Identification of a Proline-Kinked Amphipathic α-Helix Downstream from the Methyltransferase Domain of a Potexvirus Replicase and Its Role in Virus Replication and Perinuclear Complex Formation.
Ken Komatsu, Nobumitsu Sasaki, Tetsuya Yoshida, Katsuhiro Suzuki, Yuki Masujima, Masayoshi Hashimoto, Satoru Watanabe, Naoya Tochio, Takanori Kigawa, Yasuyuki Yamaji, Kenro Oshima, Shigetou Namba, Richard S Nelson, Tsutomu Arie
Author Information
Ken Komatsu: Laboratory of Plant Pathology, Graduate School of Agriculture, Tokyo University of Agriculture and Technologygrid.136594.c (TUAT), Fuchu, Japan. ORCID
Nobumitsu Sasaki: Institute of Global Innovation Research (GIR), Tokyo University of Agriculture and Technologygrid.136594.c (TUAT), Fuchu, Japan.
Tetsuya Yoshida: Laboratory of Plant Pathology, Graduate School of Agricultural and Life Sciences, The University of Tokyogrid.26999.3d, Tokyo, Japan.
Katsuhiro Suzuki: Laboratory of Plant Pathology, Graduate School of Agriculture, Tokyo University of Agriculture and Technologygrid.136594.c (TUAT), Fuchu, Japan.
Yuki Masujima: Laboratory of Plant Pathology, Graduate School of Agriculture, Tokyo University of Agriculture and Technologygrid.136594.c (TUAT), Fuchu, Japan.
Masayoshi Hashimoto: Laboratory of Plant Pathology, Graduate School of Agricultural and Life Sciences, The University of Tokyogrid.26999.3d, Tokyo, Japan.
Satoru Watanabe: Laboratory for Cellular Structural Biology, RIKEN Center for Biosystems Dynamics Research, Yokohama, Japan.
Naoya Tochio: Laboratory for Cellular Structural Biology, RIKEN Center for Biosystems Dynamics Research, Yokohama, Japan.
Takanori Kigawa: Laboratory for Cellular Structural Biology, RIKEN Center for Biosystems Dynamics Research, Yokohama, Japan.
Yasuyuki Yamaji: Laboratory of Plant Pathology, Graduate School of Agricultural and Life Sciences, The University of Tokyogrid.26999.3d, Tokyo, Japan.
Kenro Oshima: Faculty of Bioscience, Department of Clinical Plant Science, Hosei Universitygrid.257114.4, Koganei, Japan.
Shigetou Namba: Laboratory of Plant Pathology, Graduate School of Agricultural and Life Sciences, The University of Tokyogrid.26999.3d, Tokyo, Japan.
Richard S Nelson: Department of Entomology and Plant Pathology, Oklahoma State University, Stillwater, Oklahoma, USA.
Tsutomu Arie: Laboratory of Plant Pathology, Graduate School of Agriculture, Tokyo University of Agriculture and Technologygrid.136594.c (TUAT), Fuchu, Japan. ORCID
Characterized positive-strand RNA viruses replicate in association with intracellular membranes. Regarding viruses in the genus , the mechanism by which their RNA-dependent RNA polymerase (replicase) associates with membranes is understudied. Here, by membrane flotation analyses of the replicase of Plantago asiatica mosaic potexvirus (PlAMV), we identified a region in the methyltransferase (MET) domain as a membrane association determinant. An amphipathic α-helix was predicted downstream from the core region of the MET domain, and hydrophobic amino acid residues were conserved in the helical sequences in replicases of other potexviruses. Nuclear magnetic resonance (NMR) analysis confirmed the amphipathic α-helical configuration and unveiled a kink caused by a highly conserved proline residue in the α-helix. Substitution of this proline residue and other hydrophobic and charged residues in the amphipathic α-helix abolished PlAMV replication. Ectopic expression of a green fluorescent protein (GFP) fusion with the entire MET domain resulted in the formation of a large perinuclear complex, where virus replicase and RNA colocated during virus infection. Except for the proline substitution, the amino acid substitutions in the α-helix that abolished virus replication also prevented the formation of the large perinuclear complex by the respective GFP-MET fusion. Small intracellular punctate structures were observed for all GFP-MET fusions, and high-molecular-weight complexes were formed by both replication-competent and -incompetent viral replicons and thus were not sufficient for replication competence. We discuss the roles of the potexvirus-specific, proline-kinked amphipathic helical structure in virus replication and intracellular large complex and punctate structure formation. RNA viruses characteristically associate with intracellular membranes during replication. Although virus replicases are assumed to possess membrane-targeting properties, their membrane association domains generally remain unidentified or poorly characterized. Here, we identified a proline-kinked amphipathic α-helix structure downstream from the methyltransferase core domain of PlAMV replicase as a membrane association determinant. This helical sequence, which includes the proline residue, was conserved among potexviruses and related viruses in the order . Substitution of the proline residue, but not the other residues necessary for replication, allowed formation of a large perinuclear complex within cells resembling those formed by PlAMV replicase and RNA during virus replication. Our results demonstrate the role of the amphipathic α-helix in PlAMV replicase in a perinuclear complex formation and virus replication and that perinuclear complex formation by the replicase alone will not necessarily indicate successful virus replication.
