High-resolution mass spectrometric mapping of reovirus digestion.

Reovirus is an enteric virus built from eight structural proteins that form a double-layered capsid. During virus entry into cells the reovirus outermost capsid layer (composed of proteins sigma3 and mu1C) is proteolytically processed to generate first an infectious subviral particle (ISVP), then the transcriptionally active core particle. Previous studies have demonstrated that protein sigma3, the outermost protein in the viral capsid, is removed from virus particles extremely rapidly. Other studies, using the detergent tetradecyl sulfate (14SO4) in combination with the protease chymotrypsin, have shown that mu1C cleavage is not necessary for infectious viral processing. We have recently used mass spectrometry to characterize the cascade of sigma3 proteolysis in intact reovirus serotype 1 Lang (T1L) virions (Mendez et al., Virology 2003; 311: 289-304). In the present study, we use high-resolution mass spectrometry to characterize the cascade of outer capsid digestion of both T1L and the other commonly used reovirus strain (serotype 3 Dearing [T3D]), with the protease trypsin, both in the presence and absence of 14SO4. These studies indicate that digestion kinetics and specificities are determined both by virus type and by presence or absence of detergent. Presence of detergent accelerated digestion of both outer capsid proteins. In contrast to chymotrypsin digestion, which segregated sigma3 digestion from mu1 digestion, both proteins were rapidly digested by trypsin in the presence of detergent.