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

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In this protocol

Original research article

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


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, Reoviridae, Mammalian orthoreovirus, Viral entry, Conformational change, Heat inactivation


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.

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