哺乳动物正呼肠孤病毒传染性亚病毒颗粒诱导的溶血试验

Anthony J. Snyder; Pranav Danthi

doi:10.21769/BioProtoc.2701

Improve Research Reproducibility A Bio-protocol resource

提交稿件
订阅
登录
/
注册
- 个人主页
- 编辑个人信息
- 修改密码
- 退出
CN
- EN - English
- CN - 中文

Peer-reviewed

Infectious Subviral Particle-induced Hemolysis Assay for Mammalian Orthoreovirus

哺乳动物正呼肠孤病毒传染性亚病毒颗粒诱导的溶血试验

AS Anthony J. Snyder

PD Pranav Danthi email

发布: 2018年01月20日第8卷第2期 DOI: 10.21769/BioProtoc.2701 浏览次数: 5647

评审: Yannick DebingKristin ShinglerSmita Nair

PDF

Q&A

引用

Cited by

参见作者原研究论文

The authors used this protocol in:

Cover of Journal of Virology, featuring study using the protocol.

Mar 2016

实验方案合集

Cell Imaging - A Special Collection for Cell Bio 2023

相关实验方案

iSLK细胞系的常规维护与KSHV再活化操作

Ariana C. Calderón-Zavala [...] Ekaterina E. Heldwein

2025年06月05日 1197 阅读

诱导型HIV-1库削减检测（HIVRRA）：用于评估外周血单个核细胞中HIV-1潜伏库清除策略毒性与效力的快速敏感方法

Jade Jansen [...] Neeltje A. Kootstra

2025年07月20日 1544 阅读

基于转化相关重组的酵母体系组装与诱变人冠状病毒OC43基因组

Brett A. Duguay and Craig McCormick

2025年08月20日 1452 阅读

Abstract

Mammalian orthoreovirus (reovirus) utilizes pore forming peptides to penetrate host cell membranes. This step is essential for delivering its genome containing core particle during viral entry. This protocol describes an in vitro assay for measuring reovirus-induced pore formation.

Keywords: Virology (病毒学)

Reoviridae (呼肠孤病毒科)

Mammalian orthoreovirus (哺乳动物正呼肠弧病毒)

Viral entry (病毒进入)

Membrane penetration (膜穿透)

Hemolysis (溶血)

Background

Reoviruses are nonenveloped, double-stranded RNA viruses that are composed of two concentric protein shells: the inner capsid (core) and the outer capsid (Dryden et al., 1993; Zhang et al., 2005; Dermody et al., 2013). Following attachment, virions are endocytosed (Borsa et al., 1979; Ehrlich et al., 2004; Maginnis et al., 2006; Maginnis et al., 2008) and host cathepsin proteases degrade the σ3 outer capsid protein (Chang and Zweerink, 1971; Silverstein et al., 1972; Borsa et al., 1981; Sturzenbecker et al., 1987; Dermody et al., 1993; Baer and Dermody, 1997; Ebert et al., 2002). This process generates a metastable intermediate, called infectious subviral particle (ISVP), in which the cell penetration protein, µ1, is exposed (Dryden et al., 1993). Reovirus ISVPs undergo a second conformational change to deposit the genome- containing core into the host cell cytoplasm. The altered particle is called ISVP* (Chandran et al., 2002). ISVP-to-ISVP* conversion culminates in the release of µ1-derived pore forming peptides (Nibert et al., 1991; Zhang et al., 2005; Chandran et al., 2002; Odegard et al., 2004; Nibert et al., 2005; Agosto et al., 2006; Ivanovic et al., 2008). The released peptides form pores within endosomal membranes, which are thought to mediate core delivery (Agosto et al., 2006; Ivanovic et al., 2008; Zhang et al., 2009).

Many of the conformational changes that define reovirus entry can be recapitulated in vitro: (i) ISVPs are produced by digesting purified virions with chymotrypsin (Joklik, 1972; Borsa et al., 1973a), and (ii) ISVP* formation can be induced using heat (Middleton et al., 2002), large monovalent cations (Borsa et al., 1973b), µ1-derived peptides (Agosto et al., 2008), red blood cells (Chandran et al., 2002; Sarkar and Danthi, 2010), or lipids (Snyder and Danthi, 2015 and 2016). Thus, questions related to reovirus entry are studied using biochemical and cell-based approaches. In this protocol, we describe an in vitro assay that recapitulates ISVP-to-ISVP* conversion and subsequent pore formation.

Materials and Reagents

Pipette tips
1. 0.1-10 µl capacity (USA Scientific, catalog number: 1111-3700 )
2. 1-200 µl capacity (VWR, catalog number: 89079-474 )
3. 100-1,250 µl capacity (VWR, catalog number: 53508-924 )
PCR 8-well tube strips (VWR, catalog number: 20170-004 )
50 ml centrifuge tube (VWR, catalog number: 89039-660 )
1.7 ml microcentrifuge tubes (MIDSCI, catalog number: AVSS1700 )
Vacuum driven and disposable bottle top 0.22 µm filter (Merck, catalog number: SCGPT05RE )
Flat bottom, 96-well plate (Greiner Bio One International, catalog number: 655180 )
Purified reovirus stocks (see Berard and Coombs, 2009; Kobayashi et al., 2010 for propagation and purification procedures)
Crushed ice
Standard SDS-PAGE materials and reagents (e.g., 10% SDS-polyacrylamide mini gels)
Coomassie Brilliant Blue stain and destain solutions (Bio-Rad Laboratories, catalog number: 1610435 )
Citrated bovine calf blood (Colorado Serum Company, catalog number: 31023 )
Bleach (Biz4USA, Janitorial Supplies, catalog number: CLO30966CT )
2-Amino-2-(hydroxymethyl)-1,3-propanediol (Tris) (MP Biomedicals, catalog number: 02103133 )
Sodium chloride (NaCl) (Merck, catalog number: SX0420-3 )
0.1 N hydrochloric acid (Sigma-Aldrich, catalog number: 2104 )
0.1 N sodium hydroxide (Sigma-Aldrich, catalog number: 2105 )
Nα-p-tosyl-L-lysine chloromethyl ketone (TLCK)-treated chymotrypsin (Worthington Biochemical, catalog number: LS001432 )
Phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: P7626 )
Isopropyl alcohol (Avantor Performance Materials, Macron, catalog number: 3032-02 )
Dulbecco’s phosphate buffered saline (Thermo Fisher Scientific, Gibco^TM, catalog number: 21600044 )
Magnesium chloride hexahydrate (MgCl₂·6H₂O) (Sigma-Aldrich, catalog number: M9272 )
Ultrapure DNase/RNase-free distilled H₂O (Thermo Fisher Scientific, Invitrogen^TM, catalog number: 10977015 )
Triton X-100 (TX-100) (Sigma-Aldrich, catalog number: X100 )
50% bleach (see Recipes)
Virus storage buffer (VB) (see Recipes)
2 mg/ml TLCK-treated chymotrypsin (see Recipes)
100 mM phenylmethylsulfonyl fluoride (PMSF) (see Recipes)
Phosphate buffered saline supplemented with 2 mM MgCl₂ (PBS^Mg) (see Recipes)
10% Triton X-100 (TX-100) (see Recipes)

Equipment

Personal protective equipment (PPE)
1. Laboratory coat
2. Gloves
3. Eye protection
Biosafety level 2 (BSL-2) laboratory facility
BSL-2 certified tissue culture hood
Solid and liquid waste containers
Autoclave
Vacuum pump and aspirator
Ice bucket
-20 °C freezer
Micropipettes
1. 0.1-2.5 µl capacity (Eppendorf, catalog number: 3123000012 )
2. 2-20 µl capacity (Eppendorf, catalog number: 3123000039 )
3. 20-200 µl capacity (Eppendorf, catalog number: 3123000055 )
4. 100-1,000 µl capacity (Eppendorf, catalog number: 3123000063 )
Digital pH meter (VWR, model: SB70P )
Digital laboratory balance (Mettler Toledo, model: PB1502-S )
NanoDrop spectrophotometer (Thermo Fisher Scientific, Thermo Scientific^TM, model: ND-1000 )
Hot plate stirrer (VWR, catalog number: 12365-382 )
Magnetic stir bar (VWR, catalog number: 58948-273 )
Microcentrifuge (Eppendorf, model: 5424 )
Thermal cycler (Bio-Rad Laboratories, model: S1000^TM )
Temperature controlled water bath (VWR, catalog number: 89501-466 )
Gel imaging system (LI-COR, model: Odyssey^® Classic )
Microplate reader (Molecular Devices, model: FilterMax F5 Multi-Mode )
250 ml glass beaker (VWR, catalog number: 89000-204 )
1,000 ml glass beaker (VWR, catalog number: 89000-212 )
100 ml graduated cylinder (VWR, catalog number: 65000-006 )
1,000 ml graduated cylinder (VWR, catalog number: 65000-012 )
100 ml storage bottle (VWR, catalog number: 89000-234 )
1,000 ml storage bottle (VWR, catalog number: 89000-240 )
Note: This product has been discontinued.

Software

Image Studio Lite (LI-COR)
SoftMax Pro (Molecular Devices)

Procedure

English

中文翻译

文章信息

版权信息

如何引用

Snyder, A. J. and Danthi, P. (2018). Infectious Subviral Particle-induced Hemolysis Assay for Mammalian Orthoreovirus. Bio-protocol 8(2): e2701. DOI: 10.21769/BioProtoc.2701.