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Infectious Subviral Particle to Membrane Penetration Active Particle (ISVP-to-ISVP*) Conversion Assay for Mammalian Orthoreovirus

哺乳动物正呼肠孤病毒从传染性亚病毒颗粒转化为膜穿透活性颗粒（ISVP-to-ISVP*）分析

AS Anthony J. Snyder
PD Pranav Danthi

发布: 2018年01月20日第8卷第2期 DOI: 10.21769/BioProtoc.2700 浏览次数: 6802

评审: Yannick DebingKristin ShinglerSmita Nair

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Abstract

The mammalian orthoreovirus (reovirus) outer capsid undergoes a series of conformational changes prior to or during viral entry. These transitions are necessary for delivering the genome-containing core across host cell membranes. This protocol describes an in vitro assay for monitoring the transition into a membrane penetration-active form (i.e., ISVP*).

Keywords: Virology (病毒学) search Reoviridae (呼肠孤病毒科) search Mammalian orthoreovirus (哺乳动物正呼肠孤病毒) search Viral entry (病毒进入) search Conformational change (构象改变) search Heat inactivation (热灭活) search

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). ISVPs are produced in vitro by treating purified virions with chymotrypsin (Joklik, 1972; Borsa et al., 1973a). The outer capsid undergoes a second conformational change to deposit the genome-containing core into the host cell cytoplasm. As a consequence, the central δ fragment of µ1 adopts a protease sensitive conformation. The altered particle is called ISVP* (Chandran et al., 2002). ISVP-to-ISVP* conversion can be induced in vitro 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 (e.g., the relationship between particle stability and infectivity) are studied using biochemical and cell-based approaches. In this protocol, we describe an in vitro assay that recapitulates ISVP-to-ISVP* conversion.

Materials and Reagents

  1. 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 )
  2. PCR 8-well tube strips (VWR, catalog number: 20170-004 )
  3. 50 ml centrifuge tube (VWR, catalog number: 89039-660 )
  4. 1.7 ml microcentrifuge tubes (MIDSCI, catalog number: AVSS1700 )
  5. Purified reovirus stocks (see [Berard and Coombs, 2009; Kobayashi et al., 2010] for propagation and purification procedure)
  6. Crushed ice
  7. Standard SDS-PAGE materials and reagents (e.g., 10% SDS-polyacrylamide mini gels)
  8. Coomassie Brilliant Blue stain and destain solutions (Bio-Rad Laboratories, catalog number: 1610435 )
  9. Bleach (Biz4USA, Janitorial Supplies, catalog number: CLO30966CT )
  10. 2-Amino-2-(hydroxymethyl)-1,3-propanediol (Tris) (MP Biomedicals, catalog number: 02103133 )
  11. Magnesium chloride hexahydrate (MgCl2·6H2O) (Sigma-Aldrich, catalog number: M9272 )
  12. Sodium chloride (NaCl) (Merck, catalog number: SX0420-3 )
  13. 0.1 N hydrochloric acid (Sigma-Aldrich, catalog number: 2104 )
  14. N sodium hydroxide (Sigma-Aldrich, catalog number: 2105 )
  15. 4x Laemmli sample buffer (Bio-Rad Laboratories, catalog number: 1610747 )
  16. Nα-p-tosyl-L-lysine chloromethyl ketone (TLCK)-treated chymotrypsin (Worthington Biochemical, catalog number: LS001432 )
  17. Trypsin (Sigma-Aldrich, catalog number: T6567 )
  18. Phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: P7626 )
  19. Ultrapure DNase/RNase-free distilled H2O (Thermo Fisher Scientific, InvitrogenTM, catalog number: 10977015 )
  20. Isopropyl alcohol (Avantor Performance Materials, Macron, catalog number: 3032-02 )
  21. 50% bleach (see Recipes)
  22. Virus storage buffer (VB) (see Recipes)
  23. 2 mg/ml Nα-p-tosyl-L-lysine chloromethyl ketone (TLCK)-treated chymotrypsin (see Recipes)
  24. 100 mM phenylmethylsulfonyl fluoride (PMSF) (see Recipes)
  25. 1 mg/ml trypsin (see Recipes)

Equipment

  1. Personal protective equipment (PPE)
    1. Laboratory coat
    2. Gloves
    3. Eye protection
  2. Biosafety level 2 (BSL-2) laboratory facility
  3. BSL-2 certified tissue culture hood
  4. Solid and liquid waste containers
  5. Autoclave
  6. Ice bucket
  7. -20 °C freezer
  8. 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 )
  9. Digital pH meter (VWR, model: SB70P )
  10. Digital laboratory balance (Mettler Toledo, model: PB1502-S )
  11. NanoDrop spectrophotometer (Thermo Fisher Scientific, Thermo ScientificTM, model: ND-1000 )
  12. Hot plate stirrer (VWR, catalog number: 12365-382 )
  13. Magnetic stir bar (VWR, catalog number: 58948-273 )
  14. Thermal cycler (Bio-Rad Laboratories, model: S1000TM )
  15. Analog dry block heater (VWR, catalog number: 12621-110 )
  16. Thermometer (VWR, catalog number: 89095-566 )
  17. Gel imaging system (LI-COR, model: Odyssey® Classic )
  18. 1,000 ml glass beaker (VWR, catalog number: 89000-212 )
  19. 1,000 ml graduated cylinder (VWR, catalog number: 65000-012 )
  20. 1,000 ml storage bottle (VWR, catalog number: 89000-240 )
    Note: This product has been discontinued.

Software

  1. Image Studio Lite (LI-COR)

Procedure

English
中文翻译

文章信息

版权信息

© 2018 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Snyder, A. J. and Danthi, P. (2018). Infectious Subviral Particle to Membrane Penetration Active Particle (ISVP-to-ISVP*) Conversion Assay for Mammalian Orthoreovirus. Bio-protocol 8(2): e2700. DOI: 10.21769/BioProtoc.2700.

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分类

微生物学 > 微生物-宿主相互作用 > 病毒

生物化学 > 蛋白质 > 活性

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