Keisuke Komoda | Iowa State University (original) (raw)

Papers by Keisuke Komoda

Research paper thumbnail of Possible involvement of eEF1A in Tomato spotted wilt virus RNA synthesis

Virology, Nov 2014

Tomato spotted wilt virus (TSWV) is a negative-strand RNA virus in the family Bunyaviridae and pr... more Tomato spotted wilt virus (TSWV) is a negative-strand RNA virus in the family Bunyaviridae and propagates in both insects and plants. Although TSWV can infect a wide range of plant species, host factors involved in viral RNA synthesis of TSWV in plants have not been characterized. In this report, we demonstrate that the cell-free extract derived from one of the host plants can activate mRNA transcriptional activity of TSWV. Based on activity-guided fractionation of the cell-free extract, we identified eukaryotic elongation factor (eEF) 1A as a possible host factor facilitating TSWV transcription and replication. The RNA synthesis-supporting activity decreased in the presence of an eEF1A inhibitor, suggesting that eEF1A plays an important role in RNA synthesis of TSWV.

Research paper thumbnail of Expression, purification, crystallization and preliminary X-ray crystallographic study of the nucleocapsid protein of Tomato spotted silt virus

Acta Crystallographica Section F, 2013

Tomato spotted wilt virus (TSWV), which causes severe damage to various agricultural crops such a... more Tomato spotted wilt virus (TSWV), which causes severe damage to various agricultural crops such as tomato, pepper, lettuce and peanut, is a negative-stranded RNA virus belonging to the Tospovirus genus of the Bunyaviridae family. Viral genomic RNA molecules are packaged in the form of ribonucleoprotein complexes, each of which contains one viral RNA molecule that is coated with many nucleocapsid (N) proteins. Here, the expression and crystallization of TSWV N protein are reported. Native and selenomethionine-substituted crystals of N protein belonged to the same space group P21. Their unit-cell parameters were a = 66.8, b = 97.2, c = 72.0 Å, [beta] = 112.8° and a = 66.5, b = 96.3, c = 72.1 Å, [beta] = 113.4°, respectively.

Research paper thumbnail of Identification of a Ribonucleoprotein Intermediate of Tomato Mosaic Virus RNA Replication Complex Formation

Journal of Virology, 2007

The replication of eukaryotic positive-strand RNA virus genomes occurs in the membrane-bound RNA ... more 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.

Research paper thumbnail of In Vitro Translation and Replication of Tobamovirus RNA in a Cell-Free Extract of Evacuolated Tobacco BY2 Protoplasts

Research paper thumbnail of Replication of plant RNA virus genomes in a cell-free extract of evacuolated plant protoplasts

Proceedings of The National Academy of Sciences, 2004

The replication of eukaryotic positive-strand RNA virus genomes occurs through a complex process ... more The replication of eukaryotic positive-strand RNA virus genomes occurs through a complex process involving multiple viral and host proteins and intracellular membranes. Here we report a cell-free system that reproduces this process in vitro. This system uses a membrane-containing extract of uninfected plant protoplasts from which the vacuoles had been removed by Percoll gradient centrifugation. We demonstrate that the system supported translation, negative-strand RNA synthesis, genomic RNA replication, and subgenomic RNA transcription of tomato mosaic virus and two other plant positive-strand RNA viruses. The RNA synthesis, which depended on translation of the genomic RNA, produced virus-related RNA species similar to those that are generated in vivo. This system will aid in the elucidation of the mechanisms of genome replication in these viruses.

Research paper thumbnail of Subcellular localization of host and viral proteins associated with tobamovirus RNA replication

The EMBO Journal, Jan 2003

Arabidopsis TOM1 (AtTOM1) and TOM2A (AtTOM2A) are integral membrane proteins genetically identifi... more Arabidopsis TOM1 (AtTOM1) and TOM2A (AtTOM2A) are integral membrane proteins genetically identified to be necessary for efficient intracellular multiplication of tobamoviruses. AtTOM1 interacts with the helicase domain polypeptide of tobamovirus-encoded replication proteins and with AtTOM2A, suggesting that both AtTOM1 and AtTOM2A are integral components of the tobamovirus replication complex. We show here that AtTOM1 and AtTOM2A proteins tagged with green fluorescent protein (GFP) are targeted to the vacuolar membrane (tonoplast)-like structures in plant cells. In subcellular fractionation analyses, GFP-AtTOM2A, AtTOM2A and its tobacco homolog NtTOM2A were predominantly fractionated to low-density tonoplast-rich fractions, whereas AtTOM1-GFP, AtTOM1 and its tobacco homolog NtTOM1 were distributed mainly into the tonoplast-rich fractions and partially into higher-buoyant-density fractions containing membranes from several other organelles. The tobamovirus-encoded replication proteins were co-fractionated with both NtTOM1 and viral RNA-dependent RNA polymerase activity. The replication proteins were also found in the fractions containing non-membrane-bound proteins, but neither NtTOM1 nor the polymerase activity was detected there. These observations suggest that the formation of tobamoviral RNA replication complex occurs on TOM1-containing membranes and is facilitated by TOM2A.

Research paper thumbnail of Possible involvement of eEF1A in Tomato spotted wilt virus RNA synthesis

Virology, Nov 2014

Tomato spotted wilt virus (TSWV) is a negative-strand RNA virus in the family Bunyaviridae and pr... more Tomato spotted wilt virus (TSWV) is a negative-strand RNA virus in the family Bunyaviridae and propagates in both insects and plants. Although TSWV can infect a wide range of plant species, host factors involved in viral RNA synthesis of TSWV in plants have not been characterized. In this report, we demonstrate that the cell-free extract derived from one of the host plants can activate mRNA transcriptional activity of TSWV. Based on activity-guided fractionation of the cell-free extract, we identified eukaryotic elongation factor (eEF) 1A as a possible host factor facilitating TSWV transcription and replication. The RNA synthesis-supporting activity decreased in the presence of an eEF1A inhibitor, suggesting that eEF1A plays an important role in RNA synthesis of TSWV.

Research paper thumbnail of Expression, purification, crystallization and preliminary X-ray crystallographic study of the nucleocapsid protein of Tomato spotted silt virus

Acta Crystallographica Section F, 2013

Tomato spotted wilt virus (TSWV), which causes severe damage to various agricultural crops such a... more Tomato spotted wilt virus (TSWV), which causes severe damage to various agricultural crops such as tomato, pepper, lettuce and peanut, is a negative-stranded RNA virus belonging to the Tospovirus genus of the Bunyaviridae family. Viral genomic RNA molecules are packaged in the form of ribonucleoprotein complexes, each of which contains one viral RNA molecule that is coated with many nucleocapsid (N) proteins. Here, the expression and crystallization of TSWV N protein are reported. Native and selenomethionine-substituted crystals of N protein belonged to the same space group P21. Their unit-cell parameters were a = 66.8, b = 97.2, c = 72.0 Å, [beta] = 112.8° and a = 66.5, b = 96.3, c = 72.1 Å, [beta] = 113.4°, respectively.

Research paper thumbnail of Identification of a Ribonucleoprotein Intermediate of Tomato Mosaic Virus RNA Replication Complex Formation

Journal of Virology, 2007

The replication of eukaryotic positive-strand RNA virus genomes occurs in the membrane-bound RNA ... more 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.

Research paper thumbnail of In Vitro Translation and Replication of Tobamovirus RNA in a Cell-Free Extract of Evacuolated Tobacco BY2 Protoplasts

Research paper thumbnail of Replication of plant RNA virus genomes in a cell-free extract of evacuolated plant protoplasts

Proceedings of The National Academy of Sciences, 2004

The replication of eukaryotic positive-strand RNA virus genomes occurs through a complex process ... more The replication of eukaryotic positive-strand RNA virus genomes occurs through a complex process involving multiple viral and host proteins and intracellular membranes. Here we report a cell-free system that reproduces this process in vitro. This system uses a membrane-containing extract of uninfected plant protoplasts from which the vacuoles had been removed by Percoll gradient centrifugation. We demonstrate that the system supported translation, negative-strand RNA synthesis, genomic RNA replication, and subgenomic RNA transcription of tomato mosaic virus and two other plant positive-strand RNA viruses. The RNA synthesis, which depended on translation of the genomic RNA, produced virus-related RNA species similar to those that are generated in vivo. This system will aid in the elucidation of the mechanisms of genome replication in these viruses.

Research paper thumbnail of Subcellular localization of host and viral proteins associated with tobamovirus RNA replication

The EMBO Journal, Jan 2003

Arabidopsis TOM1 (AtTOM1) and TOM2A (AtTOM2A) are integral membrane proteins genetically identifi... more Arabidopsis TOM1 (AtTOM1) and TOM2A (AtTOM2A) are integral membrane proteins genetically identified to be necessary for efficient intracellular multiplication of tobamoviruses. AtTOM1 interacts with the helicase domain polypeptide of tobamovirus-encoded replication proteins and with AtTOM2A, suggesting that both AtTOM1 and AtTOM2A are integral components of the tobamovirus replication complex. We show here that AtTOM1 and AtTOM2A proteins tagged with green fluorescent protein (GFP) are targeted to the vacuolar membrane (tonoplast)-like structures in plant cells. In subcellular fractionation analyses, GFP-AtTOM2A, AtTOM2A and its tobacco homolog NtTOM2A were predominantly fractionated to low-density tonoplast-rich fractions, whereas AtTOM1-GFP, AtTOM1 and its tobacco homolog NtTOM1 were distributed mainly into the tonoplast-rich fractions and partially into higher-buoyant-density fractions containing membranes from several other organelles. The tobamovirus-encoded replication proteins were co-fractionated with both NtTOM1 and viral RNA-dependent RNA polymerase activity. The replication proteins were also found in the fractions containing non-membrane-bound proteins, but neither NtTOM1 nor the polymerase activity was detected there. These observations suggest that the formation of tobamoviral RNA replication complex occurs on TOM1-containing membranes and is facilitated by TOM2A.