Reovirus: evidence for a second step in the intracellular uncoating and transcriptase activation process

Abstract Intracellular uncoating of reovirus has been reexamined. Biochemical and electron microscopy techniques were used. Present findings demonstrate that intracellular uncoating to the level of activated transcriptase proceeds via at least two distinct steps. In the first step, intact virions are converted to subviral particles which, by several criteria, appear to be identical to intermediate subviral particles (ISVP) generated in vitro . The endogenous transcriptase in such particles is in a switched-off state. Cells were infected with ISVP in an attempt to demonstrate further uncoating. Incubation of ISVP-infected cells at 37° for an appropriate time interval converts the input ISVP, which are totally refractory to proteolytic digestion, to a form in which a single major polypeptide is either lost or becomes protease sensitive. In electron micrographs of thin sections of cells which have been infected with ISVP, and subsequently incubated at 37°, virus particles of reduced diameter can be seen within the cytoplasm. Particles with activated transcriptase can be extracted from infected cells which have been incubated at 37° for an appropriate time. Extraction of these particles requires treatment of the cell homogenate with proteinase K. No active particles can be extracted with identical treatment of infected cells which have not been incubated at 37° prior to cell homogenization. These findings strongly suggest that the intracellular uncoating of reovirus to the level of active transcriptase proceeds via a pathway which is mechanistically identical to that elucidated for uncoating and transcriptase activation in vitro .