Abstract
The replication of eukaryotic positive-strand RNA virus genomes occurs in the membrane-bound RNA replication complexes. Previously, we found that the extract of evacuolated tobacco BY-2 protoplasts (BYL) is capable of supporting the translation and subsequent replication of the genomic RNAs of plant positive-strand RNA viruses, including Tomato mosaic virus (ToMV). Here, to dissect the process that precedes the formation of ToMV RNA replication complexes, we prepared membrane-depleted BYL (mdBYL), in which the membranes were removed by centrifugation. In mdBYL, ToMV RNA was translated to produce the 130-kDa and 180-kDa replication proteins, but the synthesis of any ToMV-related RNAs did not occur. When BYL membranes were added back to the ToMV RNA-translated mdBYL after the termination of translation with puromycin, ToMV RNA was replicated. Using a replication-competent ToMV derivative that encodes the FLAG-tagged 180-kDa replication protein, it was shown by affinity purification that a complex that contained the 130-kDa and 180-kDa proteins and ToMV genomic RNA was formed after translation in mdBYL. When the complex was mixed with BYL membranes, ToMV RNA was replicated, which suggests that this ribonucleoprotein complex is an intermediate of ToMV RNA replication complex formation. We have named this ribonucleoprotein complex the "pre-membrane-targeting complex." Our data suggest that the formation of the pre-membrane-targeting complex is coupled with the translation of ToMV RNA, while posttranslationally added exogenous 180-kDa protein and replication templates can contribute to replication and can be replicated, respectively. Based on these results, we discuss the mechanisms of ToMV RNA replication complex formation.
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Claims 6
Claim · model-written summary
In membrane-depleted tobacco BY-2 protoplast extract, Tomato mosaic virus RNA was translated into the 130-kDa and 180-kDa replication proteins but no ToMV-related RNAs were synthesized.
Verbatim
e0“In mdBYL, ToMV RNA was translated to produce the 130-kDa and 180-kDa replication proteins, but the synthesis of any ToMV-related RNAs did not occur”
e1“evacuolated tobacco BY-2 protoplasts”
Claim · model-written summary
Adding BYL membranes back to ToMV RNA-translated membrane-depleted extract after translation was stopped with puromycin allowed ToMV RNA replication.
- Taxon
- tobacco BY-2 protoplastse1
- Treatment
- BYL membranes were added backe0
- Interactor
- ToMVe0pathogen
- Outcome
- ToMV RNA was replicatede0
Not reportedComparatorDirection
Verbatim
e0“When BYL membranes were added back to the ToMV RNA-translated mdBYL after the termination of translation with puromycin, ToMV RNA was replicated”
e1“evacuolated tobacco BY-2 protoplasts”
Claim · model-written summary
Affinity purification showed that a complex containing the 130-kDa and 180-kDa proteins and ToMV genomic RNA formed after translation in membrane-depleted extract.
Verbatim
e0“it was shown by affinity purification that a complex that contained the 130-kDa and 180-kDa proteins and ToMV genomic RNA was formed after translation in mdBYL”
e1“evacuolated tobacco BY-2 protoplasts”
Claim · model-written summary
The complex, when mixed with BYL membranes, supported ToMV RNA replication, which the authors take to indicate it is an intermediate of replication complex formation.
Verbatim
e0“When the complex was mixed with BYL membranes, ToMV RNA was replicated, which suggests that this ribonucleoprotein complex is an intermediate of ToMV RNA replication complex formation”
e1“evacuolated tobacco BY-2 protoplasts”
Claim · model-written summary
The authors suggest that formation of the pre-membrane-targeting complex is coupled with translation of ToMV RNA.
Verbatim
e0“Our data suggest that the formation of the pre-membrane-targeting complex is coupled with the translation of ToMV RNA”
e1“evacuolated tobacco BY-2 protoplasts”
Claim · model-written summary
Exogenous 180-kDa protein and replication templates added after translation can contribute to replication and can be replicated, respectively.
Verbatim
e0“posttranslationally added exogenous 180-kDa protein and replication templates can contribute to replication and can be replicated, respectively”
e1“evacuolated tobacco BY-2 protoplasts”