Laboratory of Plant Pathology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
Correspondence
Kazuyuki Mise
kmise{at}kais.kyoto-u.ac.jp
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ABSTRACT |
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INTRODUCTION |
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Bromoviruses are a group of icosahedral plant viruses, the genomes of which are divided into three positive-sense tripartite RNAs, designated RNA 1, RNA 2 and RNA 3 (Lane, 1981). RNAs 1 and 2 encode the 1a and 2a proteins, respectively, which are both required for genomic RNA replication in protoplasts (Kroner et al., 1989
, 1990
). RNA 3, which is not necessary for viral RNA replication, encodes the 3a protein required for virus cell-to-cell movement (Mise et al., 1993
; Schmitz & Rao, 1996
) and the coat protein (CP) translated from a subgenomic RNA designated RNA 4 (Sacher & Ahlquist, 1989
). Some interactions between bromovirus factors necessary for successful infection have been demonstrated. For instance, the 1a and 2a proteins of Brome mosaic virus (BMV) form a complex to achieve successful replication (Kao et al., 1992
; O'Reilly et al., 1997
). Moreover, BMV 1a protein must interact with the intercistronic region of BMV RNA 3 for efficient amplification of RNA 3 (French & Ahlquist, 1987
; Sullivan & Ahlquist, 1999
). Recent studies have identified sequences in the CP and viral RNAs that are required for bromovirus encapsidation (Choi et al., 2002
; Damayanti et al., 2002
). However, the interactions between bromovirus components required for movement have not been examined closely.
Reassortment is a powerful strategy with which to study the compatibility of interactions among viral components in some viruses with divided genomes, such as bromoviruses. For example, reassortants in which only RNAs 1 or 2 are exchanged between BMV and Cowpea chlorotic mottle virus (CCMV) do not allow virus genomes to accumulate to detectable levels in barley protoplasts, whereas reassortants in which RNA 3 is exchanged allow this accumulation (Allison et al., 1988). This observation reflects the importance of compatible combinations of RNAs 1 and 2, and therefore of the 1a and 2a proteins, for bromovirus replication.
Spring beauty latent virus (SBLV) is a member of the genus Bromovirus, together with BMV and CCMV (Valverde, 1985). SBLV is closely related to BMV and CCMV, and biologically active SBLV cDNA clones have been constructed (Fujisaki et al., 2003
). To further analyse the interactions between bromovirus components required for systemic infection, we created reassortants of SBLV, BMV and CCMV and examined their infectivity in Nicotiana benthamiana, which is a common systemic host of the three bromoviruses. In this paper, we show that SBLV RNA 2 in combination with heterologous bromovirus RNA 1 directs systemic infection when inoculated together with bromovirus RNA 3, but that SBLV RNA 1 in combination with heterologous bromovirus RNA 2 does not. This is the first report to demonstrate that SBLV RNA 2 directs successful virus infection in combination with RNA 1 of other virus species within the family Bromoviridae. We also show that the infectivity of these reassortants in N. benthamiana plants is critically determined by the level of virus accumulation in single cells, and discuss the interactions between bromovirus components required for virus infection.
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METHODS |
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Preparation of N. benthamiana plants and protoplasts, and virus inoculation.
N. benthamiana plants were grown as described previously (Fujisaki et al., 2003). Plants were mechanically inoculated with transcripts (0·3 µg µl-1) from cDNA clones of BMV, CCMV or SBLV, as described previously (Fujisaki et al., 2003
). Isolation of N. benthamiana protoplasts and inoculation with in vitro transcripts were performed essentially as described previously (Okuno & Furusawa, 1978
; Kroner & Ahlquist, 1992
). N. benthamiana protoplasts were prepared from plants at the eight-leaf stage or older. Protoplasts were prepared by incubation for 3·5 h at 25 °C in an isolation solution [0·6 M mannitol, 10 mM CaCl2, 1 % cellulase (Onozuka R-10; Yakult, Tokyo, Japan) and 0·05 % macerozyme (R-10; Yakult), pH 5·5]. Typically, 4·0x105 protoplasts were inoculated with 3·0 µg transcripts (RNAs 1+2+3 in total) or a DNA inoculum consisting of 8·0 µg 1a and 2a expression plasmids, 2·0 µg RNA 3 transcripts and 20 µg salmon sperm DNA (Clontech) as carrier.
RNA analysis.
A tissue printing assay of whole plants was performed as described previously (Mise et al., 1993). In protoplast experiments, total RNA was extracted as described (Kroner & Ahlquist, 1992
) from infected protoplasts at 24 h post-inoculation (p.i.). Northern blot analysis of total RNA was performed as described previously (Damayanti et al., 1999
). Positive-strand RNAs of BMV and CCMV were detected using digoxigenin (DIG)-labelled SP6 transcripts from HindIII-linearized pBSPL10 (Kaido et al., 1995
) and T3 transcripts from EcoRI-linearized pCC3RA518 (Allison et al., 1990
), respectively. To construct pSB1MC501, pSB2MC502 and pSB3MS503 for the detection of SBLV RNA 1, RNA 2 and RNA 3, respectively, the 0·7 kb ClaI/PstI fragments of pSB1TP6 and pSB2TP7, and the 0·7 kb SalI/PstI fragment of pSB3TP9, respectively, were cloned into pBluescript II KS(-) (Stratagene). These cDNA fragments correspond to the 3'-terminal regions of the genomic RNAs. Positive-strand SBLV RNAs 1, 2 and 3 were detected using DIG-labelled T7 transcripts from ClaI-linearized pSB1MC501 and pSB2MC502 and SalI-linearized pSB3MS503, respectively. Viral RNAs were detected with anti-DIG AP Fab fragment (Roche Molecular Biochemicals) and CDP-star substrate (New England Biolabs) as described (Sasaki et al., 2001). Membranes obtained were exposed to X-ray films (Fuji Photo Film) and the image of viral RNA accumulation was densitometrically analysed with the NIH Image program version 1.61 (National Institutes of Health, USA). In each quantification analysis, samples containing known amounts of transcripts from the three bromoviruses (BMV RNA 3, CCMV RNA 3 and SBLV RNAs 1, 2 and 3) were included, and the signal intensities of the samples were measured to compare the specific activity of each probe. Minor data corrections were made as appropriate, to allow proper comparisons of the various reassortant signals.
Protein analysis.
Press-blot analysis of the distribution of bromovirus CPs in N. benthamiana was performed as described previously (Takahashi et al., 2001). Briefly, inoculated leaves were hammered between two pieces of 3MM paper. Residual green colour was removed by rinsing in 2 % Triton X-100 prior to blocking with skim milk and immunodetection. Infected protoplasts were disrupted in Laemmli sample buffer (Laemmli, 1970
) and subjected to SDS-PAGE. Immunoblot analysis was carried out as described previously (Damayanti et al., 1999
), using an Immobilon-P transfer membrane (Millipore). A mouse anti-CCMV 3a protein monoclonal antibody (Sasaki et al., 2003
) was used to detect CCMV 3a protein. Moreover, because anti-BMV antiserum cross-reacts with CCMV and SBLV CPs (Fujisaki et al., 2003
), CCMV and SBLV CPs as well as BMV CP were detected using a rabbit anti-BMV antiserum (ATCC PVAS-178). The accumulation of CCMV 3a protein and CP was densitometrically quantified using the NIH Image program.
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RESULTS |
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Replication assay of reassortants in N. benthamiana protoplasts
To elucidate the mechanism determining the infectivity of the reassortants, protoplasts of N. benthamiana were inoculated with the reassortants, and the accumulation of viral RNAs was examined by Northern blot analysis. To estimate viral RNA accumulation, probes each recognizing the conserved 3'-terminal sequences of BMV RNAs (Kaido et al., 1995) or those of CCMV RNAs (Allison et al., 1990
) were used. Moreover, three individual probes recognizing the 3'-terminal sequences of SBLV RNAs 1, 2 and 3 were prepared to detect SBLV RNAs because the similarity of the 3'-terminal sequences of SBLV RNAs 1, 2 and 3 was too low (Fujisaki et al., 2003
) to cross-hybridize with a single probe in a preliminary experiment (data not shown). Therefore, a mixture of four or five probes was used to estimate viral RNA accumulation in reassortants constructed from SBLV and other bromoviruses, and equimolar amounts of B3, C3, S1, S2 and S3 were used as controls for the specific activities of these five probes. Consistent results were obtained from at least five independent experiments and representative data are shown in Fig. 2
.
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Although the S2 component directed viral RNA accumulation when inoculated with B1 or C1, the levels of total viral RNA accumulated in plants inoculated with B1S2B3 or B1S2S3, both of which inefficiently infected N. benthamiana plants (Fig. 1), were about 10-fold lower than levels detected during wild-type (wt) SBLV infection (Fig. 2a
). Because equimolar amounts of transcripts loaded as controls showed similar signal intensities (Fig. 2
), the specific activity of each probe seemed to be comparable. Therefore the band intensities of viral RNAs after infection with each reassortant directly reflected the level of viral RNA accumulation. Thus, these data indicate that the B1S2 combination functioned in viral RNA accumulation, but was inefficient compared with wild-type viruses. This suggests that the low infectivity of the reassortants containing the B1S2 combination (Fig. 1
) was caused by the low-level accumulation of viral RNAs in single cells. Total viral RNA accumulation after inoculation with C1S2C3 or C1S2S3 was around half to two-thirds the level seen during wt CCMV and SBLV infections (Fig. 2b
), which far exceeds the accumulation observed with B1S2B3 and B1S2S3. This indicates that the C1S2 combination was more compatible than the B1S2 combination, and facilitated more efficient virus multiplication in N. benthamiana protoplasts.
In this assay, all reassortants with homologous combination of RNAs 1 and 2 (B1B2S3, S1S2B3, C1C2S3 and S1S2C3) accumulated to a detectable level. However, the accumulation in protoplasts of B1B2S3 and C1C2S3, which showed poor infectivity in N. benthamiana plants, was significantly lower (Fig. 2a, b), suggesting that the low infectivity of these two reassortants was due to their inability to multiply efficiently in single cells. It is noteworthy that, during C1C2S3 infection, S3 and its subgenomic RNA 4 (S4) accumulated to quite low levels, although S3 accumulated to some degree during B1B2S3 infection. Because the combination of RNAs 1 and 2 was identical to that in wt CCMV, the low-level accumulation of viral RNAs in C1C2S3 infection may have been due to the incompatibility of C1C2 and S3.
Assay of transiently expressed 1a and 2a proteins to facilitate viral RNA accumulation in protoplasts
Bromovirus RNAs 1 and 2 encode the 1a and 2a proteins, respectively (designated B1a and B2a in BMV; C1a and C2a in CCMV; S1a and S2a in SBLV). B1a and B2a are components of viral replicase and function by interacting with each other (Kao et al., 1992). In this study, the B1S2 and C1S2 combinations directed viral RNA accumulation to a detectable level, suggesting that S2a protein interacts compatibly with B1a and C1a. In contrast, S1 failed to produce detectable accumulation of viral RNA when combined with heterologous B2 or C2. Its failure in S1B2 and S1C2 may reflect that S1a cannot form a functional replicase in combination with B2a or C2a. Alternatively, the replicase containing S1aB2a or S1aC2a may be functional but specifically unable to interact with cis-acting elements of RNA 1 and/or RNA 2 with sufficient compatibility, as assumed by Dinant et al. (1993)
in BMVCCMV reassortments. This may cause a reduction in the accumulation of RNA 1 and/or RNA 2 or in the expression of 1a and/or 2a proteins, which are indispensable for viral RNA replication. Consequently, the accumulation of total viral RNAs may be undetectable. To examine these possibilities, the 1a and 2a proteins were transiently expressed under the CaMV 35S promoter from expression plasmids (designated pB1, pB2, pC1, pC2, pS1 and pS2). The expression system using these plasmids is independent of any amplification of RNAs 1 and 2, because mRNAs transcribed from these plasmids lack both the 5' and 3' non-coding cis-acting sequences (Fig. 3
a).
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Effects of virus movement on infectivity of reassortants
A replication assay of reassortants indicated that the infectivity of the reassortants in N. benthamiana plants is critically determined by their ability to multiply in single cells. However, the possibility remains that the ability of reassortants to move locally also affects their ability to accumulate in inoculated and uninoculated leaves. For example, the systemic infectivity of C1S2S3 was much lower than that of B1S2C3, although C1S2S3 accumulated more in protoplasts than did B1S2C3 (Figs 1 and 2).
We examined the efficiency of virus movement in infections with C1S2C3, S1S2C3 or wt CCMV, which all carry the CCMV 3a MP gene and cause the accumulation of high levels of viral RNA in protoplasts (Fig. 2b). Although all three combinations efficiently infected N. benthamiana plants at 14 days p.i. (Fig. 1
), the systemic infectivity of C1S2C3 and S1S2C3 at 7 days p.i. was lower than that of wt CCMV (data not shown). Moreover, press-blot analysis demonstrated that the local spread of C1S2C3 and S1S2C3 in N. benthamiana was significantly delayed compared with that of wt CCMV and SBLV at 2 days p.i. (Fig. 4
). CCMV and SBLV had spread throughout the entire inoculated leaves at 4 days p.i., whereas the reassortants had spread only locally even at 8 days p.i. (data not shown). On the other hand, Western blot analysis demonstrated that the accumulation of movement-associated proteins, 3a protein and CP, in N. benthamiana protoplasts infected with C1S2C3 and S1S2C3 was similar to that during infection with wt CCMV (Fig. 5
a, b). These data suggest that C1S2C3 and S1S2C3 infect N. benthamiana plants inefficiently because their cell-to-cell movement is slower, rather than because the viruses multiply to lower levels in single cells, when compared with CCMV and SBLV infections.
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DISCUSSION |
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SBLV RNA 2 functions with heterologous bromovirus RNA 1 in virus multiplication in single cells
Replication assays of the reassortants demonstrated that all reassortants containing S2 components replicated and accumulated to a detectable level in N. benthamiana protoplasts, even when the combination of RNAs 1 and 2 was heterologous (Fig. 2). To our knowledge, there has been no report of reassortants with heterologous combinations of RNAs 1 and 2 from different virus species that can direct tripartite viral genome accumulation to detectable levels, except those produced between strains of a certain virus species (e.g. in bromoviruses; Shang & Bujarski, 1993
; De Jong & Ahlquist, 1995
) or between progeny derivatives containing mutations that allow them to interact compatibly (Masuta et al., 1998
). Therefore, SBLV is the first virus in the family Bromoviridae with an RNA 2 known to function in combination with RNA 1 of other virus species.
BMV 1a and 2a proteins, which are encoded by RNAs 1 and 2, respectively, must interact with each other for successful replication (Kao et al., 1992; O'Reilly et al., 1997
). This interaction has also been reported for Cucumber mosaic virus (Kim et al., 2002
) and Alfalfa mosaic virus (Maurice et al., 2001
), the genomes of which consist of three divided RNAs corresponding to RNAs 1, 2 and 3 of the bromoviruses, indicating that the 1a2a interaction is a common characteristic in the family Bromoviridae. Detectable accumulation of viral RNA was directed by B1S2 and C1S2 combinations. Moreover, S2a protein expressed from a DNA plasmid supported detectable accumulation of bromovirus RNA 3s in combination with heterologous B1a or C1a protein. These results indicate that the SBLV 2a protein interacts compatibly with heterologous 1a proteins. Immune coprecipitation assays using mutant polypeptides made in an in vitro translation system demonstrated that the N-terminal segment of BMV 2a protein interacts directly with BMV 1a protein (Kao & Ahlquist, 1992
). Similar experiments may help identify interacting domains between S2a and B1a or C1a. In contrast, no reassortants with heterologous combinations of RNAs 1 and 2 containing S1 (S1B2 and S1C2) accumulated to detectable levels. Transiently expressed SBLV 1a protein in combination with BMV or CCMV 2a protein did not support detectable accumulation of viral RNAs (Fig. 3
), suggesting that SBLV 1a protein cannot form a functional replicase with either of the heterologous 2a proteins.
Compatible combinations of RNAs 1 and 2 support efficient amplification of SBLV RNA 3
In contrast to the heterologous combinations of RNAs 1 and 2, all homologous combinations of RNAs 1 and 2 directed the detectable accumulation of viral RNAs in protoplasts, consistent with previous results (Allison et al., 1988). However, two reassortants with homologous RNAs 1 and 2, B1B2S3 and C1C2S3, showed low-level accumulations of total viral RNAs (Fig. 2a, b
). In particular, inoculation with C1C2S3 produced quite low levels of S3 and S4 accumulation. The low-level accumulation of total viral RNA during C1C2S3 infection may be caused by inefficient encapsidation with heterologous SBLV CP. However, this is unlikely to explain the S3-specific reduction in accumulation. Alternatively, the replicase formed from C1a plus C2a might not interact compatibly with cis-elements in SBLV RNA 3. On the other hand, the C1S2S3 combination accumulated to high levels (Fig. 2b
), indicating that, in addition to the C1aS2a interaction, the replicaseS3 interaction was also compatible. Taken together, these data suggest that S2a may function more efficiently than C2a in the amplification of S3.
Compatible combinations of viral genomic RNAs required for efficient virus movement
All reassortants non-infectious to N. benthamiana plants failed to accumulate to detectable levels in single cells, and most of the reassortants showing low infectivity accumulated to lower levels than wild-types. These results suggest that infectivity of reassortants is primarily determined by their ability to multiply at the single-cell level. Furthermore, significant delay in local spread of C1S2C3 and S1S2C3 when compared with the spread of wt CCMV suggests that virus cell-to-cell movement is also a crucial step determining infectivity of reassortants, because viral RNAs of C1S2C3, S1S2C3 and wt CCMV accumulated to high levels in protoplasts (Fig. 2). C1S2C3, S1S2C3 and wt CCMV have different combinations of RNAs1 and 2 but all contain the C3 component and therefore express CCMV MP and CP, and the accumulation levels of these proteins were similar in each inoculum (Fig. 5
). SBLV RNAs 1 and 2 should be functional because wt SBLV spread in inoculated leaves of N. benthamiana as efficiently as wt CCMV (Fig. 4
). Therefore, slower spread of C1S2C3 and S1S2C3 may have been caused by poor compatibility of interactions between the viral components rather than the difference in functions of an individual component. Together, these data suggest that a compatible combination with RNAs 1 and/or 2 may be required for C3 to function in virus cell-to-cell movement. Supporting this suggestion, viral genes for replication, such as the bromovirus 1a and 2a genes, function not only in viral RNA replication but also in virus movement (Traynor et al., 1991
; Gal-On et al., 1994
; Hirashima & Watanabe, 2001
). Because the local spread of C1S2C3 was slower than that of wt CCMV, an RNA 2-mediated interaction at least may be necessary.
So far, most reassortment tests have been performed to map virus functions. Therefore, reassortants were created between virus species or virus strains with distinct features (Allison et al., 1988; Shang & Bujarski, 1993
; Takahashi et al., 1994
; De Jong & Ahlquist, 1995
). However, the reassortants in this study were created from different virus species with similar infectivity in a common host plant to elucidate the compatibility of interactions between viral factors. We confirmed that compatibility is required for viral RNA replication, as reported previously (1a2a interaction), and demonstrated that S2 can function in replication heterologously with either B1 or C1. This characteristic of RNA2 made it possible for us to discover an RNA2-mediated interaction necessary for the step after viral RNA replication. Analysis of the RNA2-mediated interaction required for bromovirus movement is in progress.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 30 November 2002;
accepted 10 February 2003.
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