Open AccessNovel replication complex architecture in rubella replicon‐transfected cells
Author(s) -
Fontana Juan,
Tzeng WenPin,
Calderita Gloria,
FraileRamos Alberto,
Frey Teryl K.,
Risco Cristina
Publication year - 2007
Publication title -
cellular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.542
H-Index - 138
eISSN - 1462-5822
pISSN - 1462-5814
DOI - 10.1111/j.1462-5822.2006.00837.x
Subject(s) - biology , replicon , transfection , golgi apparatus , internal ribosome entry site , microbiology and biotechnology , virology , vacuole , endoplasmic reticulum , rna , cytoplasm , ribosome , gene , genetics , genome
Summary Rubella virus (RUB) assembles its replication complexes (RCs) in modified organelles of endo‐lysosomal origin, known as cytopathic vacuoles (CPVs). These peculiar structures are key elements of RUB factories, where rough endoplasmic reticulum, mitochondria, and Golgi are recruited. Bicistronic RUB replicons expressing an antibiotic resistance gene either in the presence or the absence of the RUB capsid (C) gene were used to study the structure of RCs in transfected cells. Confocal microscopy showed that the RUB replicase components P90 and P150 localized to CPVs, as did double‐stranded RNA (dsRNA), a marker for RNA synthesis. Electron microscopy (EM) showed that replicons generated CPVs containing small vesicles and large vacuoles, similar to CPVs from RUB‐infected cells and that the replicase proteins were sufficient for organelle recruitment. Some of these CPVs contained straight membranes. When cross‐sectioned, these rigid membranes appeared to be sheets of closely packed proteins. Immuno‐EM revealed that these sheets, apparently in contact with the cytosol, contained both P150 and P90, as well as dsRNA, and thus could be two‐dimensional arrays of functional viral replicases. Labelling of dsRNA after streptolysin‐O permeabilization showed that replication of viral genome takes place on the cytoplasmic side of CPVs. When present, C accumulated around CPVs. Mitochondrial protein P32 was detected within modified CPVs, the first demonstration of involvement of this protein, which interacts with C, with RCs.