Functional analysis of the helicase-like domain of Bamboo mosaic virus replicase

• Main Authors: Yu-Tsung Han, 韓宇聰
• Other Authors: 孟孟孝
• Format: Others
• Language: zh-TW
• Published: 2009
• Online Access: http://ndltd.ncl.edu.tw/handle/08621745780625051813

博士 === 國立中興大學 === 生物科技學研究所 === 97 === Abstract-1 The helicase-like domain of BaMV replicase possesses NTPase and RNA 5’-triphosphatase activities. In this study, mutational effects of the helicase signature motifs and residue L543 on the two activities were investigated. Either activity was inactivated by K643A-S644A, D702A, D730A, R855A, or L543P mutations. On the other hand, Q826A, D858A, and L543A had activities, in terms of kcat/Km, reduced by 5-15 folds. AMPPNP, a nonhydrolyzable ATP analogue, competitively inhibited RNA 5’-triphosphatase activity. Analogies of mutational effects on the two activities and approximation of Ki(AMPPNP) and Km(ATP) suggest that the catalytic sites of the activities are overlapped. Mutational effects on the viral accumulation in Chenopodium quinoa indicated that the activities manifested by the domain are required for BaMV survival. Results also suggest that Q826 in motif V plays an additional role in preventing tight binding to ATP, which would otherwise decrease further RNA 5’-triphosphatase, leading to demise of the virus in plant. Abstract-2 The central helicase-like domain of the 155-kDa replicase of Bamboo mosaic virus catalyzes the release of 5’-γ-phosphate from ATP and 5’-triphosphated RNA. The catalytic sites of the two reactions are overlapped with a presumably larger binding pocket for RNA. To learn about residues involved in RNA binding and their respective functions, the domain was crosslinked to a biotinylated RNA by formaldehyde. After trypsin treatment, the peptides linked to RNA were recovered and identified by mass spectrometry. Accordingly, six peptidyl regions were located and eleven residues within were subjected to mutagenesis for functional analysis. H636A, Y704A, and K706A abolished both the enzymatic activities and could not support the viral replication in protoplast. K843A dropped the enzymatic activities to 17% of WT and accumulated the viral coat protein and genomic RNA to ~20% of WT, suggesting that an enzymatic activity as low as ~20% could support a proportional viral replication in protoplasts. R597A and K610A retained ~50 and ~90% of the enzymatic activities, respectively. However, the replication of the two mutants was extremely limited in protoplasts. K603A, R628A, R645A, H794A, and R799A accumulated themselves at levels of 35-69% of WT in protoplasts. However, their fates in plants were different. K603A and R628A did not spread sufficiently in the inoculated leaves, while R645A and H794A could not support the movement from inoculated into systemic leaves. The variations in response to mutations suggest that the helicase-like domain is multifaceted involved in functions including replication, translation, and movement. Abstrat-3 Bamboo mosaic virus encodes a 155-kDa replicase that the middle region contains a helicase-like domain according to the existence of several conserved featured motifs of helicase. Previous studies showed that the helicase-like domain exhibits both ATPase and RNA 5’-triphosphotase activities, and Mg2+ is essential for both activities. Mutagenesis of conserved motifs showed these amino acids play important roles for enzymatic activities. These results suggested that the properties of this denatured and renatured protein are coincident with other SF1 helicases. Competitive inhibition also showed that the catalytic sites of helicase-like domain for hydrolyzing γ-phosphate of mononucleotide and RNA are identical to many other virus helicases. Even so, the unwinding activity of this protein have not been demonstrated yet. Tobacco mosaic virus, a member of alphavirus-like superfamily, the N-terminal truncated TMV helicase formed hexamer and exhibited double strand RNA unwinding activity. Accordingly, we also constructed the same N-terminal truncated BaMV helicase-like domain and analysis the capability to unwind double-strand RNA. The result showed that these N-terminal truncated protein reduced ATPase activity and did not show obvious unwinding activity. But the monomeric form of wild-type helicase-like domain possessed the best ATPase activity than oligmeric form and seen to have a marginal ability to unwind 3’ tail partial-duplex RNA in the presence of ATP although the signal was larger than single strand RNA. We speculate that the N-terminal regions of helicase-like domain or host factors may be essential to form properly oligomeric state and exhibit fully unwinding activity.