Reovirus virion-like particles obtained by recoating infectious subvirion particles with baculovirus-expressed σ3 protein: An approach for analyzing σ3 functions during virus entry

Judit Jané-Valbuena, Max L. Nibert, Stephan M. Spencer, Stephen B. Walker, Timothy S. Baker, Ya Chen, Victoria E. Centonze, Leslie A. Schiff

Research output: Contribution to journalArticlepeer-review

42 Scopus citations

Abstract

Structure-function studies with mammalian reoviruses have been limited by the lack of a reverse-genetic system for engineering mutations into the viral genome. To circumvent this limitation in a partial way for the major outer-capsid protein σ3, we obtained in vitro assembly of large numbers of virion-like particles by binding baculovirus-expressed σ3 protein to infectious subvirion particles (ISVPs) that lack σ3. A level of σ3 binding approaching 100% of that in native virions was routinely achieved. The σ3 coat in these recoated ISVPs (rcISVPs) appeared very similar to that in virions by electron microscopy and three-dimensional image reconstruction. rcISVPs retained full infectivity in murine L cells, allowing their use to study σ3 functions in virus entry. Upon infection, rcISVPs behaved identically to virions in showing an extended lag phase prior to exponential growth and in being inhibited from entering cells by either the weak base NH4Cl or the cysteine proteinase inhibitor E-64. rcISVPs also mimicked virions in being incapable of in vitro activation to mediate lysis of erythrocytes and transcription of the vital mRNAs. Last, rcISVPs behaved like virions in showing minor loss of infectivity at 52°C. Since rcISVPs contain virion-like levels of σ3 but contain outer-capsid protein μ1/μ1C mostly cleaved at the δ-φ junction as in ISVPs, the fact that rcISVPs behaved like virions (and not ISVPs) in all of the assays that we performed suggests that σ3, and not the δ-φ cleavage of μ1/μ1C, determines the observed differences in behavior between virions and ISVPs. To demonstrate the applicability of rcISVPs for genetic studies of protein functions in reovirus entry (an approach that we call recoating genetics), we used chimeric σ3 proteins to localize the primary determinants of a strain-dependent difference in σ3 cleavage rate to a carboxy-terminal region of the ISVP- bound protein.

Original languageEnglish (US)
Pages (from-to)2963-2973
Number of pages11
JournalJournal of virology
Volume73
Issue number4
DOIs
StatePublished - Apr 1999

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