td>Corning</td>
			<td>3603</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">384 Well Flat Clear Bottom Black Polystyrene TC-Treated Microplates</td>
			<td>Corning</td>
			<td>3712</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Trypsin, 2X, Sterile, Irradiated</td>
			<td>Worthington Biochemical Corp.</td>
			<td>TRLVMF</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Phosphate-Buffered Saline (PBS)</td>
			<td>Gibco</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Dimethyl Sulfoxide, Cell Culture (DMSO)</td>
			<td>American Bioanalytical</td>
			<td>AB03091</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">1-&#946;-D-Arabinofuranosylcytosine&#160; (AraC), MW= 280 g/mol</td>
			<td>SigmaAldrich Co</td>
			<td>C6645</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">isatin &#946; -thiosemicarbazone (IBT), MW= 234 g/mol</td>
			<td>Fisher Scientific</td>
			<td>NC9075202</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Rifampicin, MW= 823 g/mol</td>
			<td>SigmaAldrich Co</td>
			<td>R3501</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">ST-246, MW= 376 g/mol</td>
			<td>SIGA Labs, Corvallis, OR</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">8% Paraformaldehyde (formaldehyde) aqueous solution</td>
			<td>Electron Microscopy Sciences</td>
			<td>157-8</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">TempPlate RT optically clear film</td>
			<td>USA Scientific</td>
			<td>2978-2700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Opti-MEM Reduced Serum Medium</td>
			<td>Gibco</td>
			<td>31985-070</td>
			<td></td>
		</tr>
	</tbody>
</table>

vaccinia reporter viruses for quantifying viral function at all stages of gene expression

痘病毒是一个家族的双链DNA病毒，包括有源人类病原体如猴痘，软疣contagiousum和Contagalo病毒。该系列还包括天花病毒，天花。由于痘病毒复制的复杂性，许多问题仍然对他们的基因表达的策略。在这篇文章中，我们描述的重组痘苗病毒，使之在高通量形式病毒基因表达的单一和多级实时测量的概念和用法。这是通过使用光谱不同的荧光蛋白作为报告的每个病毒复制的三个阶段启用。这些病毒提供了高的信号噪声比，同时保持阶段特异性表达模式，使板为基础的检测和病毒繁殖和复制的显微观察。这些工具具有用途抗病毒药发现，病毒 - 宿主相互作用的研究，和进化的生物迟缓。

因为这些病毒的典型用法的示例，三重荧光记者病毒被用来抑制的几个定义良好的痘病毒抑制剂的点进行比较。 将HeLa细胞接种在组织培养物处理的黑色壁透明平底96孔板中并孵育过夜。汇合的单层细胞感染在感染使用任一三重记者病毒的多重10（MOI）（TRPV， 表1）或启动子的金星（PLV）所详述的板图（ 图1）上。加入含有治疗感染媒体取得0.1％DMSO，3....

Watch this Scientific Journal Video about Vaccinia Reporter Viruses for Quantifying Viral Function at All Stages of Gene Expression at JoVE.com

Vaccinia Reporter Viruses for Quantifying Viral Function at All Stages of Gene Expression

我们描述了一种荧光报告牛痘病毒，使病毒的感染性和基因表达的实时测量通过的光谱不同的荧光报告基团的阶段特异性表达的用法。我们详细的板基础的方法准确地确定在哪个病毒复制受到影响响应小分子抑制阶段。

牛痘病毒记者量化病毒功能在基因表达的各个阶段

Poxviruses are a family of double stranded DNA viruses that include active human pathogens such as monkeypox, molluscum contagiousum, and Contagalo virus. ...

Research

JoVE Journal

Immunology and Infection

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Multivirus特异性T细胞的生成，预防/治疗异基因造血干细胞移植后的病毒感染

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully ...

科研

免疫与感染

'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens

“发光法”的分类检测细菌病原体的记者噬菌体

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. ...

Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays

所有流感的定量分析类型使用通用抗体的一种病毒Hemagglutinins和Neuraminidases，在简单的狭缝杂交分析

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle ...

Cost-effective Method for Microbial Source Tracking Using Specific Human and Animal Viruses

微生物源使用特定的人类和动物病毒的追踪成本效益的方法

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant ...

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 ...

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

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 ...

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

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 ...

Mouse Fetal Liver Culture System to Dissect Target Gene Functions at the Early and Late Stages of Terminal Erythropoiesis

小鼠胎肝文化系统解剖靶基因功能的终端红细胞生成的早期和晚期

Erythropoiesis involves a dynamic process that begins with committed erythroid burst forming units (BFU-Es) followed by rapidly dividing erythroid colony ...

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

重组流感疫苗的嵌合体小鼠模型鼻腔给药研究黏膜免疫

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...