JoVE Journal > Immunology and Infection
Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
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Immunology and Infection

高灵敏检测方法的沙粒，细胞附着的测量

Published: March 02, 2016
doi:
10.3791/53682
,
1Department of Medicine, Division of Immunobiology,University of Vermont, 2Department of Microbiology & Molecular Genetics,University of Vermont

Summary

The first step in the arenavirus life cycle is attachment of viral particles to host cells. We report a quantitative (q)RT-PCR-based assay for ultrasensitive detection and quantitation of arenavirus attachment events.

Abstract

Arenaviruses are a family of enveloped RNA viruses that cause severe human disease. The first step in the arenavirus life cycle is attachment of viral particles to host cells. While virus-cell attachment can be measured through the use of virions labeled with biotin, radioactive isotopes, or fluorescent dyes, these approaches typically require high multiplicities of infection (MOI) to enable detection of bound virus. We describe a quantitative (q)RT-PCR-based assay that measures Junin virus strain Candid 1 attachment via quantitation of virion-packaged viral genomic RNA. This assay has several advantages including its extreme sensitivity and ability to measure attachment over a large dynamic range of MOIs without the need to purify or label input virus. Importantly, this approach can be easily tailored for use with other viruses through the use of virus-specific qRT-PCR reagents. Further, this assay can be modified to permit measurement of particle endocytosis and genome uncoating. In conclusion, we describe a simple, yet robust assay for highly sensitive measurement of arenavirus-cell attachment.

Introduction

沙粒病毒是那些主要保持在啮齿类动物中自然1-3笼罩，单链RNA病毒家族。虽然这些病毒通常建立在啮齿动物水库1,2无症状，持续感染，可导致人类3严重疾病。重要的致病品种包括新世界胡宁病毒（JUNV）4和旧世界拉沙病毒5，分别是阿根廷出血热在南美和拉沙热的病原体在非洲。淋巴细胞脉络丛脑膜炎病毒（LCMV），家庭6的原型病毒，具有全球分销并负责多种疾病状态，包括在免疫活性个体6无菌性脑膜炎，在发育中的胎儿7严重的先天缺陷，或在免疫抑制高致死以下固体器官移植8,9个人。缺乏美国食品和药物管理局（FDA）批准的疫苗或打击这些病毒有效的抗病毒药物凸显迫切需要制定新的策略来治疗针对这些新兴/再出现的病原体。

的沙粒病毒基因组由两个单链RNA区段，所述大（L）（〜7.2 kb的）和小的（S）（〜3.6kb的）段10。而L区段编码的病毒聚合酶（L）和基质蛋白（Z） – 在S段编码病毒核蛋白（NP）和包膜糖蛋白（GP）。的L和S的基因组RNA区段（vRNAs）被打包成病毒颗粒10-12。沙粒病毒感染的最初步骤是病毒颗粒的附着通过病毒GP和其相应的宿主细胞受体的，为JUNV，包括人类的转铁蛋白受体1（hTfR1）13,14之间的相互作用对宿主细胞。以下附件，JUNV颗粒通过网格蛋白介导的内吞作用15取入细胞。颗粒然后投放到晚期内体，其中该隔室的低pH触发的GP 16,17的激活。一旦被激活，在GP-编码的融合肽插入到内体膜，病毒和内体膜之间引发熔合。成功融合结果在释放病毒粒子包装vRNAs的进入细胞质，在那里它们作为用于基因组复制和转录的模板。当产生病毒结构蛋白和vRNAs的足够数量时，新粒子组装和从感染细胞芽完成生命周期18。

为了促进新战略的发现到治疗目标致病沙粒病毒，我们集团一直专注于主机因素是主机的病毒的繁殖至关重要，但可有可无的鉴定。在这种情况下，限定由这种宿主因素控制病毒生命周期的精确阶段（s）是必要的引导的抗病毒策略的发展。在此，我们描述了基于RT-PCR的定量（q）的测定法可用于测量用于识别选定的宿主的蛋白和/或抗病毒分子是否影响病毒进入19的该初始阶段的目的沙粒病毒-细胞附着。衡量沙粒病毒细胞附着替代方法有特色生物素标记20，荧光染料21，或者放射性同位素22的病毒体。缺点这些方法可包括用于大量输入病毒的，使用放射性的（ 例如高感染复数（MOI）的多重），和/或附加的处理步骤以制备输入病毒（ 例如浓缩和/或病毒的标签）的要求。特别是，使用高MOI的使得难以区分生产性与非生产性附着事件15,23。这里描述的基于定量RT-PCR的检测可以用尽可能少的20个牌匾检测附件事件明台病毒（PFU就能），换算成0.0004的MOI为48孔板。因此，测定的强灵敏度克服了高感染复数的要求以及任何病毒标签或浓度的步骤。此外，该测定可以是ⅰ）适于测量病毒体的内吞作用以及病毒基因组的释放入细胞质以下融合和ii）适于通过病毒特异性的qRT-PCR试剂的发展与其他病毒使用（例如，参见24-27）。

Protocol

注意：这是非常关键的，所描述的协议，采用严格的预PCR技术（见28关于如何建立一个-PCR实验室前和如何使用好-PCR技术前进行实验的精彩概述）进行。至关重要的是，所有的试剂和仪器都是免费的含有定量PCR靶序列扩增的DNA，并有适当的控制到位，以检测这种污染。该协议的概述示于图1。 1.准备在48孔板媒体和种子细胞通过加入50ml制备含有10％胎牛血清，1％青?…

Representative Results

为了证明qPCR分析的灵敏度和动态范围，如在该协议的第4部分中所述编码JUNV C＃1（范围5-5×10 7个拷贝）的NP基因的质粒的已知量经受的qPCR。该测定是敏感的，能够可靠地检测作为目标核酸的5个拷贝（ 图2）少。此外，该测定的动态范围大并且， 如图2，可覆盖至少7日志模板。虽然未示出，我们已检测出核酸的多达5×10 9个拷贝。 </p…

Discussion

我们描述的协议是简单的和健壮提供了以下关键的考虑因素观察。首先，严格预-PCR技术必须使用（参见第28一个很好的回顾）。至关重要的是，所有的试剂和仪器都是免费的含有定量PCR靶序列扩增的DNA，并有适当的控制到位，以检测这种污染。第二，对于协议，病毒，细胞和介质的病毒 – 细胞附着部分必须在4℃下保持，以防止表面结合的病毒粒子的内吞作用。第三，收集用于RNA提取的细?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

我们感谢马库斯·塔利，弥敦道罗伊，克里斯托弗·齐格勒，艾米莉布鲁斯，便雅悯王为通过赠款T32 AI055402（JK），R21 AI088059（JB），以及P20RR021905有益的探讨和美国国立卫生研究院支持这些研究（JB） 。

Materials

DMEM Life Technologies 11965-118
Penicillin-Streptomycin Life Technologies 15140-163 100x
HEPES Buffer Solution Life Technologies 15630-130 100x
PBS pH 7.4 Life Technologies 10010-023 1x
Vero E6 cells American Type Culture Collection CRL-1586
Costar 48-well plate Corning 3548
RNeasy mini kit Qiagen 74106 do not mix buffers RLT or  RW1 with bleach
QIAshredder homogenizer Qiagen 79654
Taqman Universal PCR Master Mix Life Technologies 4326614
Multiscribe Reverse Transcriptase (50U/µl) Life Technologies 4311235
dNTP Mixture (10 mM; 2.5 mM each dNTP) Life Technologies N808-0260
GeneAmp 10X PCR Buffer II and 25 mM MgCl2 solution Life Technologies N808-0010
RNase Inhibitor (5000U/100 µl) Life Technologies N808-0119
RT primer 5’-AAGGGTTTAAAAATGGTAGCAGAC-3’ Integrated DNA Technologies 250 nmol DNA oligo standard desalting
Forward qPCR primer  5’-CATGGAGGTCAAACAACTTCCT-3’ Life Technologies 4304971 standard desalting
Reverse qPCR primer 5’-GCCTCCAGACATGGTTGTGA-3’ Life Technologies 4304971 standard desalting
TaqMan Probe 5’-6FAM-ATGTCATCGGATCCTT-MGBNFQ-3’ Life Technologies 4316033 HPLC purified
MicroAmp Fast Optical 96-well reaction plate (0.1 mL) Life Technologies 4346906
StepOnePlus Real-Time PCR System Life Technologies 4376598 or other qPCR instrument
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Tags

ArenavirusVirus AttachmentVirus EndocytosisVirus FusionVero E6 CellsJunin Candid 1 VirusFocus Forming UnitsPlaque Forming UnitsRNA IsolationCell Lysis
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Klaus, J. P., Botten, J. Highly Sensitive Assay for Measurement of Arenavirus-cell Attachment. J. Vis. Exp. (109), e53682, doi:10.3791/53682 (2016).
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