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Plant |
Instituto de Biología Molecular y Celular de Plantas, Universidad Politecnica de Valencia-CSIC, Avenida de los Naranjos s/n, 46022 Valencia, Spain1
Institute of Molecular Plant Sciences, Gorlaeus Laboratories, Leiden University, PO Box 9502, 2300 RA Leiden, The Netherlands2
Author for correspondence: John F. Bol. Fax +31 71 527 4469. e-mail J.BOL{at}chem.LeidenUniv.nl
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; Jaspars, 1999 ). Although the overall sequence similarity between AMV and ilarviruses is relatively low, the 3' termini of the viral RNAs contain similar stem–loop structures flanked by AUGC sequences that represent specific binding sites for homologous as well as heterologous CPs of AMV and ilarviruses (Zuidema & Jaspars, 1985 ; Houser-Scott et al., 1994 ; Reusken & Bol, 1996 ; Reusken et al., 1994 ). CPs of AMV and ilarviruses are interchangeable in initiation of infection by these viruses (van Vloten-Doting, 1975 ; Gonsalves & Garnsey, 1975 ; Gonsalves & Fulton, 1977 ). In addition to its role in the initiation of infection and encapsidation of viral RNA, AMV CP is required for plus-strand RNA accumulation and cell-to-cell movement of the virus (van der Vossen et al., 1994 ; de Graaff et al., 1995 ; Neeleman & Bol, 1999 ; Tenllado & Bol, 2000 ).
AMV and PNRSV are phylogenetically closely related (Sánchez-Navarro & Pallás, 1997 ). Previously, we have replaced the MP and CP genes in AMV RNA 3 by the corresponding PNRSV genes and studied the replication of the chimeric RNAs in transgenic P12 tobacco plants and protoplasts (Sánchez-Navarro et al., 1997 ). P12 plants express the AMV P1 and P2 proteins and can be infected with AMV RNA 3 without a requirement for CP in the inoculum (Taschner et al., 1991 ). PNRSV CP could substitute for all functions of AMV CP in the replication cycle, and PNRSV MP and CP mediated a reduced level of cell-to-cell transport of chimeric RNAs in plants, although tobacco is a non-permissive host for PNRSV (Sánchez-Navarro et al., 1997 ). In the present study, we analysed the cis-acting sequences in PNRSV RNA 3 that are recognized by the AMV RNA-dependent RNA polymerase (RdRp) by exchanging the 5'-untranslated regions (UTRs) and 3'-UTRs of RNA 3 of the two viruses. The 5'-UTRs of AMV and PNRSV RNA 3 are 345 and 176 nucleotides (nt) long, respectively, and do not show significant sequence identity. The 3'-UTRs of AMV and PNRSV RNA 3 are 183 and 171 nt long, respectively, and show an overall similarity of 42%. The chimeric RNAs, shown in Fig. 1(A), were used as templates in in vitro polymerase assays with purified AMV RdRp, and the replication of the chimeras was analysed in P12 protoplasts and plants.
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). Plasmid pUC3m1 contains a cDNA clone of PNRSV RNA 3 (NcM1 isolate; Aparicio et al., 1999 ). To engineer plasmid pUC3m1 (Fig. 1A
), total RNAs extracted from PNRSV-infected cucumber plants were subjected to reverse transcription and subsequent PCR amplification (RT–PCR) using an antisense primer complementary to the terminal 18 nt of viral RNA 3 plus an extra PstI site (Sánchez-Navarro & Pallás, 1997 ). PCR was carried out with the antisense primer described above and a sense primer containing a HindIII site, the T7 RNA polymerase sequence promoter and the first 18 nt of PNRSV RNA 3 (PV96 isolate). Construct pAMV-5P was made by amplification of the PNRSV 5'-UTR sequence from plasmid pUC3m1 using specific primers flanked with PvuII and NcoI sites. The resulting fragment with the T7 promoter and the PNRSV 5'-UTR was then inserted in plasmid pAL3NcoP3 previously digested with PvuII and NcoI. To engineer construct pPNRSV-5A, a PNRSV cDNA 3 fragment lacking the 5'-UTR region was amplified from plasmid pUC3m1 using appropriate primers bordered with NcoI and PstI sites. The PCR product was digested with the corresponding restriction enzymes and introduced in the pAL3NcoP3 construct. Construct pPNRSV-3A was created by amplification of the AMV 3'-UTR from pAL3NcoP3 using specific primers flanked with XbaI and PstI sites. The fragment was introduced in the pUC3m1 construct using an XbaI site after the CP stop-codon and the PstI site located at the end of the cDNA clone. In the same way, construct pAMV-3P was engineered by amplification of the PNRSV 3'-UTR and introduction of the PCR fragment in plasmid pAL3NcoP3 using KpnI and PstI sites that flank the AMV 3'-UTR. Plasmids containing wt AMV cDNA 3, PNRSV cDNA 3 and the chimeric AMV–PNRSV constructs were linearized with PstI and transcribed with T7 RNA polymerase as described previously (van der Kuyl et al., 1991 ). Inoculation of plants and protoplasts with the transcripts was done as described by Neeleman & Bol (1999) .
Fig. 1(B) shows the autoradiogram of an agarose gel run with 32P-labelled minus-strand RNA 3 products that were synthesized by the purified AMV RdRp (de Graaff et al., 1995 ) in an in vitro polymerase assay when the chimeric RNAs were used as templates. The template activity of PNRSV RNA 3 (Fig. 1B, lane 7) was 18% of the activity of AMV RNA 3 (Fig. 1B, lane 1). In AMV RNA 3, the promoter for in vitro minus-strand RNA synthesis is located in the 3'-terminal 166 nt (van Rossum et al., 1997 ). The two chimeras with the AMV 3'-UTR showed a 100% level of template activity (Fig. 1B, lanes 5 and 6) whereas the chimeras with the PNRSV 3'-UTR, i.e. AMV-3P and PNRSV-5A, showed a template activity of 16 and 35%, respectively (Fig. 1B, lanes 3 and 4). When the 3'-UTR sequences were used as templates instead of full-length RNA 3, template activity of the PNRSV 3'-UTR (Fig. 1C, lane 2) was 20% of that of AMV (Fig. 1C, lane 1). AMV RNA 3 ends with the sequence AUGC whereas PNRSV RNA 3 ends with AAGC. When this AAGC sequence was changed to AUGC, the template activity of the 3'-UTR of PNRSV (Fig. 1C, lane 3) increased to 83% of that of AMV.
Fig. 2 shows the accumulation of viral RNAs in P12 protoplasts inoculated with the chimeric RNAs. For the detection of plus-strand RNAs, the Northern blots were hybridized to DIG-labelled riboprobes (Pallás et al., 1999 ) complementary to the 3'-UTR of AMV (Fig. 2A) or PNRSV (Fig. 2B). Panels (A) and (B) of Fig. 2 were developed for the same time to permit a comparison of the signals. The 42% sequence similarity between the two 3'-UTRs was too low to permit cross-hybridization of the probes under the conditions used. Minus-strand RNAs in the protoplasts were detected by using a mixture of plus-strand probes corresponding to the MP genes of AMV and PNRSV (Fig. 2C). The accumulation of AMV RNA 3 is shown as a control in Fig. 2(A), lane 1. (The minor band at the top of the gel may represent an aggregate or incompletely melted double-stranded RNA.) Only chimeras AMV-3P and PNRSV-5A (Fig. 2B, lanes 3 and 4) induced accumulation of plus-strand RNAs at levels similar to the control. The relatively low amounts of plus-strand RNA detectable in protoplasts inoculated with PNRSV-3A (Fig. 2A, lane 5), AMV-5P (Fig. 2A, lane 6) and PNRSV RNA 3 (Fig. 2B, lane 7) probably represent fragments of inoculum RNAs as these RNAs are shorter than full-length wild-type or chimeric RNA 3. The signals observed in lanes 1, 3 and 4 of Fig. 2(A, B) may have to be corrected for similar background levels. Fig. 2(C) shows that plus-strand AMV RNA 3 (2142 nt), PNRSV RNA 3 (1951 nt) and chimeric inoculum RNAs are all transcribed into complementary minus-strand RNAs by the transgenic AMV RdRp. Particularly, the results with chimera AMV-5P demonstrate that minus-strand RNA 3 synthesis (Fig. 2C, lane 6) is independent of de novo plus-strand RNA 3 synthesis (Fig. 2A, lane 6). Previously, we have shown that minus-strand RNA 3 transcribed in P12 protoplasts from inoculum RNA 3 is sufficient to direct wild-type levels of asymmetric plus-strand RNA 3 synthesis (Neeleman & Bol, 1999 ). Apparently, the AMV RdRp recognized the 3'-UTR of PNRSV RNA 3 both in vitro (Fig. 1B, C) and in vivo (Fig. 2C). However, only chimeras that contained the 5'-UTR of AMV RNA 3 (AMP-3P, PNRSV-5A) were able to direct de novo plus-strand RNA synthesis in vivo (Fig. 2B). This indicates that the AMV RdRp does not recognize promoter sequences for plus-strand RNA synthesis in the 5'-UTR of PNRSV RNA 3.
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shows the accumulation of viral RNAs in P12 plants. Seven days after inoculation of plants with the chimeric RNAs, RNA was extracted from inoculated leaves and analysed by Northern blot hybridization using a mixed probe, detecting both AMV and PNRSV sequences. Subliminal infections confined to single cells are not detectable in this assay (van der Vossen et al., 1994 ). In addition to the control with AMV RNA 3 (Fig. 3, lane 1), only the chimera consisting of PNRSV RNA 3 with the 5'-UTR replaced by the 5'-UTR of AMV was able to accumulate in plants (Fig. 3, lane 4). This chimera (PNRSV-5A) encodes the MP and CP of PNRSV, which were previously shown to mediate cell-to-cell transport in tobacco (Sánchez-Navarro et al., 1997 ). It remains to be determined why these two proteins do not permit cell-to-cell movement of PNRSV in tobacco. One possibility is that PNRSV triggers a host response that prevents the virus from infecting tobacco. Surprisingly, the chimera consisting of AMV RNA 3 with the 3'-UTR replaced by the 3'-UTR of PNRSV (pAMV-3P) accumulated in protoplasts but not in plants (Fig. 3, lane 3). Previously, we showed that various AMV–PNRSV chimeras were encapsidated by CPs of either virus but these chimeras all contained the 3'-UTR of AMV (Sánchez-Navarro et al., 1997 ). We have not yet analysed the possibility that a defect in encapsidation of pAMV-3P affects cell-to-cell movement. However, AMV CP mutants have been described that do not form stable virions but do move from cell to cell (Tenllado & Bol, 2000 ; J. A. Sánchez-Navarro & J. F. Bol, unpublished). Alternatively, an interaction of the AMV CP with the 3'-UTR of PNRSV could be incompatible with a putative role in cell-to-cell transport or the chimera could activate a host response that blocks movement.
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). Rather, our data support the notion that between virus species from one genus or species from different genera of one family of plant viruses, minus-strand promoter sequences are more conserved than plus-strand promoters. In the genus Tobravirus the RdRp encoded by RNA 1 of Tobacco rattle virus (TRV) replicates RNA 2 of Pea early browning virus only when a 5' non-coding sequence of this RNA is replaced by the corresponding non-coding sequence of TRV RNA 2 (Mueller et al., 1997 ). In the family Bromoviridae, replacement of the 3'-UTR of RNA 3 of Brome mosaic virus (BMV) by the corresponding 3'-UTR of Cucumber mosaic virus (CMV) did not affect replication of the chimeric RNA by the BMV RdRp (Rao & Grantham, 1994 ). The 3'-UTR of CMV is also recognized by the RdRp of Tomato aspermy virus (Teycheney et al., 2000 ). In the family Potyviridae, the 3'-UTR of Lettuce mosaic virus is recognized by the RdRp of several potyviruses (Teycheney et al., 2000 ). Cis-acting elements in the 5'-UTR of AMV RNA 3 have been partially characterized (van der Vossen & Bol, 1996 ). A comparison of the 5'-UTRs of AMV and PNRSV may provide further insight in cis-acting elements that are essential for plus-strand promoter activity. |
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References |
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Bol, J. F. (1999). Alfalfa mosaic virus and ilarviruses: involvement of coat protein in multiple steps of the replication cycle. Journal of General Virology 80, 1089-1102.[Medline]
de Graaff, M., Man in’t Veld, M. R. & Jaspars, E. M. (1995). In vitro evidence that the coat protein of alfalfa mosaic virus plays a direct role in the regulation of plus and minus RNA synthesis: implications for the life-cycle of alfalfa mosaic virus. Virology 208, 583-589.[Medline]
Gonsalves, D. & Fulton, R. W. (1977). Activation of prunus necrotic ringspot virus and rose mosaic virus by RNA 4 components of some ilarviruses. Virology 81, 398-407.[Medline]
Gonsalves, D. & Garnsey, S. M. (1975). Infectivity of heterologous RNA–protein mixtures from alfalfa mosaic, citrus leaf rugose, citrus variegation and tobacco streak viruses. Virology 67, 319-326.[Medline]
Houser-Scott, F., Baer, M. L., Liem, F. K., Cai, J. M. & Gerke, L. (1994). Nucleotide sequence and structural determinants of specific binding of coat protein or coat protein peptides to the 3'-untranslated region of alfalfa mosaic virus RNA 4. Journal of Virology 68, 2194-2205.
Jaspars, E. M. J. (1999). Genome activation in alfamo- and ilarviruses. Archives of Virology 144, 843-863.[Medline]
Mueller, A.-M., Mooney, A. L. & MacFarlane, S. A. (1997). Replication of in vitro tobravirus recombinants shows that the specificity of template recognition is determined by 5' non-coding but not by 3' non-coding sequences. Journal of General Virology 78, 2085-2088.[Abstract]
Neeleman, L. & Bol, J. F. (1999). Cis-acting functions of alfalfa mosaic virus proteins involved in replication and encapsidation of viral RNA. Virology 254, 324-333.[Medline]
Pallás, V., Sánchez-Navarro, J. A. & Diez, J. (1999). In vitro evidence for RNA binding properties of the coat protein of Prunus necrotic ringspot ilarvirus and their comparison to related and unrelated viruses. Archives of Virology 144, 797-803.[Medline]
Rao, A. L. N. & Grantham, G. L. (1994). Amplification in vivo of brome mosaic virus RNAs bearing 3' noncoding region from cucumber mosaic virus. Virology 204, 478-481.[Medline]
Reusken, C. B. E. M. & Bol, J. F. (1996). Structural elements of the 3' terminal coat protein binding site in alfalfa mosaic virus RNAs. Nucleic Acids Research 24, 2660-2665.
Reusken, C. B. E. M., Neeleman, L. & Bol, J. F. (1994). The 3' untranslated region of alfalfa mosaic virus RNA 3 contains at least two independent binding sites for viral coat protein. Nucleic Acids Research 22, 1346-1353.
Sánchez-Navarro, J. A. & Pallás, V. (1997). Evolutionary relationships in the ilarviruses: nucleotide sequence of Prunus necrotic ringspot RNA 3. Archives of Virology 142, 749-763.[Medline]
Sánchez-Navarro, J. A., Reusken, C. B. E. M., Bol, J. F. & Pallás, V. (1997). Replication of alfalfa mosaic virus RNA 3 with movement and coat protein genes replaced by corresponding genes of Prunus necrotic ringspot ilarvirus. Journal of General Virology 78, 3171-3176.[Abstract]
Taschner, P. E. M., van der Kuyl, A. C., Neeleman, L. & Bol, J. F. (1991). Replication of an incomplete alfalfa mosaic virus genome in plants transformed with viral replicase genes. Virology 181, 445-450.[Medline]
Tenllado, F. & Bol, J. F. (2000). Genetic dissection of the multiple functions of alfalfa mosaic virus coat protein in viral RNA replication, encapsidation, and movement. Virology 268, 29-40.[Medline]
Teycheney, P. Y., Aaziz, R., Dinant, S., Salánki, K., Tourneur, C., Balázs, E., Jacquemond, M. & Tepfer, M. (2000). Synthesis of (-)-strand RNA from the 3' untranslated region of plant viral genomes expressed in transgenic plants upon infection with related viruses. Journal of General Virology 81, 1121-1126.
van der Kuyl, A. C., Neeleman, L. & Bol, J. F. (1991). Role of alfalfa mosaic virus coat protein in regulation of the balance between viral plus and minus strand RNA synthesis. Virology 185, 496-499.[Medline]
van der Vossen, E. A. G. & Bol, J. F. (1996). Analysis of cis-acting elements in the 5' leader sequence of alfalfa mosaic virus RNA 3. Virology 220, 539-543.[Medline]
van der Vossen, E. A. G., Neeleman, L. & Bol, J. F. (1993). Role of the 5' leader sequence of alfalfa mosaic virus RNA 3 in replication and translation of the viral RNA. Nucleic Acids Research 6, 1361-1367.
van der Vossen, E. A. G., Neeleman, L. & Bol, J. F. (1994). Early and late functions of alfalfa mosaic virus coat protein can be mutated separately. Virology 202, 891-903.[Medline]
van Rossum, C. M. A., Neeleman, L. & Bol, J. F. (1997). Comparison of the role of 5' terminal sequences of alfalfa mosaic virus RNAs 1, 2 and 3 in viral RNA replication. Virology 235, 333-341.[Medline]
van Vloten-Doting, L. (1975). Coat protein is required for infectivity of tobacco streak virus: biological equivalence of the coat proteins of tobacco streak and alfalfa mosaic viruses. Virology 65, 215-225.[Medline]
Zuidema, D. & Jaspars, E. M. J. (1985). Specificity of RNA and coat protein interaction in alfalfa mosaic virus and related viruses. Virology 140, 342-350.
Received 10 November 2000;
accepted 3 January 2001.
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