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Infectious Subviral Particle-induced Hemolysis Assay for Mammalian Orthoreovirus   

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Original research article

A brief version of this protocol appeared in:
Journal of Virology
Mar 2016

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, Reoviridae, Mammalian orthoreovirus, Viral entry, Membrane penetration, Hemolysis

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.

Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: 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.
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