Metadata about: Abstract Reovirus is an enteric virus comprising eight structural proteins that form a double-layered capsid. During reovirus entry into cells, the outermost capsid layer (composed of proteins σ3 and μ1C) is proteolytically processed to generate an infectious subviral particle (ISVP) that is subsequently uncoated to produce the transcriptionally active core particle. Kinetic studies suggest that protein σ3 is rapidly removed from virus particles and then protein μ1C is cleaved. Initial cleavage of μ1C has been well described and generates an amino (N)-terminal δ peptide and a carboxyl (C)-terminal φ peptide. However, cleavage and removal of σ3 is an extremely rapid event that has not been well defined. We have treated purified reovirus serotype 1 Lang virions with a variety of endoproteases. Time-course digestions with chymotrypsin, Glu-C, pepsin, and trypsin resulted in the initial generation of two peptides that were resolved in SDS–PAGE and analyzed by in-gel tryptic digestion and MALDI-Qq-TOFMS. Most tested proteases cut σ3 within a “hypersensitive” region between amino acids 217 and 238. In addition, to gain a better understanding of the sequence of subsequent proteolytic events that result in generation of reovirus subviral particles, time-course digestions of purified particles were performed under physiologic salt conditions and released peptide fragments ranging from 500 to 5000 Da were directly analyzed by MALDI-Qq-TOFMS. Trypsin digestion initially released a peptide that corresponded to the C-terminus of μ1C, followed by a peptide that corresponded to amino acids 214–236 of the σ3 protein. Other regions of μ1C were not observed until protein σ3 was completely digested. Similar experiments with Glu-C indicated the hypersensitive region of σ3 was cut first when virions were treated at pH values of 4.5 or 7.4, but treatment of virions with pepsin at pH 3.0 released different σ3 peptides, suggesting acid-induced conformational changes in this outer capsid protein. These studies also revealed that the N-terminus of σ3 is acetylated.

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