Identification and functional characterization of regions that can be crosslinked to RNA in the helicase-like domain of BaMV replicase.
The helicase-like domain of the Bamboo mosaic virus replicase catalyzes the release of 5'-gamma-phosphate from both ATP and 5'-triphosphated RNA by an identical set of catalytic residues with a presumably larger binding pocket for RNA. In this study, the peptidyl regions involved in RNA binding were mapped by reversible formaldehyde crosslinking and mass spectrometry. Eleven residues within these regions were examined by mutational analysis. H636A, Y704A, and K706A greatly diminished the enzymatic activities and were unable to support the viral replication in Nicotiana benthamiana protoplasts. K843A decreased activity toward the RNA substrate to 17% of WT, and approximately 20% replication efficiency was retained in protoplasts. R597A and K610A retained approximately 50 and approximately 90% of the enzymatic activities, respectively. However, replication in protoplasts of these mutants was extremely limited. Proteins with the mutations K603A, R628A, R645A, H794A, and R799A were present at levels 30-69% of WT in protoplasts. However, the fates of these mutations in plants were different. Viral cell-to-cell movement was limited by the K603A and R628A mutations, while systemic movement was restricted by R645A and H794A. The implications of the helicase-like domain in the viral replication and movement are discussed.
Han
Yu-Tsung
YT
Graduate Institute of Biotechnology, National Chung Hsing University, 250 Kuo-Kuang Rd, Taichung, Taiwan 40227, ROC.
Hsu
Yau-Heiu
YH
Lo
Chia-Wen
CW
Meng
Menghsiao
M
D000595:Amino Acid Sequence; D027465:Chenopodium quinoa / Q000821:virology; D003432:Cross-Linking Reagents; D005557:Formaldehyde; D008969:Molecular Sequence Data; D009029:Mosaic Viruses / Q000235:genetics* / Q000502:physiology; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011523:Protoplasts / Q000821:virology; D020365:RNA Helicases / Q000235:genetics / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics* / Q000378:metabolism; D014026:Nicotiana / Q000821:virology; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism; D014779:Virus Replication
D003432:Cross-Linking Reagents; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D014764:Viral Proteins; D005557:Formaldehyde; D012324:RNA-Dependent RNA Polymerase; D020365:RNA Helicases