TY - JOUR
T1 - Authentic in vitro replication of two tombusviruses in isolated mitochondrial and endoplasmic reticulum membranes
AU - Xu, Kai
AU - Huang, Tyng Shyan
AU - Nagy, Peter D.
PY - 2012/12
Y1 - 2012/12
N2 - Replication of plus-stranded RNA viruses takes place on membranous structures derived from various organelles in infected cells. Previous works with Tomato bushy stunt tombusvirus (TBSV) revealed the recruitment of either peroxisomal or endoplasmic reticulum (ER) membranes for replication. In case of Carnation Italian ringspot tombusvirus (CIRV), the mitochondrial membranes supported CIRV replication. In this study, we developed ER and mitochondrion-based in vitro tombusvirus replication assays. Using purified recombinant TBSV and CIRV replication proteins, we showed that TBSV could use the purified yeast ER and mitochondrial preparations for complete viral RNA replication, while CIRV preferentially replicated in the mitochondrial membranes. The viral RNA became partly RNase resistant after~40 to 60 min of incubation in the purified ER and mitochondrial preparations, suggesting that assembly of TBSV and CIRV replicases could take place in the purified ER and mitochondrial membranes in vitro. Using chimeric and heterologous combinations of replication proteins, we showed that multiple domains within the replication proteins are involved in determining the efficiency of tombusvirus replication in the two subcellular membranes. Altogether, we demonstrated that TBSV is less limited while CIRV is more restricted in utilizing various intracellular membranes for replication. Overall, the current work provides evidence that tombusvirus replication could occur in vitro in isolated subcellular membranes, suggesting that tombusviruses have the ability to utilize alternative organellar membranes during infection that could increase the chance of mixed virus replication and rapid evolution during coinfection.
AB - Replication of plus-stranded RNA viruses takes place on membranous structures derived from various organelles in infected cells. Previous works with Tomato bushy stunt tombusvirus (TBSV) revealed the recruitment of either peroxisomal or endoplasmic reticulum (ER) membranes for replication. In case of Carnation Italian ringspot tombusvirus (CIRV), the mitochondrial membranes supported CIRV replication. In this study, we developed ER and mitochondrion-based in vitro tombusvirus replication assays. Using purified recombinant TBSV and CIRV replication proteins, we showed that TBSV could use the purified yeast ER and mitochondrial preparations for complete viral RNA replication, while CIRV preferentially replicated in the mitochondrial membranes. The viral RNA became partly RNase resistant after~40 to 60 min of incubation in the purified ER and mitochondrial preparations, suggesting that assembly of TBSV and CIRV replicases could take place in the purified ER and mitochondrial membranes in vitro. Using chimeric and heterologous combinations of replication proteins, we showed that multiple domains within the replication proteins are involved in determining the efficiency of tombusvirus replication in the two subcellular membranes. Altogether, we demonstrated that TBSV is less limited while CIRV is more restricted in utilizing various intracellular membranes for replication. Overall, the current work provides evidence that tombusvirus replication could occur in vitro in isolated subcellular membranes, suggesting that tombusviruses have the ability to utilize alternative organellar membranes during infection that could increase the chance of mixed virus replication and rapid evolution during coinfection.
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U2 - 10.1128/JVI.00973-12
DO - 10.1128/JVI.00973-12
M3 - Article
C2 - 22973028
AN - SCOPUS:84869236220
SN - 0022-538X
VL - 86
SP - 12779
EP - 12794
JO - Journal of Virology
JF - Journal of Virology
IS - 23
ER -