New Intermediate Subviral Particles in the In Vitro Uncoating of Reovirus Virions by Chymotrypsin

Abstract

Reovirus virions, grown in suspension cultures of L cells and extensively purified by density gradient and velocity gradient centrifugation after their release from cell debris by fluorocarbon extraction, are characterized by a mean particle diameter of 73 nm and a density in CsCl of 1.36 to 1.37 g/cm 3 . Treatment of intact virions by chymotrypsin (CHT) digestion in vitro converts them to subviral particles (SVP) having characteristics which are determined by the species of monovalent cation present during the digestion. In the presence of Cs + ions, CHT converts the virions to SVP of mean diameter 51 nm and density 1.43 to 1.44 g/cm 3 . In the presence of K + ions, the conversion is to SVP of diameter 51 nm and density 1.39 to 1.40 g/cm 3 . The SVP made in the presence of either Cs + or K + possess an extremely active RNA polymerase and nucleoside triphosphate phosphohydrolase (NTPase) activity in vitro and are resistant to further digestion by CHT. Treatment of intact virions with CHT in the presence of Na + or Li + ions results in their conversion to SVP of mean diameter 64 nm and density 1.37 to 1.38 g/cm 3 . Such SVP are not active in in vitro RNA synthesis or NTP hydrolysis and are resistant to further digestion by CHT even during prolonged exposure to high concentrations of enzyme. Addition of Cs + or K + ions to the digestion mixture allows conversion of the 64-nm diameter SVP to 51-nm diameter SVP in which the RNA polymerase and NTPase are active in vitro. Analysis of the proteins present in intact virions and in the different SVP reveals clear differences which indicate that the conversions are accomplished by removal or cleavage of particular species of polypeptides.

A Forward Genetic Strategy Reveals Destabilizing Mutations in the Ebolavirus Glycoprotein That Alter Its Protease Dependence during Cell Entry

“…Analogous to furin-mediated priming of many class I viral fusion proteins, CatL-mediated cleavage of the Nipah paramyxovirus glycoprotein precursor, F 0 , to F 1 plus F 2 occurs in virus producer cells, liberating an N-terminal fusion peptide in F 2 and sensitizing F for fusion triggering (48). Like EBOV GP, the nonenveloped mammalian reovirus capsid undergoes extensive proteolytic disassembly during entry; 600 copies of the 3 protector protein are degraded by CatL and CatB to expose the 1 penetration protein (8,17,19). Unlike the EBOV GP 17K intermediate, the Nipah virus and reovirus intermediates no longer require cysteine protease activity to enter cells (1,13,57).…”

Selection and characterization of a reovirus mutant with improved thermostability

“…T1L, T1L/T3D M2, T1L μ1 M258I, T1L σ3 S344P, T1L μ1 M258I σ3 S344P, or T1L/T3D M2 μ1 M258I σ3 S344P virions (2×10 12 particles/ml) were digested with 200 μg/ml TLCK ( N α- p -tosyl-L-lysine chloromethyl ketone)-treated chymotrypsin (Worthington Biochemical) in a total volume of 100 μl for 20 min at 32°C (30, 31). The reactions were then incubated on ice for 20 min and quenched by the addition of 1 mM phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich).…”

“…2A) (29). The resulting intermediate is called the infectious subviral particle (30). Proteolytic disassembly (virion-to-ISVP conversion) is recapitulated in vitro by treating purified virions with exogenous protease (29–31).…”

The reovirus μ1 protein contributes to the environmental stability of virions

“…T1L, T1L/T3D M2, T1L/T3D M2 μ1 E89A, or T1L/T3D M2 μ1 D371A virions (2×10 12 particles/ml) were digested with 200 μg/ml TLCK ( N α- p -tosyl-L-lysine chloromethyl ketone)-treated chymotrypsin (Worthington Biochemical) in a total volume of 100 μl for 20 min at 32°C (36, 37). The reactions were then incubated on ice for 20 min and quenched by the addition of 1 mM phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich).…”