Name: arbovirus infections as screening tools for the identification of viral immunomodulators and host antiviral factors
Uploaded: 2018-09-13T14:30:00
Description: Watch this Scientific Journal Video about Arbovirus Infections As Screening Tools for the Identification of Viral Immunomodulators and Host Antiviral Factors at JoVE.com

危险品得到了德克萨斯大学西南医学中心资助的学者计划提供的资金支持。作者感谢迈克尔 Whitt (田纳西州大学健康科学中心) 和肖恩. 惠兰 (哈佛医学院) 提供 VSV-DsRed 和 VSV-卢克。作者还感谢加里 Luker (密歇根大学医学院) 为 VACV--FL-GFP 菌株的亲切礼物。

1. 一般毒蛾(LD652) 细胞和病毒培养
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LD652 细胞培养与电镀
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<li>培养蛾LD652 细胞, 维持生长培养基 (材料表) 中的细胞单层, 在正常大气下孵化27摄氏度。保持细胞在10厘米组织培养处理的菜肴和通过细胞后, 达到80% 融合。</li>
		<li>对板, 去除黏附的 LD652 细胞从板材通过吹打媒介反复地到单层 (这些细胞不要求 trypsinization 去除), 并且稀释样品在生?...

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在这里, 我们描述了简单的荧光和发光的检测, 以筛选条件, 拯救 VSV 复制在限制性的鳞翅目细胞培养。VSV 在鳞翅目细胞中的流产感染在 VSV 基因表达的检测中产生了良好的信噪比。例如, 裂解物从单 VSV 感染中检测到的 LU 信号比模拟感染的裂解物高1000倍, 然而这些信号在72小时的时间内只改变了大约两个。相比之下, VSV 的复合感染与 VACV 增强的 LU 信号相比, 300 倍于单 VSV-卢克感染 72 hpi。因此, VSV 救...

病毒在特定宿主中进行高效复制的能力, 部分取决于支持病毒输入和复制1的宿主细胞因子的可用性。病毒宿主范围也可以由病毒抵御细胞抗病毒防御的能力决定, 否则会阻碍病毒复制2,3。这是这些复杂的病毒宿主交互的结果, 最终决定病毒是否能够在特定主机上完成其生命周期。鉴于病毒复制的出现可能会对宿主造成致病性后果, 制定实验策略以进一步了解可能导致流产和生产效率平衡的关键病毒宿主相互作用至关重要。感染。阐明病毒宿主相互作用的分子特征将有助于制定新的和替代的抗病毒治疗策略。
随着 RNA 干扰 (核糖核酸)4、5和基因组编辑工具 (CRISPR-Cas9、锌指核酸、TALENs)6、7的出现, 改变细胞因子在全基因组范围内的表达, 并探讨这些改变对病毒复制的影响。事实上, 在无脊椎动物和脊椎动物宿主细胞类型中进行了大量的基于 rna 和基因组编辑的屏幕, 揭示了病毒宿主相互作用的新方面8、9、10,11,12. 这些屏幕通常使用病毒编码的记者, 如萤火虫荧光素酶 (卢克) 或荧光蛋白 (e., GFP, DsRed), 提供了方便的手段定量评估病毒基因表达作为一个读数病毒复制9,12。这一策略允许研究人员确定宿主因素, 它们要么促进或拮抗病毒复制, 在病毒报告信号9,12中分别增加或减少。然而, 在绝大多数情况下, 这些屏幕使用的病毒是很好地适应了他们正在研究的宿主细胞类型。虽然这一策略对于理解病毒病原体及其自然宿主之间的进化关系是很重要的, 但对于它们在揭示宿主抗病毒因子方面的应用却构成了根本的担忧。在这些情况下, 正在寻找病毒报告器上的信号增强, 或者是一种通常会阻碍病毒复制的细胞因子的失活。首先, 如果病毒已经能够在控制条件下检查的宿主细胞中进行强力复制, 则屏幕的动态范围 (即区分背景和增强的病毒报告信号的能力) 可能会受到限制。第二, 这个问题进一步复杂化的情况下, 病毒是良好的适应宿主细胞, 并有效地对抗主机防御路径, 是在屏幕上的目标。
由于上述对传统病毒宿主交互筛选方法的关注, 我们开发了一种新的模式来研究病毒宿主相互作用, 利用鳞翅目昆虫细胞中自然流产的虫媒病毒感染。这一策略来源于一项观察, 即研究良好的人类虫媒病毒, VSV, 在来自吉普赛蛾 (L. 蛾)13的细胞中经历了一次流产感染。VSV 是自然传播的 dipteran 昆虫 (即, 沙蝇) 的哺乳动物宿主, 并已证明实验性感染广泛的无脊椎动物和脊椎动物宿主在细胞培养和体内14。VSV 的 11 kb 负感单链 RNA 基因组编码五 subgenomic 基因, 它们分别转化为构成包络病毒的蛋白质。然而, VSV 反向遗传系统已经允许创建复制能力的菌株编码卢克或荧光蛋白, 除了五自然 VSV 基因产品15,16,17。由于这些记者的蛋白质没有纳入 VSV 病毒, 他们提供了一个方便的读数 VSV 基因表达, 发生后进入。我们以前曾表明, 使用 VSV 菌株编码 GFP 或卢克, VSV 基因表达严重限制在 LD652 细胞的进入, VSV 的滴度不会增加72小时后感染 (hpi)。相比之下, LD652 细胞与 VSV 和哺乳动物痘, 痘苗病毒 (VACV) 的复合感染, 导致在 VSV 基因表达和效价的这一时间点的对数增加。VACV 在 LD652 细胞感染中经历早期基因表达、DNA 复制和晚期基因表达, 但 VACV 复制周期最终由于不完全病毒形态发生18而流产。VACV 编码 > 200 蛋白的大 ~ 192 kb DNA 基因组, 其中许多显示免疫调节特性, 通过抑制宿主免疫应答19来促进病毒复制。因此, 我们假设 VACV 复合感染在 LD652 细胞中 VSV 复制的 "营救" 可能是由 VACV 免疫调节剂引起的, 抑制L. 蛾响应通常限制 VSV 复制。为支持这一点, LD652 细胞与宿主 RNA 聚合酶 II 抑制剂放线菌 D 的治疗也拯救 VSV 在 LD652 细胞复制, 表明转录依赖主机响应阻止 VSV 复制后进入13。
上述观察表明, LD652 细胞对 VSV 感染的自然限制性, 在筛选增强 VSV 编码的报告信号的条件时可能提供相对较低的背景 (即那些抑制宿主抗病毒防御)。在这里, 我们提供的方法, 以荧光或卢克为基础的化验, 以筛选条件, 以缓解 VSV 限制鳞翅目细胞。首先, 我们展示了如何使用这些检测方法来识别维拉利编码的免疫调节因子, 在复合感染实验中或通过候选病毒因子的异位表达打破 VSV 限制。举例来说, 我们说明了我们如何使用这些筛选技术来识别痘编码的 A51R 蛋白作为一种新的免疫调节因子家族, 在没有其他痘因素13的情况下拯救 VSV 复制。其次, 我们说明了如何在限制性 VSV-LD652 细胞感染中进行 rna 干扰筛选, 以直接识别虫媒病毒限制13中所涉及的真核宿主因素。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>6-well tissue culture plates</td>
			<td>CELLTREAT</td>
			<td>229106</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well tissue culture plates</td>
			<td>CELLTREAT</td>
			<td>229124</td>
			<td></td>
		</tr>
		<tr>
			<td>10 cm tissue culture dishes</td>
			<td>Corning</td>
			<td>C430167</td>
			<td></td>
		</tr>
		<tr>
			<td>Grace&#8217;s Insect Medium</td>
			<td>Sigma</td>
			<td>G8142</td>
			<td></td>
		</tr>
		<tr>
			<td>EX-CELL 420</td>
			<td>Sigma</td>
			<td>14420C</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum - Optima</td>
			<td>Atlanta Biologicals</td>
			<td>S12450</td>
			<td></td>
		</tr>
		<tr>
			<td>Growth medium</td>
			<td></td>
			<td></td>
			<td>1:1 mixture of Grace&#39;s Insect Medium and EX-Cell 420 Serum-Free Medium also containing 1 % antibiotic-antimycotic solution and 10 % Fetal bovine serum</td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic Solution (100&#215;)</td>
			<td>Sigma</td>
			<td>A5955</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate Buffered Saline (DPBS)</td>
			<td>Sigma</td>
			<td>D8662</td>
			<td></td>
		</tr>
		<tr>
			<td>Serum Free Media (SFM)</td>
			<td>Thermo Fisher</td>
			<td>10902096</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytosine arabinoside</td>
			<td>Sigma</td>
			<td>C1768</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfection reagent</td>
			<td>Thermo Fisher</td>
			<td>10362100</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning cellgro DMSO (Dimethyl Sulfoxide)</td>
			<td>Corning</td>
			<td>25950CQC</td>
			<td></td>
		</tr>
		<tr>
			<td>Reporter lysis buffer 5X</td>
			<td>Promega</td>
			<td>E3971</td>
			<td></td>
		</tr>
		<tr>
			<td>Luciferase Assay Reagent</td>
			<td>Promega</td>
			<td>E1483</td>
			<td></td>
		</tr>
		<tr>
			<td>96-Well Microplates</td>
			<td>Corning</td>
			<td>3915</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-FLAG antibody</td>
			<td>Wako</td>
			<td>014-22383</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-firefly luciferase antibody</td>
			<td>Abcam</td>
			<td>ab21176</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-actin antibody</td>
			<td>Sigma</td>
			<td>A2066</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-VSV M</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Dr. John Connor (Boston University)</td>
		</tr>
		<tr>
			<td>Mouse anti-VACV I3L</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Dr. David Evans (University of Alberta)</td>
		</tr>
		<tr>
			<td>8-well Chambered dish</td>
			<td>Lab-Tek II</td>
			<td>155409</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell viability dye</td>
			<td>Thermo Fisher</td>
			<td>C12881</td>
			<td></td>
		</tr>
		<tr>
			<td>FLUOstar microplate reader</td>
			<td>BMG Labtech</td>
			<td>FLUOstar</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Olympus</td>
			<td>FV10i-LIV</td>
			<td></td>
		</tr>
		<tr>
			<td>Image analysis software</td>
			<td>Olympus</td>
			<td>v1.18</td>
			<td>cellSens software</td>
		</tr>
		<tr>
			<td>Eppendorf 5702 ventilated centrifuge</td>
			<td>Eppendorf</td>
			<td>22628102</td>
			<td></td>
		</tr>
		<tr>
			<td>Odyssey Fc Infrared Imaging System</td>
			<td>Li-COR Biosciences</td>
			<td>Odyssey Fc</td>
			<td></td>
		</tr>
		<tr>
			<td>LD652 cells</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Dr. Basil Arif (Natural Resources Canada)</td>
		</tr>
		<tr>
			<td>BSC-40 cells</td>
			<td>ATCC</td>
			<td>CRL-2761</td>
			<td></td>
		</tr>
		<tr>
			<td>BHK cells</td>
			<td>ATCC</td>
			<td>CCL-10</td>
			<td></td>
		</tr>
		<tr>
			<td>HeLa cells</td>
			<td>ATCC</td>
			<td>CCL-2</td>
			<td></td>
		</tr>
		<tr>
			<td>BSC-1 cells</td>
			<td>ATCC</td>
			<td>CCL-26</td>
			<td></td>
		</tr>
		<tr>
			<td>in vitro transcription and purification kit</td>
			<td>Thermo Fisher</td>
			<td>AM1626</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR purification kit</td>
			<td>Qiagen</td>
			<td>28104</td>
			<td></td>
		</tr>
	</tbody>
</table>

arbovirus infections as screening tools for the identification of viral immunomodulators and host antiviral factors

RNA 干扰-和基因组编辑为基础的筛选平台已广泛用于识别宿主细胞因子, 限制病毒复制。然而, 这些屏幕通常进行的细胞, 自然纵容的病毒病原体正在研究中。因此, 在控制条件下的病毒的健壮复制可能会限制这些屏幕的动态范围。此外, 如果病毒适应宿主并能够抵抗抗病毒防御, 这些屏幕可能无法轻易识别出限制病毒复制的细胞防御通路。在本文中, 我们描述了一个新的范式, 以探索病毒宿主相互作用, 通过使用的屏幕, 中心的自然流产感染的虫媒病毒, 如水泡性口炎病毒 (VSV)。尽管 VSV 能够在广泛的 dipteran 昆虫和哺乳动物寄主中进行复制, VSV 还是经历了进入后, 在鳞翅目昆虫 (毒蛾) 的各种细胞系中流产后的感染。然而, 当宿主细胞抗病毒防御被破坏时, 这些流产的 VSV 感染可以被 "拯救"。我们描述如何 VSV 菌株编码方便的报告基因和限制性的L. 蛾细胞系可以配对设置屏幕, 以确定宿主因素参与虫媒病毒限制。此外, 我们还显示了这些筛选工具的效用, 以确定维拉利编码因素, 拯救 VSV 复制期间复合感染或通过异位表达, 包括那些编码的哺乳动物病毒。VSV 复制在L. 蛾细胞中的自然限制在筛选促进 VSV 救援的条件时提供了高信噪比, 从而使使用简单的荧光和荧光检测方法能够监测VSV 复制的更改。这些方法对于了解宿主抗病毒反应与病毒免疫规避因素之间的相互作用是有价值的。

作为活体细胞成像应用监测 VSV 救援 VACV 复合感染的例子, LD652 细胞被镀在一个8井腔内的盘子里, 然后在 VACV--GFP (语言 = 25) 的存在或不存在的情况下, 在 VSV-DsRed (语言 = 1) 中被模拟感染或感染。因为 VSV-DsRed 表达 DsRed 作为一个自由的蛋白质, 并没有融合到结构 VSV 蛋白 (图 1A), 它只是检测后, VSV 进入和基因表达启动。所有细胞都被标记为细胞活性染料, ?...

Watch this Scientific Journal Video about Arbovirus Infections As Screening Tools for the Identification of Viral Immunomodulators and Host Antiviral Factors at JoVE.com

Arbovirus Infections As Screening Tools for the Identification of Viral Immunomodulators and Host Antiviral Factors (Video) | JoVE

在这里, 我们提出了识别 1) 病毒编码的免疫调节剂的协议, 以促进虫媒病毒复制和 2) 限制虫媒病毒复制的真核宿主因素。这些荧光和发光的方法, 使研究人员能够迅速获得定量读数的虫媒病毒复制在简单的检测, 低信噪比。

虫媒病毒感染作为鉴定病毒免疫调节剂和宿主抗病毒因子的筛选工具

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. ...

Research

JoVE Journal

Immunology and Infection

Arbovirus Infections As Screening Tools for the Identification of Viral Immunomodulators and Host Antiviral Factors

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells (Video) | JoVE

RNAi筛选参与宿主因素牛痘病毒感染使用果蝇细胞

Viral pathogens represent a significant public health threat; not only can viruses cause natural epidemics of human disease, but their potential use in ...

科研

免疫与感染

Introducing Shear Stress in the Study of Bacterial Adhesion

Introducing Shear Stress in the Study of Bacterial Adhesion (Video) | JoVE

在细菌粘附的研究剪应力

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

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

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

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, ...

VIGS-Mediated Forward Genetics Screening for Identification of Genes Involved in Nonhost Resistance

VIGS-Mediated Forward Genetics Screening for Identification of Genes Involved in Nonhost Resistance (Video) | JoVE

VIGS介导的正向遗传学筛选参与非寄主抗性基因鉴定


	Nonhost disease resistance of plants against bacterial pathogens is controlled by complex defense pathways. Understanding this mechanism is important ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants (Video) | JoVE

生成和多表型高内涵筛选<em&gt;贝氏柯克斯体</em&gt;转座子突变体

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds

Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds (Video) | JoVE

早期的病毒进入测定的鉴定和抗病毒化合物的评价

Cell-based systems are useful for discovering antiviral agents. Dissecting the viral life cycle, particularly the early entry stages, allows a mechanistic ...

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) | 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. ...

Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface

Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface (Video) | JoVE

人胎盘及蜕膜器官培养研究在母胎界面感染

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is ...

Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells

Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells (Video) | JoVE

在参与宿主因素的高通量筛选基于显微镜的检测方法<em&gt;布鲁氏菌</em&gt; Hela细胞感染

Brucella species are facultative intracellular pathogens that infect animals as their natural hosts. Transmission to humans is most commonly caused by ...