Reovirus virion-like particles obtained by recoating infectious subvirion particles with baculovirus-expressed sigma 3 protein: an approach for analyzing sigma 3 functions during virus entry

Structure-function studies with mammalian reoviruses have been limited by t he lack of a reverse-genetic system for engineering mutations into the vira l genome. To circumvent this limitation in a partial way for the major oute r-capsid protein sigma 3, we obtained in vitro assembly of large numbers of virion-like particles by binding baculovirus-expressed sigma 3 protein to infectious subvirion particles (ISVPs) that lack sigma 3. A level of sigma 3 binding approaching 100% of that in native virions was routinely achieved . The sigma 3 coat in these recoated ISVPs (rcISVPs) appeared very similar to that in virions by electron microscopy and three-dimensional image recon struction. rcISVPs retained full infectivity in murine L cells, allowing th eir use to study sigma 3 functions in virus entry. Upon infection, rcISVPs behaved identically to virions in showing an extended lag phase prior to ex ponential growth and in being inhibited from entering cells by either the w eak base NH4Cl or the cysteine proteinase inhibitor E-64. rcISVPs also mimi cked virions in being incapable of in vitro activation to mediate lysis of erythrocytes and transcription of the viral mRNAs. Last, rcISVPs behaved li ke virions in showing minor loss of infectivity at 52 degrees C. Since rcIS VPs contain virion-like levels of sigma 3 but contain outer-capsid protein mu 1/mu 1C mostly cleaved at the delta-phi junction as in ISVPs, the fact t hat rcISVPs behaved like virions (and not ISVPs) in all of the assays that we performed suggests that sigma 3, and not the delta-phi cleavage of mu 1/ mu 1C, determines the observed differences in behavior between virions and ISVPs. To demonstrate the applicability of rcISVPs for genetic studies of p rotein functions in reovirus entry (an approach that we call recoating gene tics), we used chimeric sigma 3 proteins to localize the primary determinan ts of a strain-dependent difference in sigma 3 cleavage rate to a carboxy-t erminal region of the ISVP-bound protein.