Promotion of Bamboo Mosaic Virus Accumulation in Nicotiana benthamiana by 5'→3' Exonuclease NbXRN4.

Bamboo mosaic virus (BaMV) has a 6.4-kb (+) sense RNA genome with a 5' cap and a 3' poly(A) tail. ORF1 of this potexvirus encodes a 155-kDa replication protein responsible for the viral RNA replication/transcription and 5' cap formation. To learn more about the replication complex of BaMV, a protein preparation enriched in the 155-kDa replication protein was obtained from Nicotiana benthamiana by a protocol involving agroinfiltration and immunoprecipitation. Subsequent analysis by SDS-PAGE and mass spectrometry identified a handful of host proteins that may participate in the viral replication. Among them, the cytoplasmic exoribonuclease NbXRN4 particularly caught our attention. NbXRN4 has been shown to have an antiviral activity against Tomato bushy stunt virus and Tomato mosaic virus. In Arabidopsis, the enzyme could reduce RNAi- and miRNA-mediated RNA decay. This study found that downregulation of NbXRN4 greatly decreased BaMV accumulation, while overexpression of NbXRN4 resulted in an opposite effect. Mutations at the catalytically essential residues abolished the function of NbXRN4 in the increase of BaMV accumulation. Nonetheless, NbXRN4 was still able to promote BaMV accumulation in the presence of the RNA silencing suppressor P19. In summary, the replication efficiency of BaMV may be improved by the exoribonuclease activity of NbXRN4.

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.

Mutational analysis of a helicase motif-based RNA 5'-triphosphatase/NTPase from bamboo mosaic virus.

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 k(cat)/K(m), reduced by 5- to 15-fold. AMPPNP, a nonhydrolyzable ATP analogue, competitively inhibited RNA 5'-triphosphatase activity. Analogies of mutational effects on the two activities and approximation of K(i(AMPPNP)) and K(m(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.

mRNA guanylation catalyzed by the S-adenosylmethionine-dependent guanylyltransferase of bamboo mosaic virus.

The S-adenosylmethionine-dependent guanylyltransferase of bamboo mosaic virus belongs to a novel class of mRNA capping enzymes distantly conserved in Alphavirus-like superfamily. The reaction sequence of the viral enzyme has been proposed comprising steps of 1) binding of GTP and S-adenosylmethionine, 2) formation of m7GTP and S-adenosylhomocysteine, 3) formation of the covalent (Enzyme-m7GMP) intermediate, and 4) transfer of m7GMP from the intermediate to the RNA acceptor. In this study the acceptor specificity of the viral enzyme was characterized. The results show that adenylate or guanylate with 5'-diphosphate group is an essential feature for acceptors, which can be RNA or mononucleotide, to receive m7GMP. The transfer rate of m7GMP to guanylate is greater than to adenylate by a factor of approximately 3, and the K(m) value for mononucleotide acceptor is approximately 10(3)-fold higher than that for RNA. The capping efficiency of the viral genomic RNA transcript depends on the length of the transcript and the formation of a putative stem-loop structure, suggesting that mRNA capping process may participate in regulating the viral gene expression.

Critical residues for GTP methylation and formation of the covalent m7GMP-enzyme intermediate in the capping enzyme domain of bamboo mosaic virus.

Open reading frame 1 of Bamboo mosaic virus (BaMV), a Potexvirus in the alphavirus-like superfamily, encodes a 155-kDa replicase responsible for the formation of the 5' cap structure and replication of the viral RNA genome. The N-terminal domain of the viral replicase functions as an mRNA capping enzyme, which exhibits both GTP methyltransferase and S-adenosylmethionine (AdoMet)-dependent guanylyltransferase activities. We mutated each of the four conserved amino acids among the capping enzymes of members within alphavirus-like superfamily and a dozen of other residues to gain insight into the structure-function relationship of the viral enzyme. The mutant enzymes were purified and subsequently characterized. H68A, the mutant enzyme bearing a substitution at the conserved histidine residue, has an approximately 10-fold increase in GTP methyltransferase activity but completely loses the ability to form the covalent m(7)GMP-enzyme intermediate. High-pressure liquid chromatography analysis confirmed the production of m(7)GTP by the GTP methyltransferase activity of H68A. Furthermore, the produced m(7)GTP sustained the formation of the m(7)GMP-enzyme intermediate for the wild-type enzyme in the presence of S-adenosylhomocysteine (AdoHcy), suggesting that the previously observed AdoMet-dependent guanylation of the enzyme using GTP results from reactions of GTP methylation and subsequently guanylation of the enzyme using m(7)GTP. Mutations occurred at the other three conserved residues (D122, R125, and Y213), and H66 resulted in abolition of activities for both GTP methylation and formation of the covalent m(7)GMP-enzyme intermediate. Mutations of amino acids such as K121, C234, D310, W312, R316, K344, W406, and K409 decreased both activities by various degrees, and the extents of mutational effects follow similar trends. The affinity to AdoMet of the various BaMV capping enzymes, except H68A, was found in good correlations with not only the magnitude of GTP methyltransferase activity but also the capability of forming the m(7)GMP-enzyme intermediate. Taken together with the AdoHcy dependence of guanylation of the enzyme using m(7)GTP, a basic working mechanism, with the contents of critical roles played by the binding of AdoMet/AdoHcy, of the BaMV capping enzyme is proposed and discussed.