Arg-16 and Arg-21 in the N-terminal region of the triple-gene-block protein 1 of Bamboo mosaic virus are essential for virus movement

Ming-Kuem Lin1, Ban-Yang Chang2, Jia-Teh Liao1, Na-Sheng Lin3 and Yau-Heiu Hsu1

1 Graduate Institute of Biotechnology, National Chung Hsing University, Taichung 402, Taiwan
2 Graduate Institute of Biochemistry, National Chung Hsing University, Taichung 402, Taiwan
3 Institute of Botany, Academia Sinica, Taipei 115, Taiwan

Correspondence
Yau-Heiu Hsu
yhhsu{at}nchu.edu.tw


   ABSTRACT
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The protein encoded by the first gene of the triple gene block (TGBp1) of potexviruses is required for movement of the viruses. It has been reported that single Arg->Ala substitutions at position 11, 16 or 21 of TGBp1 of Bamboo mosaic virus (BaMV) eliminate its RNA-binding activity, while substitutions at position 16 or 21 only affect its NTPase activity (Liou et al., Virology 277, 336–344, 2000). However, it remains unclear whether these Arg->Ala substitutions also affect the movement of BaMV in plants. To address this question, six mutants of BaMV, each containing either a single- or a double-alanine substitution at Arg-11, Arg-16 and Arg-21 of TGBp1, were constructed and used to infect Chenopodium quinoa and Nicotiana benthamiana. We found that all of the BaMV mutants were able to replicate in protoplasts of N. benthamiana. However, only the mutant with an Arg-11->Ala substitution in TGBp1 remained capable of movement from cell to cell in plants. Mutants with Arg-16, Arg-21 or both Arg-16 and Arg-21 of TGBp1 replaced with alanine were defective in virus movement. This defect was suppressed when a wild-type TGBp1 allele was co-introduced into the cells using a novel satellite replicon. The ability to trans-complement the movement defect by the wild-type TGBp1 strongly suggests that the Arg->Ala substitution at position 16 or 21 of TGBp1, which diminishes the RNA-binding and NTPase activities of TGBp1, also eliminates the capability of BaMV to move from cell to cell in host plants.


   INTRODUCTION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The movement of plant viruses from cell to cell and over long distances requires the assistance of virus-encoded movement proteins (MPs) (reviewed by Atabekov & Taliansky, 1990; Gilbertson & Lucas, 1996). Several mechanisms, including the Tobacco mosaic virus (TMV)-like and tubule-based mechanisms, have been reported for intercellular transport of plant viruses (Carrington et al., 1996). For each mechanism, the MPs involved seem to possess different functions. In the TMV-like mechanism, the MP is able to increase the size-exclusion limit (SEL) of the plasmodesmata (Wolf et al., 1989), to bind non-specifically to single-stranded RNA (ssRNA) (Citovsky et al., 1990, 1992) and to interact with the cytoskeleton through which the viral movement complexes are transported to the plasmodesmata (Carrington et al., 1996; Heinlein et al., 1995; McLean et al., 1995). MPs participating in the tubule-based mechanism are able to form tubules through cell walls and/or plasmodesmata (Perbal et al., 1993; Storms et al., 1995; van Lent et al., 1990; Wieczorek & Sanfacon, 1993), to interact with virions assembled in the cytoplasm and to transfer virions through the tubules via specific MP–capsid protein (CP) complexes (Wellink & van Kammen, 1989).

The three proteins encoded by the triple gene block (TGBps) of potexviruses are essential for the cell-to-cell movement of viruses of this group (Angell et al., 1996; Beck et al., 1991). However, TGBps are structurally and functionally different from the MPs described above. Among the three TGB proteins, TGBp1 appears to exist in soluble and cytoplasmic inclusion forms. The soluble form of TGBp1 binds cooperatively to ssRNA, possesses ATPase (Rouleau et al., 1994; Wung et al., 1999) and RNA helicase activities (Kalinina et al., 2002), increases the SEL of plasmodesmata (Angell et al., 1996; Lough et al., 1998) and forms a ribonucleoprotein complex (Lough et al., 1998, 2000). TGBp1 is also a gene silencing suppressor, which prevents spread of the gene silencing signal in Nicotiana benthamiana (Voinnet et al., 2000). TGBp1 can move from cell to cell (Lough et al., 1998; Yang et al., 2000) and mediates cell-to-cell movement of TGBp2 and TGBp3 (Krishnamurthy et al., 2002). Further analysis of the soluble TGBp1 of Bamboo mosaic virus (BaMV) has revealed that Arg-11, Arg-16 and Arg-21 in the N-terminal region of this protein are essential for its RNA-binding activity (Liou et al., 2000). Although BaMV TGBp1 belongs to the RNA helicase superfamily I (Kadare & Haenni, 1997; Morozov et al., 1999; Liou et al., 2000), Arg-16 and Arg-21, which are located outside the seven conserved motifs of the superfamily I RNA helicase, are also critical for the NTPase activity of TGBp1 (Liou et al., 2000). TGBp1s of potexviruses are in fact mainly associated with cytoplasmic inclusions in infected tissues (Chang et al., 1997; Davies et al., 1993; Rouleau et al., 1994). The TGBp1 obtained from the cytoplasmic inclusions also possesses NTP-binding and NTPase activities but lacks the RNA-binding activity (Liou et al., 2000).

TGBp2 and TGBp3 both contain stretches of hydrophobic amino acids and are associated with cell wall and membrane fractions both in vivo and in vitro (Donald et al., 1993; Hefferon et al., 1997; Morozov et al., 1990, 1991; Niesbach-Klosgen et al., 1990; Solovyev et al., 2000). Using a microinjection and transient expression strategies, TGBp2 was shown to assist the movement of the ribonucleoprotein complex of potexviruses (Lough et al., 1998) and to be capable of increasing the SEL of the plasmodesmata (Tamai & Meshi, 2001). However, contradictory results were obtained for TGBp3. It has been reported to be required for the movement of White clover mosaic virus (WClMV) (Lough et al., 1998) but is not absolutely required for the movement of potato virus X (Tamai & Meshi, 2001). Recently, Krishnamurthy et al. (2003) and Mitra et al. (2003) have demonstrated that TGBp2 and TGBp3 are endoplasmic reticulum (ER)-targeted proteins and that ER targeting is important for virus movement.

A satellite RNA (satBaMV) has been found to associate with the BaMV-V isolate (Lin & Hsu, 1994). The satBaMV is a linear RNA molecule 836 nucleotides in length, excluding the poly(A) tail, and encodes a 20 kDa non-structural protein (P20). The satBaMV can be used as a plant expression vector since P20 is not essential for satellite replication and can be replaced with other proteins such as chloramphenicol acetyltransferase (Lin et al., 1996).

In this study, we analysed the relationship between the RNA-binding and NTPase activities of TGBp1 and the capability of BaMV to move from cell to cell. With the aid of the infectious cDNA clones of BaMV-S and a satellite replicon based on satBaMV (Lin et al., 1996), we have demonstrated that Arg-16 and Arg-21 in the N-terminal region of the BaMV TGBp1, residues that are essential for the RNA-binding and NTPase activities of TGBp1 (Wung et al., 1999; Liou et al., 2000), are also crucial for cell-to-cell movement of BaMV in host plants.


   METHODS
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Infectious BaMV and satBaMV cDNA clones.
The plasmid pCB is an infectious clone of BaMV-S (EMBL/GenBank accession no. AF018156). Expression of the BaMV genome in this plasmid is under the transcriptional control of the 35S promoter of Cauliflower mosaic virus (CaMV). Plasmid pCBG is a derivative of pCB; a green fluorescent protein gene (gfp) (Sheen et al., 1995) has been inserted between the two open reading frames of TGBp3 and CP in the BaMV genome and is controlled by a duplicated subgenomic promoter for CP expression (J.-T. Liao & Y.-H. Hsu, unpublished data). The recombinant BaMV bearing the gfp gene in pCBG can be transcribed from the 35S promoter of CaMV. Plasmid pCBSF4, derived from pBSF4 (Lin et al., 1996), is an infectious cDNA clone of the satBaMV, in which expression of the cDNA sequence is driven by the 35S promoter as described for the pCass plasmid (Ding et al., 1995).

Construction of mutant BaMV and satellite replicons.
Each of the mutant BaMV with Arg->Ala substitution(s) in TGBp1 was derived from pCBG by site-directed mutagenesis (Sambrook et al., 1989). Primers used for mutagenesis are shown in Table 1. pR11A, pR16A and pR21A are plasmids that contain a single Arg->Ala substitution at amino acid positions 11, 16 and 21 of TGBp1, respectively. Plasmids pR11/16A, pR11/21A and pR16/21A containing double Arg->Ala substitutions were derived from pR11A or pR16A. To construct pSat28, the P20-coding sequence of the satBaMV in pCBSF4 was replaced with the TGBp1-coding sequence amplified from pCBG using primers P28N and P28C (Table 1). The plasmid pSat28f was a mutant clone of pSat28, in which a single-base deletion was observed at position 7 of the TGBp1-coding sequence, resulting in a shift of reading frame from the third amino acid of TGBp1.


View this table:
[in this window]
[in a new window]
 
Table 1. Primers used in this study

 
Inoculation of plants with the cDNA clones of BaMV.
Methods used for inoculation of N. benthamiana or Chenopodium quinoa with clones of BaMV were as previously reported (Lin & Hsu, 1994; Lin et al., 1996), except that each leaf was inoculated with 1 µg plasmid DNA instead of RNA transcript. Methods for total protein extraction and Western blot analysis were as previously reported (Lin et al., 1996; Chang et al., 1997) except that an ECL detection system was adopted (Amersham).

Protoplast preparation, inoculation and Northern and Western blot analyses of viral products.
Methods for preparation and inoculation of protoplasts were as previously described (Cheng & Tsai, 1999) except that plasmid DNA, rather than RNA transcript, was used as the inoculum. In brief, the protoplasts (2x105) prepared from mesophyll cells of N. benthamiana were inoculated with 10 µg plasmid DNA. Methods for Northern and Western blot analyses of viral RNA and proteins in infected protoplasts were as previously described (Cheng & Tsai, 1999).

FluorImage and confocal laser scanning microscopy of the BaMV-infected leaves.
Fluorescent images of leaves infected with the wild-type BaMV or the TGBp1 mutants were obtained with a FluorImager (Molecular Dynamics, model 595) with an excitation filter of 488 nm and an emission filter using calibration files. Fluorescent images for epidermal and mesophyll cells were obtained through a confocal laser-scanning microscope (Zeiss LSM410). Laser illumination at 488 nm (argon ion laser) was recorded through a bandpass filter normally used for the detection of fluorescence. Image analysis and display (adjustments in contrast, brightness, etc.) were all performed using Adobe PhotoShop.

RT-PCR and cDNA sequencing.
Total RNAs were extracted from the inoculated leaves of C. quinoa and N. benthamiana as previously described (Lin et al., 1996). Reverse transcription was then carried out to synthesize the first strand cDNA of TGBp1 using primer 5081 (Table 1) complementary to the 3' end of the genomic or subgenomic RNA for TGBp1. Following that, 30 PCR cycles were performed using the forward primer (3572R) and the reverse primer (4818), which are complementary to the cDNA sequence of TGBp1. The PCR products were finally sequenced using primer 3759R with the IRDye 800 terminator sequencing protocol designed for the LI-COR IR2 automated DNA sequencer.


   RESULTS
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Replacement of Arg-16, Arg-21 or both Arg-16 and Arg-21 of TGBp1 with alanine affects cell-to-cell movement of BaMV
To determine whether there is any relationship between the RNA-binding and NTPase activities of TGBp1 and the capability of BaMV to move from cell to cell, six plasmid clones of mutant BaMV, each containing a single- or a double-alanine substitution at Arg-11, Arg-16 and Arg-21 of TGBp1, were constructed (see Methods) and designated pR11A, pR16A, pR21A, pR11/16A, pR16/21A and pR11/21A, according to the arginine residue(s) replaced (Fig. 1). Inoculation of C. quinoa and N. benthamiana with each of the mutant TGBp1 BaMV plasmids or the wild-type TGBp1-containing plasmid, pCBG, was then performed. Since an expressible gfp gene had been inserted in between the coding sequences of TGBp3 and CP in the BaMV genome (see Methods), we expected to see spread of green fluorescence if the mutant BaMV was able to replicate and move from cell to cell. As shown in Fig. 2, spread of green fluorescence was only observed in leaves of N. benthamiana 10 days (Fig. 2A) and C. quinoa 5 days (Fig. 2B) post-inoculation (p.i.) with pCBG or pR11A. No fluorescence spread was detected when plant leaves were inoculated with the other mutant BaMVs. These results indicated that only the R11A mutant is able to move from cell to cell and that the movement function is eliminated for the other mutant BaMVs. Since the alanine codon (GCA) remained unchanged in the progeny of the R11A mutant present in the inoculated leaves of C. quinoa (Fig. 3) and in both the inoculated (data not shown) and systemic leaves of N. benthamiana (Fig. 3), we concluded that R11A-TGBp1 is functional for cell-to-cell movement.



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 1. The N-terminal amino acid sequences of TGBp1 of the wild-type (Wt) and mutant BaMVs. The positions of arginine (R) being replaced with alanine (A) are indicated above the amino acid sequence of the wild-type TGBp1.

 


View larger version (83K):
[in this window]
[in a new window]
 
Fig. 2. Fluorescence images of the leaves of N. benthamiana and C. quinoa inoculated with the plasmid clones of wild-type and mutant BaMVs. (A) Fluorescence graphs of leaves from N. benthamiana 10 days p.i. (B) Fluorescence graphs of leaves from C. quinoa 5 days p.i. The plasmid used to inoculate the plant leaves is indicated above each image.

 


View larger version (52K):
[in this window]
[in a new window]
 
Fig. 3. Nucleotide sequences of the progenies of the mutant BaMV containing R11A in TGBp1. Total RNA was extracted from the inoculated leaves of C. quinoa or from the systemic leaves of N. benthamiana. RT-PCR and cDNA sequencing were performed to examine the sequences of the R11A progenies. The corresponding nucleotide sequence of TGBp1 from Ala-11 to Arg-16 (R16) is indicated on the left, with Ala-11 boxed.

 
The inability of the R16A, R21A, R11/16A, R11/21A or R16/21A mutants to move from cell to cell was also evidenced by the lack of fluorescence spread between adjacent cells monitored by a confocal laser scanning microscope (Fig. 4). Cell-to-cell spread of fluorescence was undetectable, even in epidermal cells infected with the five BaMV mutants; it was restricted to a single cell (Fig. 4E and F). In contrast, the spread of green fluorescence between adjacent cells was easily detected in the epidermal (data not shown) and mesophyll cells of C. quinoa infected with pCBG (Fig. 4C) or pR11A (Fig. 4D). The restriction of movement of the mutant BaMVs containing R16A, R21A, R11/16A, R11/21A or R16/21A in TGBp1 was further corroborated by the absence of viral CP in leaves of both C. quinoa and N. benthamiana 10 days p.i. (Fig. 5A and B).



View larger version (55K):
[in this window]
[in a new window]
 
Fig. 4. Fluorescence of the inoculated leaves of C. quinoa as viewed using a confocal laser-scanning microscope. Fluorescence in epidermal cells inoculated with water (A), pCB (B), pR16A (E), pR21A (F) or fluorescence in mesophyll cells inoculated with pCBG (C) or pR11A (D) 5 days p.i. Bar, 50 µm.

 


View larger version (38K):
[in this window]
[in a new window]
 
Fig. 5. Western blot analyses of CP and fluorescence spread in leaves inoculated with the plasmid clones of wild-type or mutant BaMVs. (A, B) CP in the inoculated leaves of C. quinoa (A) and N. benthamiana (B) 10 days p.i. (C, D) CP in the systemic leaves of N. benthamiana infected with the wild-type or mutant BaMVs 15 (C) and 30 (D) days p.i. The total proteins extracted from the inoculated or systemic leaves harvested at the indicated time points were separated by SDS-PAGE, blotted onto PVDF membrane and probed with polyclonal antiserum against CP (Lin & Chen, 1991). The plasmid clone of BaMV used to infect the leaves is shown above each panel. M, CP from purified BaMV. (E, F) Fluorescence images of the systemic leaves of N. benthamiana infected with the wild-type pCBG or pR11A 15 (E) and 30 (F) days p.i.

 
Similar to the result observed in inoculated leaves, spread of fluorescence was observed in systemic leaves of N. benthamiana inoculated with pCBG or pR11A (Fig. 5E and F), but not in systemic leaves of N. benthamiana inoculated with any of the remaining five BaMV mutants (data not shown). Since a similar level of green fluorescence or CP was observed in both the inoculated (Figs 2, 4 and 5A and B) and systemic leaves (Fig. 5C–F) infected with pCBG or pR11A, we believe that the Arg-11->Ala substitution of TGBp1 had, at most, only a slight effect on the capability of cell-to-cell and long-distance movement of BaMV.

BaMV mutants defective in cell-to-cell movement are able to replicate and synthesize TGBp1
Several explanations could account for the failure of mutant BaMVs with TGBp1 Arg->Ala substitutions at either position 16, 21 or both 16 and 21 to move from cell to cell. One possibility is that the Arg->Ala substitution(s) directly renders the mutant TGBp1 defective in movement function. Alternatively, it could be that these TGBp1 mutations interfere with virus replication. Finally, it is possible that the synthesis and accumulation of TGBp1 in leaves infected with the mutant BaMVs are blocked for as yet unknown reasons. To rule out the latter two possibilities, the replication of viral RNA and the synthesis of TGBp1 were analysed in protoplasts of N. benthamiana infected with each of the movement-defective BaMVs. As shown in Fig. 6(A), similar patterns of genomic and subgenomic RNAs of BaMV were observed in protoplasts inoculated with the wild-type or mutant BaMVs, indicating that the wild-type and mutant BaMVs possess similar replication activities. In addition, similar amounts of TGBp1 were also detected in protoplasts infected with the wild-type and mutant BaMVs (Fig. 6B). These results indicated that the movement-defective BaMVs are able to replicate and to synthesize TGBp1 in the plant cells. Taken together, these results demonstrate that the inability of the mutant BaMVs with Arg->Ala substitutions at position 16, 21 or both 16 and 21 of TGBp1 to move from cell to cell must be attributed to the loss of certain important TGBp1 functions.



View larger version (30K):
[in this window]
[in a new window]
 
Fig. 6. Northern and Western blot analyses of viral products in protoplasts of N. benthamiana inoculated with plasmid clones of the wild-type or mutant BaMVs. (A) Northern blot analysis of viral RNA. Total RNAs extracted from BaMV-infected protoplasts at 2 days p.i. were separated on a 1 % agarose gel, blotted onto a nylon membrane and probed with 32P-labelled RNA complementary to the 3' end of the BaMV genome (Lin et al., 1993). The various RNAs are as indicated in the right and left margins. Lane 1, viral RNA of BaMV; lane 2, mock inoculation; lanes 3–7, RNA samples extracted from protoplasts inoculated with pCBG, pR11A, pR16A, pR21A and pR11/21A, respectively. (B) Western blot analysis of TGBp1. Total proteins were extracted from the BaMV-infected protoplasts at 2 days p.i., separated by 12 % SDS-PAGE, blotted onto a PVDF membrane and probed with polyclonal antiserum against TGBp1 (Chang et al., 1997). The numbers shown in the left margin indicate the apparent Mr in kDa. Lane 1, TGBp1 of BaMV purified from an E. coli overexpression system (Chang et al., 1997); lanes 2–7, as in (A).

 
Complementation of the movement defect of the mutant BaMVs by a satellite replicon harbouring the wild-type TGBp1
To illustrate further that the movement defect of the mutant BaMVs containing R16A and/or R21A in TGBp1 was indeed due to the loss of TGBp1 function, a satellite replicon, pSat28, capable of expressing the wild-type TGBp1, was co-inoculated with equal amounts of pR16A or pR21A into N. benthamiana leaves. In parallel, co-inoculation of pR16A or pR21A with a mutant satellite replicon, pSat28f, harbouring a frameshifted TGBp1, was also conducted. The latter treatment served as a negative control for the complementation assay since no intact and functional TGBp1 could be expressed. As expected, no spread of fluorescence was detected in leaves co-inoculated with pR16A and pSat28f or with pR21A and pSat28f (Fig. 7A). In contrast, fluorescence spread was easily detected when pR16A or pR21A was co-inoculated with pSat28 (Fig. 7A). To verify that the complementation of the movement defect by pSat28 was due to the synthesis and accumulation of wild-type TGBp1, the TGBp1 content of the inoculated leaves was analysed. TGBp1 was absent from leaves inoculated with pR16A or pR21A alone (Fig. 7B, lanes 2 and 3) or further co-inoculated with pSat28f (Fig. 7B, lanes 4 and 5). However, TGBp1 was clearly observed in leaves that were co-inoculated with pR16A and pSat28 or with pR21A and pSat28 (Fig. 7B, lanes 6 and 7). Taken together, these results indicated that introduction of a wild-type TGBp1 allele to the leaves rescues the mutant TGBp1 defect and enables the mutant BaMV to move from cell to cell. Since the mutant TGBp1s are devoid of the RNA-binding and NTPase activities and can be complemented by the wild-type TGBp1, it is assumed that the RNA-binding and NTPase activities are required for the movement of BaMV.



View larger version (104K):
[in this window]
[in a new window]
 
Fig. 7. Effects of the satellite replicons, pSat28 and pSat28f, on the movement and synthesis of TGBp1 in leaves infected with the mutant BaMVs. (A) Effect of satellite replicons on the movement of R16A and R21A mutants of BaMV. The fluorescent images of leaves of N. benthamiana inoculated with pR16A and pR21A with or without the satellite replicon 10 days p.i. are shown. (B) Synthesis of TGBp1 in the presence of the satellite replicon. The total proteins extracted from the leaves as shown in (A) were separated by 12 % SDS-PAGE, blotted onto a PVDF membrane and probed with the polyclonal antiserum against TGBp1. Lane 1, recombinant TGBp1 purified from E. coli; lanes 2 and 3, samples from leaves inoculated with pR16A and pR21A, respectively; lanes 4–7, samples from leaves co-inoculated with pSat28f and pR16A, pSat28f and pR21A, pSat28 and pR16A, and pSat28 and pR21A, respectively.

 

   DISCUSSION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
It has been shown by deletion analyses that the TGBp1 homologues of potexviruses play important roles in assisting virus movement between plant cells (Beck et al., 1994; Angell et al., 1996; Lough et al., 1998; Verchot et al., 1998). With the aid of infectious plasmid clones of BaMV, we have shown that a single or double Arg->Ala substitution at positions 16 and 21, which diminishes the RNA-binding and ATP-utilizing activities of TGBp1 (Wung et al., 1999; Liou et al., 2000), also eliminates the ability of BaMV to move from cell to cell. Furthermore, our ability to trans-complement the mutant TGBp1 with its wild-type counterpart supports the idea that the RNA-binding activity and the NTPase activity of TGBp1 are necessary for the movement of potexviruses from cell to cell.

Complementation has long been used as a strategy to confirm the function of a specific gene. Strategies for complementation of a defective gene in a plant virus include co-bombardment of plants with the defective virus together with a plasmid expressing the target gene (Lough et al., 2001; Morozov et al., 1997; Tamai & Meshi, 2001), mixed infection of plants with the defective virus and a viral RNA replicon (Bleykasten-Grosshans et al., 1997), and microinjection of the target protein into a transgenic plant lacking the corresponding gene (Lough et al., 1998). The new strategy adopted in the present study involves the use of a novel satellite replicon, pSat28. The presence of TGBp1 and the spread of BaMV in N. benthamiana leaves co-infected with the mutant BaMVs and pSat28 (Fig. 7A) demonstrated that the satellite replicon strategy works as well as other strategies for complementation analyses.

There are several possible explanations for how the loss of ATPase and RNA-binding activities of TGBp1 could interfere with the movement of the mutant BaMVs. It is highly possible that the loss of the ATPase activity of TGBp1 leads to inactivation of its helicase activity, which is essential for unwinding the duplex region of the viral RNA (Kalinina et al., 2002). In addition, loss of RNA-binding activity of a TGBp1 mutant would probably negatively affect the ability of the mutant TGBp1 to form a streamlined nucleoprotein complex with the viral RNA suitable for movement (Lough et al., 1998, 2000). Thus, the movement process of the virus, at least in the initial stage of transport, is probably hampered by the loss of each of these two activities. However, it is also possible that the defective TGBp1 is unable to carry out steps in the movement process subsequent to the streamlining of viral RNA. For example, the TGBp1 mutations could prevent the viral ribonucleoprotein complex from interacting with TGBp2, TGBp3 or some other host protein so that the movement of viral nucleic acid along the cytoskeleton and toward the plasmodesmata is blocked. Furthermore, loss of the ATPase activity may inhibit the ability of TGBp1 to increase the SEL of plasmodesmata (Lough et al., 1998) and thus decrease the efficiency of viral transport across the cell wall. Further studies are required to clarify these possibilities.

Since the R11A TGBp1, while retaining its NTPase activity, has significantly reduced RNA-binding activity (Liou et al., 2000; Wung et al., 1999), we originally predicted that this mutant would be unable to move from cell to cell. However, the R11A mutant was shown to be able to move in the present study (Figs 2, 4 and 5). Because no reversion was detected in the progenies of the R11A mutant (Fig. 3), we conclude that the R11A TGBp1 mutant is able to fulfil movement function. Probably, the residual RNA-binding activity of the R11A TGBp1 is enough to assist virus movement. Alternatively, there may be some other factors that are able to enhance the RNA-binding activity of the mutant TGBp1 in vivo. The ability of the TGBp1 protein of WClMV to form ribonucleoprotein complexes with the viral CP (Lough et al., 1998, 2000) raises the possibility that CP may be one such factor. Other possibilities are interactions with TGBp2 and TGBp3 (Krishnamurthy et al., 2002).


   ACKNOWLEDGEMENTS
 
This work was supported by the National Science Council Project Grants NSC88-2311-B-005-004-B11 and NSC89-2311-B-005-008, Taiwan, Republic of China.


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Angell, S. M., Davies, C. & Baulcombe, D. C. (1996). Cell-to-cell movement of potato virus X is associated with a change in the size-exclusion limit of plasmodesmata in trichome cells of Nicotiana clevelandii. Virology 216, 197–201.[CrossRef][Medline]

Atabekov, J. G. & Taliansky, M. E. (1990). Expression of a plant virus-coded transport function by different viral genomes. Adv Virus Res 38, 201–247.[Medline]

Beck, D. L., Guilford, P. J., Voot, D. M., Andersen, M. T. & Forster, R. L. S. (1991). Triplet gene block proteins of white clover mosaic potexvirus are required for transport. Virology 183, 695–702.[Medline]

Beck, D. L., Van Dolleweerd, C. J., Lough, T. J., Balmori, E., Voot, D. M., Andersen, M. T., O'Brien, I. E. W. & Forster, R. L. S. (1994). Disruption of virus movement confers broad-spectrum resistance against systemic infection by plant viruses with a triple gene block. Proc Natl Acad Sci U S A 91, 10310–10314.[Abstract/Free Full Text]

Bleykasten-Grosshans, C., Guilley, H., Bouzoubaa, S., Richards, K. E. & Jonard, G. (1997). Independent expression of the first two triple gene block proteins of beet necrotic yellow vein virus complements virus defective in the corresponding gene but expression of the third protein inhibits viral cell-to-cell movement. Mol Plant Microbe Interact 10, 240–246.

Carrington, J. C., Kassachau, K. D. Y., Mahajan, S. K. & Schaad, M. C. (1996). Cell-to-cell movement and long-distance transport of virus in plants. Plant Cell 8, 1669–1681.[Free Full Text]

Chang, B. Y., Lin, N. S., Liou, D. Y., Chen, J. P., Liou, G. G. & Hsu, Y. H. (1997). Subcellular localization of the 28 kDa protein of the triple-gene-block of bamboo mosaic potexvirus. J Gen Virol 78, 1175–1179.[Abstract]

Cheng, C. P. & Tsai, C. H. (1999). Structural and functional analysis of the untranslated region of bamboo mosaic potexvirus genomic RNA. J Mol Biol 288, 555–565.[CrossRef][Medline]

Citovsky, V., Knorr, D., Schuster, G. & Zambryski, P. (1990). The P30 movement protein of tobacco mosaic virus is a single stranded nucleic acid binding protein. Cell 60, 637–647.[Medline]

Citovsky, V., Wong, M. L., Shaw, A. L., Prasad, B. V. V. & Zambryski, P. (1992). Visualization and characterization of tobacco mosaic virus movement protein binding to single stranded nucleic acid. Plant Cell 4, 397–411.[Abstract/Free Full Text]

Davies, C., Hills, G. & Baulcombe, D. C. (1993). Subcellular localization of the 25-kDa protein encoded in the triple gene block of potato virus X. Virology 197, 166–175.[CrossRef][Medline]

Ding, S. W., Rathjen, J. P., Li, W. X., Swanson, R., Healy, H. & Symons, R. H. (1995). Efficient infection from cDNA clones of cucumber mosaic cucumovirus RNAs in a new plasmid vector. J Gen Virol 76, 459–464.[Abstract]

Donald, R. G. K., Zhou, H. & Jackson, A. O. (1993). Serological analysis of barley stripe mosaic virus-encoded proteins in infected barley. Virology 195, 659–668.[CrossRef][Medline]

Gilbertson, R. L. & Lucas, W. J. (1996). How do viruses traffic on the "vascular highway"? Trends Plant Sci 1, 260–268.[CrossRef]

Hefferon, K. L., Doyle, S. & AbouHalder, M. G. (1997). Immunological detection of the 8K protein of potato virus X (PVX) in cell walls of PVX-infected tobacco and transgenic potato. Arch Virol 142, 425–433.[CrossRef][Medline]

Heinlein, M., Epel, B. L., Padgett, H. S. & Beachy, R. N. (1995). Interaction of tobamovirus movement proteins with the plant cytoskeleton. Science 270, 1983–1985.[Abstract]

Kadare, G. & Haenni, A. L. (1997). Virus-encoded helicase. J Virol 71, 2583–2590.[Free Full Text]

Kalinina, N. O., Rakitina, D. V., Solovyev, A. G., Schiemann, J. & Morozov, S. Yu. (2002). RNA helicase activity of the plant virus movement proteins encoded by the first gene of the triple gene block. Virology 296, 321–329.[CrossRef][Medline]

Krishnamurthy, K., Mitra, R., Payton, M. & Verchot-Lubicz, J. (2002). Cell-to-cell movement of the PVX 12K, 8K, or coat proteins may depend on the host, leaf developmental stage, and the PVX 25K protein. Virology 300, 269–281.[CrossRef][Medline]

Krishnamurthy, K., Heppler, M., Mitra, R., Blancaflor, E., Payton, M., Nelson, R. S. & Verchot-Lubicz, J. (2003). The potato virus X TGBp3 protein associates with the ER network for virus cell-to-cell movement. Virology 309, 135–151.[CrossRef][Medline]

Lin, N. S. & Chen, C. C. (1991). Association of bamboo mosaic virus (BaMV) and BaMV-specific electron-dense crystalline bodies with chloroplasts. Phytopathology 81, 1551–1555.

Lin, N. S. & Hsu, Y. H. (1994). A satellite RNA associated with bamboo mosaic potexvirus. Virology 202, 707–714.[CrossRef][Medline]

Lin, N. S., Chai, Y. J., Huang, T. Y., Chang, T. Y. & Hsu, Y. H. (1993). Incidence of bamboo mosaic potexvirus in Taiwan. Plant Dis 77, 448–450.

Lin, N. S., Lee, Y. S., Lin, B. Y., Lee, C. W. & Hsu, Y. H. (1996). The open reading frame of bamboo mosaic potexvirus satellite RNA is not essential for its replication and can be replaced with a bacterial gene. Proc Natl Acad Sci U S A 93, 3138–3142.[Abstract/Free Full Text]

Liou, D. Y., Hsu, Y. H., Wung, C. H., Lin, N. S. & Chang, B. Y. (2000). Functional analyses and identification of two arginine residues essential to the ATP-utilizing activity of the triple gene block protein 1 of bamboo mosaic potexvirus. Virology 277, 336–344.[CrossRef][Medline]

Lough, T. J., Shash, K., Xoconostle-Cazares, B., Hofstra, K. R., Beck, D. L., Balmori, E., Forster, R. L. S. & Lucas, W. J. (1998). Molecular dissection of the mechanism by which potexvirus triple gene block proteins mediate cell-to-cell transport of infectious RNA. Mol Plant Microbe Interact 11, 801–814.

Lough, T. J., Netzler, N. E., Emerson, S. J., Sutherland, P., Carr, F., Beck, D. L., Lucas, W. J. & Forster, R. L. S. (2000). Cell-to-cell movement of potexviruses: evidence for a ribonucleoprotein complex involving the coat protein and first triple gene block protein. Mol Plant Microbe Interact 13, 962–974.[Medline]

Lough, T. J., Emerson, S. J., Lucas, W. J. & Forster, R. L. S. (2001). Trans-complementation of long-distance movement of white clover mosaic virus triple gene block (TGB) mutants: phloem-associated movement of TGBp1. Virology 288, 16–28.

McLean, B. G., Zupan, J. & Zambryski, P. C. (1995). Tobacco mosaic virus movement protein associates with the cytoskeleton in tobacco cells. Plant Cell 7, 2101–2114.[Abstract/Free Full Text]

Mitra, R., Krishnamurthy, K., Blancaflor, E., Payton, M., Nelson, R. S. & Verchot-Lubicz, J. (2003). The potato virus X TGBp2 protein association with the endoplasmic reticulum plays a role in but is not sufficient for viral cell-to-cell movement. Virology 312, 35–48.[CrossRef][Medline]

Morozov, S. Y., Miroshnichenko, N. A., Zelenina, D. A., Fedorkin, O. N., Solovijev, A. G., Lukasheva, L. I. & Atabekov, J. G. (1990). Expression of RNA transcripts of potato virus X full-length and subgenomic cDNAs. Biochimie 72, 677–684.[CrossRef][Medline]

Morozov, S. Y., Miroshnichenko, N. A., Solovyev, A. G., Zelenina, D. A., Fedorkin, O. N., Lukasheva, L. I., Grachev, S. A. & Chernov, B. K. (1991). In vitro membrane binding of the translation products of the carlavirus 7-kDa protein gene. Virology 183, 782–785.[Medline]

Morozov, S. Y., Fedorkin, O. N., Juttner, G., Schiemann, J., Baulcombe, D. C. & Atabekov, J. G. (1997). Complementation of a potato virus X mutant mediated by bombardment of plant tissues with cloned viral movement protein genes. J Gen Virol 78, 2077–2083.[Abstract]

Morozov, S. Y., Solovyev, A. G., Kalinina, N. O., Fedorkin, O. N., Samuilova, O. V., Schiemann, J. & Atabekov, J. G. (1999). Evidence for two nonoverlapping functional domains in the potato virus X 25K movement protein. Virology 260, 55–63.[CrossRef][Medline]

Niesbach-Klosgen, U., Guilley, H., Jonard, G. & Richards, K. (1990). Immunodetection in vivo of beet necrotic yellow vein virus-encoded protein. Virology 178, 52–61.[Medline]

Perbal, M. C., Thomas, C. L. & Maule, A. J. (1993). Cauliflower mosaic virus gene product (P1) forms tubular structures which extend from the infected protoplasts. Virology 195, 281–285.[CrossRef][Medline]

Rouleau, M., Smith, R. J., Bancroft, J. B. & Mackie, G. A. (1994). Purification, properties, and subcellular localization of foxtail mosaic potexvirus 26-kDa protein. Virology 204, 254–265.[CrossRef][Medline]

Sambrook, J., Fritsch, E. F. & Maniatis, T. A. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.

Sheen, J., Hwang, S., Niwa, Y., Kobayashi, H. & Galbraith, D. W. (1995). Green-fluorescent protein as a new vital marker in plant cells. Plant J 8, 777–784.[CrossRef][Medline]

Solovyev, A. G., Stroganova, T. A., Zamyatnin, A. A., Fedorkin, O. N., Schiemann, J. & Morozov, S. Y. (2000). Subcellular sorting of small membrane-associated triple gene block proteins: TGBp3-assisted targeting of TGBp2. Virology 269, 113–127.[CrossRef][Medline]

Storms, M. M. H., Kormelink, R., Peters, D., van Lent, J. W. M. & Goldbach, R. W. (1995). The nonstructural NSm protein of tomato spotted wilt virus induces tubular structures in plant and insect cells. Virology 214, 485–493.[CrossRef][Medline]

Tamai, A. & Meshi, T. (2001). Cell-to-cell movement of potato virus X: the role of p12 and p8 encoded by the second and third open reading frames of the triple gene block. Mol Plant Microbe Interact 14, 1158–1167.[Medline]

van Lent, J., Wellink, J. & Goldbach, R. W. (1990). Evidence for the involvement of the 58K and 48K proteins in the intercellular movement of cowpea mosaic virus. J Gen Virol 71, 219–223.

Verchot, J., Angell, S. M. & Baulcombe, D. C. (1998). In vivo translation of the triple gene block of potato virus X (PVX) requires two mRNAs. J Virol 72, 8316–8320.[Abstract/Free Full Text]

Voinnet, O., Lederer, C. & Baulcombe, D. C. (2000). A viral movement protein prevents spread of the gene silencing signal in Nicotiana benthamiana. Cell 103, 157–167.[Medline]

Wellink, J. & van Kammen, A. (1989). Cell-to-cell transport of cowpea mosaic virus requires both the 58K/48K proteins and the capsid protein. J Gen Virol 70, 2279–2286.

Wieczorek, A. & Sanfacon, H. (1993). Characterization and subcellular localization of tobacco ringspot nepovirus putative movement protein. Virology 194, 734–742.[CrossRef][Medline]

Wolf, S., Deom, C. M., Beachy, R. N. & Lucas, W. J. (1989). Movement protein of tobacco mosaic virus modifies plasmodesmatal size-exclusion limit. Science 246, 377–379.

Wung, C. H., Hsu, Y. H., Liou, D. Y., Hung, W. C., Lin, N. S. & Chang, B. Y. (1999). Identification of the RNA-binding sites of the triple gene block protein 1 of bamboo mosaic potexvirus. J Gen Virol 80, 1119–1126.[Abstract]

Yang, Y., Ding, B., Baulcombe, D. C. & Verchot, J. (2000). Cell-to-cell movement of the 25K protein of potato virus X is regulated by three other viral proteins. Mol Plant Microbe Interact 13, 599–605.[Medline]

Received 19 June 2003; accepted 2 October 2003.