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Short Communication |
Departamento de Biotecnología, ETSI Agrónomos, Universidad Politécnica de Madrid, 28040 Madrid, Spain
Correspondence
Fernando García-Arenal
fernando.garciaarenal{at}upm.es
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ABSTRACT |
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MAIN TEXT |
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). The 3a protein is the movement protein (MP) with RNA-binding and plasmodesmata-gating abilities, and is able to traffic itself and RNA through plasmodesmata (Vaquero et al., 1994; Ding et al., 1995; Li & Palukaitis, 1996). The role of the CP in cell-to-cell movement is unclear. RNA accumulation in protoplasts was diminished considerably for CP deletion mutants and it was proposed that the CP is necessary to protect the (+)-sense viral RNA from degradation in the host cytoplasm (Boccard & Baulcombe, 1993; Suzuki et al., 1991). It could be that CP protection of the viral RNA would be necessary for efficient cell-to-cell movement. Additionally, cell-to-cell movement requires specific compatibility of the MP and CP of different cucumoviruses (Salánki et al., 1997, 2004), suggesting that both proteins interact for this function. Both the MP and the CP are required also for long-distance movement through the phloem and the CP has host-specific determinants for access to the phloem (Palukaitis & García-Arenal, 2003).
Cucumber mosaic virus (CMV) and Tomato aspermy virus (TAV) are two cucumoviruses that differ in several properties. The three-dimensional structure of their particles has been resolved, showing high similarity (Smith et al., 2000; Lucas et al., 2002), which agrees with the ability for in vitro assembly of stable particles built of CMV and TAV CP subunits (Chen et al., 1995). Some important differences in the biology of CMV and TAV have been mapped to the CP by using reassortant and chimeric viruses (Palukaitis & García-Arenal, 2003). One such difference is the ability of CMV, but not of TAV, to infect cucumber plants. Both viruses replicate in cucumber protoplasts and move from cell to cell in the inoculated leaf, but only CMV infects cucumber systemically. The CP of CMV is required for access to the sieve elements in cucumber and can complement the systemic movement of TAV (Taliansky & García-Arenal, 1995; Thompson & García-Arenal, 1998).
Hybrid viruses exchanging CP regions between CMV and TAV were engineered with three aims: to explore the viability of hybrid CPs and their capacity to form stable particles, to explore the compatibility of the heterologous MP and CP for cell-to-cell movement and to identify determinants in the CP of CMV for systemic infection of cucumber plants. Seven CP chimeras were engineered within a full-length cDNA clone of 1-TAV RNA3 [clone p13 of Moreno et al. (1997)]. To minimize alteration of the CP structure, points for sequence exchange were in loops of the CP 
-barrel structure. Four restriction sites were chosen for sequence exchange, located at the indicated nucleotide positions (numbered as in GenBank accession no. AJ277269
[GenBank]
): XbaI (nt 1222, in the IR 4 nt upstream of the CP ORF), NheI, EagI (nt 1404 and 1679, in the CP ORF) and SphI [nt 1906, in the 3' untranslated region (UTR), 13 nt downstream of the CP ORF]. Six primers were designed to PCR-amplify specific regions of Fny-CMV RNA3: XD had an XbaI restriction site 5' to a sequence identical to nt 1257–1276, ND had an NheI site 5' to a sequence identical to nt 1440–1457, NR had an NheI site 5' to a sequence complementary to nt 1414–1439, ED had an EagI site 5' to a sequence identical to nt 1701–1721, ER had an EagI site 5' to a sequence complementary to nt 1683–1700 and SR had an SphI site 5' to a sequence complementary to nt 1902–1920 (position numbers as in GenBank accession no. D10538
[GenBank]
). By using combinations of these primers on a clone of Fny-CMV RNA3, fragments limited by nt 1257–1433 (fragment A), 1434–1699 (fragment B), 1700–1921 (fragment C), 1257–1699 (fragment AB), 1434–1921 (fragment BC) and 1257–1921 (fragment ABC) were amplified, cloned into pGEM-T (Promega), sequenced and subcloned into p13 previously digested with the corresponding restriction enzymes. Thus, the following seven chimeras were obtained: A, B, C, AB, AC, BC and ABC (Fig. 1). All hybrid RNA3 had the 5' and 3' UTRs, plus the 3a ORF and IR of 1-TAV, but chimeras C, AC, BC and ABC had the first 7 nt of the 3' UTR from CMV, resulting in the alteration of GACA to CGTG at positions 1887–1890 of 1-TAV RNA3. The chimeric nature of the clones was confirmed by partial nucleotide-sequence determination.
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were transcribed from cDNA clones (Rizzo & Palukaitis, 1990; Moreno et al., 1997) by using an AmpliCap kit (Epicentre), 5 µg of each transcript was used to transfect 5x105 protoplasts per treatment (Moreno et al., 1997) and nucleic acids were extracted 24 h post-inoculation. After 1 % agarose-gel electrophoresis, Northern blots were hybridized with a mixture of 32P-labelled RNA probes complementary to the 3' UTRs of CMV and TAV (positions 1920–2391 or 1933–2215 of the respective RNA3). All hybrid RNA3 were able to replicate in tobacco protoplasts with F1+F2 to similar levels (Fig. 2a). Hence, neither the hybrid nature of the CP ORF nor the four point mutations introduced in the 3' UTR of chimeras C, AC and ABC affected the capacity of the hybrids to replicate, compared with T3.
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) were not significant (P
0.10 in a 
2 test). New tobacco plants were inoculated with sap from transcript-infected plants and all plants became infected. Particles of F1F2F3 and F1F2T3 were purified efficiently by the methods described by Lot et al. (1972) for CMV and Habili & Francki (1974) for TAV, respectively. Particles of F1F2A and F1F2B could be purified by using the TAV procedure, whilst the CMV method gave very poor yields (not shown). Thus, the hybrid CPs A and B were able to assemble into stable particles. The chimeric nature of the viruses that infected tobacco plants was checked by nucleotide-sequence determination (not shown). Inoculation with the other chimeras did not result in symptom expression and virus accumulation was not detected in inoculated or in upper leaves (Table 1), showing that they were unable to infect tobacco leaves.
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), but were defective for systemic movement (Schmitz & Rao, 1998), which is thought to occur as particles (Blackman et al., 1998). Accordingly, hybrids A and B infected tobacco systemically and assembled into particles. We do not know the assembly ability of the other hybrid CPs, but none infected the inoculated leaves or became systemic, and it could be predicted that the CP of ABC, identical to that of Fny-CMV, should be competent for assembly. This suggests that, for our chimeras, there is no correlation between CP assembly capacity and infectivity. By using other CMV and TAV strains, Salánki et al. (1997) have shown that a chimera having the MP of TAV and the CP of CMV was not infectious with CMV RNAs 1 and 2. The non-infectivity of ABC agrees with these results. From analyses of hybrid MP and CP, Salánki et al. (2004) suggested that compatibility between the C-terminal 29 aa of the MP and the C-terminal two-thirds of the CP is required for cell-to-cell movement. The electrostatic potential of the CP would determine that interaction. Our results agree with the first hypothesis, as hybrid A, in which the C terminus of the MP and the C-terminal two-thirds of the CP are from TAV, was infectious. However, our results show a more complex scenario: the infectivity of hybrid B suggests that only the C-terminal one-third of the CP should be compatible with the C terminus of the MP, but in that case, hybrid AB also should be infectious. Thus, our results indicate an effect of aa 1–59 (fragment A) on aa 60–148 (fragment B), affecting CP structure and/or electrostatic potential and, thus, CP–MP compatibility.
A second set of seven chimeras was constructed in which the 3a ORF of 1-TAV was partly replaced by that of Fny-CMV. For convenience, the XcmI and BsaAI sites of p13 (positions 188 and 952, respectively) were used. Primers XcD, with an XcmI site and a sequence identical to nt 218–234, and BR, with a BsaAI site and a sequence complementary to nt 956–975, were used to PCR-amplify the fragment delimited by nt 218–975 of Fny-CMV RNA3, which was cloned in the XcmI and BsaAI sites of p13 and in the cDNA clones of chimeras A, B, C, AB, AC, BC and ABC, yielding chimeras T3* and A*, B*, C*, AB*, AC*, BC* and ABC* (Fig. 1). Only F1F2T3*, F1F2A* and F1F2B* infected tobacco plants, but infectivity was reduced significantly (P<0.003) compared with F1F2F3, F1F2A and F1F2B, and virus accumulation was limited to the inoculated leaves (Table 1). The chimeric nature of RNA3 of the viruses that infected tobacco plants was checked by nucleotide-sequence determination (not shown). The reduced infectivity of T3*, A* and B* compared with that of T3, A and B could be due to the hybrid MP, although similarity of the N-terminal 33 aa of Fny-CMV and 1-TAV MPs is high (91 % similarity, 73 % identity). Also, the chimeras of this second series have the TAV IR 5'-most 20 nt replaced by the CMV IR 5'-most 14 nt. Although no regulatory sequence has been described in this part of the IR (Palukaitis & García-Arenal, 2003), an effect of this substitution in RNA3 replication cannot be discarded. Interestingly, and at odds with Salánki et al. (2004), substituting the MP of TAV for that of CMV did not change the infectivity of the CP hybrids. In particular, hybrid A* was infectious, in contrast to the homologous R3SPT of Salánki et al. (2004), stressing the complexity of MP–CP functional interactions.
Northern blots of total RNA extracts from inoculated leaves suggested that accumulation of F1F2F3, F1F2T3, F1F2A or F1F2B was higher than that of F1F2T3*, F1F2A* and F1F2B* (Fig. 2b). Hence, maybe there is a relationship between rate of systemic movement and virus accumulation in inoculated leaves, with a threshold for the virus to become systemic. It could also be that the chimeric MP was non-functional for systemic movement, but competent for cell-to-cell movement, a possibility suggested by other MP mutants (Li et al., 2001).
The 15 chimeras above, plus F1F2F3 and F1F2T3, were also inoculated into cucumber cotyledons. None of the chimeras were detected in the cotyledons or leaves of inoculated plants. As expected, F1F2T3 was detected in the inoculated cotyledons, but not in upper leaves, and only F1F2F3 became systemic (not shown). Cucumber plants were also inoculated with F1F2A and F1F2B virions purified from tobacco plants, with the same results (not shown), indicating that failure to infect cucumbers was not due to low transcript infectivity. Hence, neither aa 1–50 nor 60–148 of CMV CP are sufficient for cucumber infection.
In summary, our results show that hybrid CPs exchanging the N-terminal and central one-thirds of CMV and TAV CP can assemble into stable particles, and extend previous results on the compatibility of MP–CP for cell-to-cell movement, showing a more complex picture than considered previously. MP–CP compatibility occurs with hybrids competent for particle assembly, suggesting that interaction of MP and virus particles may be required at some point in the virus life cycle. Our results also show that the compatibility of MP and CP conditions the viability of CP hybrids, limiting their use to map CP-associated differences between CMV and TAV.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 26 January 2006;
accepted 7 March 2006.
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