Ban-Yang Chang

Identification of the RNA-binding sites of the triple gene block protein 1 of bamboo mosaic potexvirus

The triple gene block protein 1 (TGBp1) encoded by open reading frame 2 of bamboo mosaic potexvirus (BaMV) was overexpressed in Escherichia coli and purified in order to test its RNA-binding activity. UV crosslinking assays revealed that the RNA-binding activity was present mainly in the soluble fraction of the refolded TGBp1. The binding activity was nonspecific and salt concentration-dependent: activity was present at 0-50 mM NaCl but was almost abolished at 200 mM. The RNA-binding domain was located by deletion mutagenesis to the N-terminal 3-24 amino acids of TGBp1. Sequence alignment analysis of the N-terminal 25 amino acids of the TGBp1 homologues of potexviruses identified three arginine residues. Arg-to-Ala substitution at any one of the three arginines eliminated most of the RNA-binding activity, indicating that they were all critical to the RNA-binding activity of the TGBp1 of BaMV.

The Stable Association of Virion with the Triple-gene-block Protein 3-based Complex of Bamboo mosaic virus

The triple-gene-block protein 3 (TGBp3) of Bamboo mosaic virus (BaMV) is an integral endoplasmic reticulum (ER) membrane protein which is assumed to form a membrane complex to deliver the virus intracellularly. However, the virus entity that is delivered to plasmodesmata (PD) and its association with TGBp3-based complexes are not known. Results from chemical extraction and partial proteolysis of TGBp3 in membrane vesicles revealed that TGBp3 has a right-side-out membrane topology; i.e., TGBp3 has its C-terminal tail exposed to the outer surface of ER. Analyses of the TGBp3-specific immunoprecipitate of Sarkosyl-extracted TGBp3-based complex revealed that TGBp1, TGBp2, TGBp3, capsid protein (CP), replicase and viral RNA are potential constituents of virus movement complex. Substantial co-fractionation of TGBp2, TGBp3 and CP, but not TGBp1, in the early eluted gel filtration fractions in which virions were detected after TGBp3-specific immunoprecipitation suggested that the TGBp2- and TGBp3-based complex is able to stably associate with the virion. This notion was confirmed by immunogold-labeling transmission electron microscopy (TEM) of the purified virions. In addition, mutational and confocal microscopy analyses revealed that TGBp3 plays a key role in virus cell-to-cell movement by enhancing the TGBp2- and TGBp3-dependent PD localization of TGBp1. Taken together, our results suggested that the cell-to-cell movement of potexvirus requires stable association of the virion cargo with the TGBp2- and TGBp3-based membrane complex and recruitment of TGBp1 to the PD by this complex.

Topological properties of the triple gene block protein 2 of Bamboo mosaic virus.

The triple gene block protein 2 (TGBp2) of Bamboo mosaic virus (BaMV) has been proposed to be a transmembrane protein; however, its features remain unclear. Here, we used biochemical approaches to determine its topological properties. Our data reveal that TGBp2 is mainly associated with the endoplasmic reticulum membrane. The resistance of TGBp2 in proteoliposomes, prepared from both the BaMV-infected tissues and in vitro reconstitution system, to both chemical extraction and trypsin digestion confirmed that it is indeed an integral membrane protein. On the basis of the minor change in the size of the major stable TGBp2-derived tryptic fragment from the monomeric TGBp2, as well as the sensitivity of the cysteine residues at the C-terminal tail of TGBp2 to maleimide modification, we suggest that TGBp2 adopts a topology with both its short N- and C-terminal tails exposed to the outer surface of the endoplasmic reticulum. Moreover, TGBp2 is able to self-assemble as revealed by the significant increase in multimeric TGBp2 when the TGBp2-containing proteoliposomes were treated with chemical crosslinker or oxidation agent.

Subgenomic RNAs of bamboo mosaic potexvirus-V isolate are packaged into virions.

Purified virions of bamboo mosaic potexvirus-V isolate (BaMV-V) were found to contain three major RNA species, the 6.4 kb genomic RNA and two RNAs of 2.0 and 1.0 kb, in addition to associated satellite RNA (0.85 kb). Results of Northern blot hybridization, primer extension analysis and cDNA sequencing showed that the packaged 2.0 and 1.0 kb RNAs of BaMV-V were subgenomic RNAs. In contrast, in the BaMV-O isolate, only genomic RNA was packaged and encapsidated subgenomic RNAs were not detectable. The transcription initiation sites for the 2.0 and 1.0 kb subgenomic RNAs of BaMV-V were located 1 1 and 16 nt upstream of the initiation codon of open reading frames (ORFs) 2 and 5, respectively. The 2.0 and 1.0 kb subgenomic RNAs functioned as messengers for the ORF2 protein and capsid protein, respectively. Packaging of the 1.0 kb subgenomic RNAs resulted in the formation of rod-shaped particles about 70 nm in length. Our results indicate that BaMV isolates have evolved distinctly for packaging of subgenomic RNAs.

The Nucleolar Fibrillarin Protein Is Required for Helper Virus-Independent Long-Distance Trafficking of a Subviral Satellite RNA in Plants

Bamboo mosaic virus satellite RNA can move autonomously in a fibrillarin-dependent manner in the absence of its helper virus in Nicotiana benthamiana. RNA trafficking plays pivotal roles in regulating plant development, gene silencing, and adaptation to environmental stress. Satellite RNAs (satRNAs), parasites of viruses, depend on their helper viruses (HVs) for replication, encapsidation, and efficient spread. However, it remains largely unknown how satRNAs interact with viruses and the cellular machinery to undergo trafficking. Here, we show that the P20 protein of Bamboo mosaic potexvirus satRNA (satBaMV) can functionally complement in trans the systemic trafficking of P20-defective satBaMV in infected Nicotiana benthamiana. The transgene-derived satBaMV, uncoupled from HV replication, was able to move autonomously across a graft union identified by RT-qPCR, RNA gel blot, and in situ RT-PCR analyses. Coimmunoprecipitation experiments revealed that the major nucleolar protein fibrillarin is coprecipitated in the P20 protein complex. Notably, silencing fibrillarin suppressed satBaMV-, but not HV-, phloem-based movement following grafting or coinoculation with HV. Confocal microscopy revealed that the P20 protein colocalized with fibrillarin in the nucleoli and formed punctate structures associated with plasmodesmata. The mobile satBaMV RNA appears to exist as ribonucleoprotein (RNP) complex composed of P20 and fibrillarin, whereas BaMV movement proteins, capsid protein, and BaMV RNA are recruited with HV coinfection. Taken together, our findings provide insight into movement of satBaMV via the fibrillarin-satBaMV-P20 RNP complex in phloem-mediated systemic trafficking.

The core-independent promoter-specific interaction of primary sigma factor

Previous studies have led to a model in which the promoter-specific recognition of prokaryotic transcription initiation factor, sigma (σ), is core dependent. Most σ functions were studied on the basis of this tenet. Here, we provide in vitro evidence demonstrating that the intact Bacillus subtilis primary sigma, σA, by itself, is able to interact specifically with promoter deoxyribonucleic acid (DNA), albeit with low sequence selectivity. The core-independent promoter-specific interaction of the σA is −10 specific. However, the promoter −10 specific interaction is unable to allow the σA to discern the optimal promoter spacing. To fulfill this goal, the σA requires assistance from core RNA polymerase (RNAP). The ability of σ, by itself, to interact specifically with promoter might introduce a critical new dimension of study in prokaryotic σ function.

Viral elements and host cellular proteins in intercellular movement of Bamboo mosaic virus

As a member of the genus Potexvirus, Bamboo mosaic virus (BaMV) also belongs to the plant viruses that encode triple gene block proteins (TGBps) for intercellular movement within the host plants. Recent studies of the movement mechanisms of BaMV have revealed similarities and differences between BaMV and other potexviruses. This review focuses on the general aspects of viral and host elements involved in BaMV movement, the interactions among these elements, and the possible pathways for intra- and intercellular trafficking of BaMV. Major features of BaMV trafficking that have not been demonstrated in other potexviruses include: (i) the involvement of replicase, (ii) fine regulation by coat protein phosphorylation, (iii) the key roles played by TGBp3, (iv) the use of virions as the major transported form, and (v) the involvement of specific host factors, such as Ser/Thr kinase-like protein of Nicotiana benthamiana. We also highlight areas for future study that will provide a more comprehensive understanding of the detailed interactions among viral movement proteins and host factors, as well as the regulatory mechanisms of virus movement. Finally, a model based on the current knowledge is proposed to depict the diverse abilities of BaMV to utilize a wide range of mechanisms for efficient intercellular movement.

Structural and functional properties of a Bacillus subtilis temperature-sensitive σA factor

Bacillus subtilis DB1005 is a temperature‐sensitive (Ts) sigA mutant containing double‐amino‐acid substitutions (I198A and I202A) on the hydrophobic face of the promoter −10 binding helix of σA factor. We have analyzed the structural and functional properties of this mutant σA factor both in vivo and in vitro. Our data revealed that the Ts σA factor possessed predominantly a multimeric structure which was prone to aggregation at restrictive temperature. The extensive aggregation of the Ts σA resulted in a very low core‐binding activity of the Ts σA factor and a markedly reduced σA‐RNA polymerase activity in B. subtilis DB1005, suggesting that extensive aggregation of the Ts σA is the main trigger for the temperature sensitivity of B. subtilis DB1005. Partial proteolysis, tryptophan fluorescence and 1‐anilinonaphthalene‐8‐sulfonate–binding analyses revealed that the hydrophobic face of the promoter −10 binding helix and also the hydrophobic core region of the Ts σA factor were readily exposed on the protein surface. This hydrophobic exposure provides an important cue for mutual interaction between molecules of the Ts σA and allows the formation of multimeric Ts σA. Our results also indicate that Ile‐198 and Ile‐202 on the hydrophobic face of the promoter −10 binding helix are essential to ensure the correct folding and stabilization of the functional structure of σA factor. Proteins 2000;40:613–622.

The cysteine residues at the C-terminal tail of Bamboo mosaic virus triple gene block protein 2 are critical for efficient plasmodesmata localization of protein 1 in the same block

The movement of some plant viruses are accomplished by three proteins encoded by a triple gene block (TGB). The second protein (TGBp2) in the block is a transmembrane protein. This study was aimed to unravel the mechanism underlying the relatively inefficient cell-to-cell movement of Bamboo mosaic virus (BaMV) caused by amino acid substitutions for the three Cys residues, Cys-109, Cys-112 and Cys-119, at the C-terminal tail of TGBp2. Results from confocal microscopy revealed that substitutions of the three Cys residues of TGBp2, especially Cys-109 and Cys-112, would reduce the efficiency of TGBp2- and TGBp3-dependent PD localization of TGBp1. Moreover, there is an additive effect of the substitutions on reducing the efficiency of PD localization of TGBp1. These results indicate that the Cys residues in the C-terminal tail region of TGBp2 participate in the TGBp2- and TGBp3-dependent PD localization of TGBp1, and thus influence the cell-to-cell movement capability of BaMV.

The core-independent promoter-specific binding of Bacillus subtilis σB

Bacillus subtilis σD is an alternative σ factor that possesses a core‐independent promoter −10 element binding specificity despite the lack of a distinct footprint on its cognate promoter. We wished to determine whether this property is common to alternative σ factors. To this end, we over‐expressed B. subtilis σB in Escherichia coli and analyzed its DNA binding ability by electrophoretic mobility shift assay and DNase I footprinting. The major complex formed by σB and its cognate promoter DNA is heparin‐sensitive. However, in contrast to the −10 element binding specificity observed for B. subtilis σD, the promoter binding of σB is specific for the −35 element. These and other results clearly demonstrate that alternative σ factors possess different promoter‐binding characteristics, and make core‐independent contributions to recognition of their cognate promoters.