RESUMO
Positive-strand RNA viruses, such as coronaviruses, induce cellular membrane rearrangements during replication to form replication organelles allowing for efficient viral RNA synthesis. Infectious bronchitis virus (IBV), a pathogenic avian Gammacoronavirus of significant importance to the global poultry industry, has been shown to induce the formation of double membrane vesicles (DMVs), zippered endoplasmic reticulum (zER) and tethered vesicles, known as spherules. These membrane rearrangements are virally induced; however, it remains unclear which viral proteins are responsible. In this study, membrane rearrangements induced when expressing viral non-structural proteins (nsps) from two different strains of IBV were compared. Three non-structural transmembrane proteins, nsp3, nsp4, and nsp6, were expressed in cells singularly or in combination and the effects on cellular membranes investigated using electron microscopy and electron tomography. In contrast to previously studied coronaviruses, IBV nsp4 alone is necessary and sufficient to induce membrane pairing; however, expression of the transmembrane proteins together was not sufficient to fully recapitulate DMVs. This indicates that although nsp4 is able to singularly induce membrane pairing, further viral or host factors are required in order to fully assemble IBV replicative structures. This study highlights further differences in the mechanism of membrane rearrangements between members of the coronavirus family.
Assuntos
Membrana Celular/metabolismo , Membrana Celular/virologia , Interações Hospedeiro-Patógeno , Vírus da Bronquite Infecciosa/fisiologia , Proteínas não Estruturais Virais/metabolismo , Animais , Linhagem Celular , Membrana Celular/ultraestrutura , Galinhas , Fibroblastos/virologia , Microscopia EletrônicaRESUMO
Positive-strand RNA (+RNA) viruses rearrange cellular membranes during replication, possibly in order to concentrate and arrange viral replication machinery for efficient viral RNA synthesis. Our previous work showed that in addition to the conserved coronavirus double membrane vesicles (DMVs), Beau-R, an apathogenic strain of avian Gammacoronavirus infectious bronchitis virus (IBV), induces regions of ER that are zippered together and tethered open-necked double membrane spherules that resemble replication organelles induced by other +RNA viruses. Here we compared structures induced by Beau-R with the pathogenic lab strain M41 to determine whether membrane rearrangements are strain dependent. Interestingly, M41 was found to have a low spherule phenotype. We then compared a panel of pathogenic, mild and attenuated IBV strains in ex vivo tracheal organ culture (TOC). Although the low spherule phenotype of M41 was conserved in TOCs, each of the other tested IBV strains produced DMVs, zippered ER and spherules. Furthermore, there was a significant correlation for the presence of DMVs with spherules, suggesting that these structures are spatially and temporally linked. Our data indicate that virus induced membrane rearrangements are fundamentally linked to the viral replicative machinery. However, coronavirus replicative apparatus clearly has the plasticity to function in different structural contexts.