Characterization of the RNA-binding properties of the triple-gene-block protein 2 of Bamboo mosaic virus
© Hsu et al; licensee BioMed Central Ltd. 2009
Received: 05 March 2009
Accepted: 07 May 2009
Published: 07 May 2009
The triple-gene-block protein 2 (TGBp2) of Bamboo mosaic virus (BaMV) is a transmembrane protein which was proposed to be involved in viral RNA binding during virus transport. Here, we report on the RNA-binding properties of TGBp2. Using tyrosine fluorescence spectroscopy and UV-crosslinking assays, the TGBp2 solubilized with Triton X-100 was found to interact with viral RNA in a non-specific manner. These results raise the possibility that TGBp2 facilitates intracellular delivery of viral RNA through non-specific protein-RNA interaction.
Bamboo mosaic virus (BaMV) is a single-stranded, positive-sense RNA virus. Its genomic RNA has three partially overlapping open reading frames, called triple gene block (TGB), located between the coding sequences for the replicase and capsid protein . The TGB-encoded proteins are referred to as TGBp1, TGBp2 and TGBp3 according to their positions  and are required for virus movement in the host plant [3–6]. The TGB proteins are found in several different viral genera. On the basis of amino acid sequence comparisons of the TGB proteins, the TGB-containing viruses have been classified into hordei-like and potex-like viruses . Bamboo mosaic virus is a potex-like virus.
The functions of each TGB protein have been investigated. TGBp2 is an integral membrane protein with two transmembrane helices  and a topology with both its N- and C-terminal tails exposed to the outer surface of endoplasmic reticulum (ER) and the central loop in the lumen of ER [9, 10]. Inhibition of virus movement by mutations disrupting the transmembrane helices of Potato virus X (PVX) TGBp2 indicated that ER association is important for the functioning of TGBp2 (8). Moreover, the PVX TGBp2 is able to induce the formation of granular vesicles derived from the ER, which align on actin filaments . Mutations in the central loop region of PVX TGBp2 eliminate the formation of granular vesicles and inhibit the cell-to-cell movement of virus . In addition, the PVX TGBp2 is able to increase the size exclusion limit of plasmodesmata (PD) , probably through its association with host interacting proteins (TIPs) which in accompany with β-1, 3-glucanase regulate callose degradation .
The membrane-associated TGBp2 is thought to assist the intracellular transport of the viral ribonucleoprotein (RNP) complex to the PD by a subcellular translocation process via cytoskeleton and is assumed to function through protein-protein or protein-RNA interactions [15, 16]. The RNA-binding activity of a thioredoxin-fused Potato mop-top virus (PMTV) TGBp2 has been detected using Northwestern blot . However, RNA binding of TGBp2 in aqueous solution has not been studied. To confirm that TGBp2 is able to bind viral RNA and to gain insight into the RNA-binding properties of TGBp2, we prepared unfused TGBp2  and His6-tagged TGBp2 of BaMV to characterize their RNA-binding properties using tyrosine fluorescence spectroscopy and zero-length UV-crosslinking assay.
To determine whether the unfused TGBp2 binds viral RNA in a specific or non-specific manner, two non-viral RNAs (the mRNAs of sigA and flgM genes from Bacillus subtilis) were synthesized in vitro using the same method as described above. The ability of TGBp2 to bind the two bacterial mRNAs (Figure 2B) indicated that TGBp2 interacts with RNA in a non-specific manner.
Primers used for the construction of pJC2N and site-directed mutagenesis of His6-TGBp2
Sequences of primers (from 5' to 3')
ATCAGAAAGCTT AAGAAGGAGATATACATATGCACCACCACCACCACCAC GACCAGCCTCTTCATCTG
It has been reported that aromatic amino acid residues can interact directly with single-stranded nucleic acids either by polar interactions or planar stacking with the exposed bases [17, 23, 24]. To test whether this is also true for tyrosine residues in TGBp2, we replaced the tyrosine residue(s) in the central loop (residues 54, 63, or 70) or C-terminal tail (residue 105) of His6-TGBp2 with alanine and analyzed the effects of these mutations on RNA binding of His6-TGBp2. No significant effect of tyrosine mutation on RNA binding of His6-TGBp2 was observed (Figure 3B), indicating that the tyrosine residues in both the central loop and C-terminal tail domains of TGBp2 are also not directly involved in non-specific RNA binding of TGBp2.
The lack of detectable effect of Arg- or Lys-to-Ala substitutions and Tyr-to-Ala substitutions on non-specific RNA binding of His6-TGBp2 (Figure 3) suggested that it is not specific amino acid residues but conformational property of TGBp2, which is responsible for the non-specific interaction between TGBp2 and viral RNA. On the basis of the known topological properties of TGBp2 , we propose that the self-assembly of TGBp2 through helical packing of transmembrane helices and/or disulfide linkages among the C-terminal tails of TGBp2 help to provide the amino acid residues at both the N- and C-terminal tails of TGBp2, which are exposed to the outer surface of the ER-derived granule vesicles, with a non-specific RNA-binding conformation.
The non-specific RNA binding of TGBp2 also raises the question of "how the non-specific RNA binding of TGBp2 leads to specific transport of viral RNA". It is unlikely that the functional specificity of TGBp2 is conferred by the protein components of viral RNP since TGBp1 and CP do not influence the RNA-binding property of TGBp2 (data not shown). More likely, some accessory proteins, such as TGBp3  and/or certain unknown host factors associated with TGBp2 in the granular vesicles, play the role. The finding that the functional specificity of non-specific RNA-binding proteins can be achieved by assistance from the components of a regulatory complex may support this idea .
This research was supported by National Science Council of Republic of China Grant NSC 94-2311-2752-B-005-011-PAE and NSC96-2752-B-005-009-PAE.
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