United States Department of Agriculture - Agricultural Research Service and Department of Plant Pathology, University of Nebraska, 344 Keim Hall, Lincoln, NE 68583, USA
Correspondence
Roy French
rfrench{at}unlnotes.unl.edu
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Compared with other potyviral gene products the function of P3 is less well understood. The P3 cistron encodes a protein that is excised from the polyprotein by proteinase activities of HC-Pro at the amino-proximal end (Oh & Carrington, 1989; Carrington & Herndon, 1992
) and NIa at the carboxy-proximal end (Carrington & Dougherty, 1987
; Dougherty et al., 1989
). Insertional mutagenesis of the Tobacco vein mottling virus (TVMV) P3 cistron abolished infectivity and detectable replication in protoplasts (Klein et al., 1994
). Immunogold-labelled antibodies raised against the TVMV P3 protein localized to the cylindrical inclusions (CIs) of infected cells, suggesting that the P3 protein interacts with the potyviral CI protein during early stages of inclusion formation (Rodríguez-Cerezo et al., 1993
). In vitro binding assays (Merits et al., 1999
) determined that the Potato virus A (PVA) P3 protein interacted with viral-encoded proteins of the putative replication complex (CI, NIa-VPg, NIa-Pro and NIb), although in yeast two-hybrid assays the same researchers reported that PVA P3 protein interacted only with NIb (Merits et al., 1999
). Similar in vitro and in vivo interaction assays revealed that P3 of Wheat streak mosaic virus (WSMV) is capable of binding to itself, P1, HC-Pro and CI (Choi et al., 2000a
).
The potyviral P3 cistron also affects pathogenesis. Symptom severity phenotype of Plum pox virus (PPV) on Nicotiana clevelandii mapped to the P3-6K1 region but was independent of virus accumulation (Sáenz et al., 2000). The ability of Pea seed-borne mosaic virus (PSbMV) to infect a resistant pea line was dependent upon the P3-6K1 region being derived from a strain of PSbMV able to overcome the resistance (Johansen et al., 2001
). Similar experimental strategies have been used to map determinants of Turnip mosaic virus (TuMV) symptom severity and avirulence on Brassica napus lines (Jenner et al., 2002
, 2003
; Suehiro et al., 2004
) and the ability to infect radish (Suehiro et al., 2004
) to the P3 cistron. Collectively, these studies identify the P3 cistron as a key component of hostviral interactions specifying both pathogenicity and virulence.
WSMV is the type species of the genus Tritimovirus within the family Potyviridae, with a genome organization typical of a monopartite potyvirus (Stenger et al., 1998). Five strains of WSMV have been completely sequenced (Stenger et al., 1998
; Choi et al., 2001
; Rabenstein et al., 2002
). Sequence comparisons among divergent WSMV strains revealed a substitutional cold-spot within the 3'-proximal half of the P3 cistron (Choi et al., 2001
) that was atypically devoid of synonymous substitutions compared with the rest of the WSMV genome. A similar analysis (Choi et al., 2001
) revealed that five PVA strains (Kekarainen et al., 1999
) also retained high intraspecies sequence conservation within the 3'-proximal half of the P3 cistron. Because the 3'-proximal half of the P3 cistron displayed a high degree of conservation within but not between species, we hypothesized that this region of the genome may contain a species-specific RNA element that is retained among divergent species of the family. In this report, we describe mutational analyses of the WSMV-Sidney-81 genome, in which multiple synonymous substitution mutations were introduced into the putative P3 RNA element. Alterations of viral phenotype associated with synonymous substitutions in the P3 cistron were observed, providing evidence for the existence of an internal RNA element in the WSMV genome, and suggests that other potyviruses retaining high intraspecies sequence conservation in this region also may bear an RNA element with similar function.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
The SphI fragment of each mutagenized plasmid was completely sequenced to verify each mutant construct. Sequencing of mutagenized subclones revealed that two mutagenesis products contained additional unintended mutations. One plasmid (pP3M1) bore the same synonymous mutations as pP3M17 and one additional non-synonymous substitution, resulting in the alteration of an aspartic acid codon to an alanine codon (Fig. 1b). A second plasmid (pP3M3) bore the same synonymous mutations as pP3M322 but also incurred two separate single base deletions such that a frame-shift was introduced into the polyprotein open reading frame (ORF) (Fig. 1b
). The SphI fragment of each mutagenized subclone was used to replace the wild-type SphI fragment of pACYC-WSMV to produce pWSMV-P3M11, pWSMV-P3M17, pWSMV-P3M29, pWSMV-P3M322, pWSMV-P3M4, pWSMV-P3M1, pWSMV-P3M3 and pWSMV-CIM1. The SphI fragment of mutagenized subclones was also used to replace the wild-type SphI fragment of pWSMV-GUS-S1RN, to place select mutations in a WSMV genome capable of expressing GUS. The resulting plasmids were designated pWSMV-G1-P3M17, pWSMV-G1-P3M29, pWSMV-G1-P3M322, pWSMV-G1-P3M4 and pWSMV-G1-P3M3.
To assess the effect of single synonymous substitutions in the P3 cistron, the four mutations present in pP3M11 (Fig. 1) were generated individually by site-directed mutagenesis of the SphI subclone bearing WSMV nt 23734289, verified by nucleotide sequencing, and used to replace the wild-type SphI fragment of pWSMV-GUS-S1RN. The resulting plasmids were designated pWSMV-G125 (A2755 to T), pWSMV-G217 (A2761 to G), pWSMV-G313 (C2770 to T) and pWSMV-G41 (A2779 to G).
Coupled in vitro transcription/translation and immunoprecipitation.
In vitro transcription/translation products were synthesized from full-length WSMV cDNA sequences using the TNT SP6/wheat germ extract system (Promega) as per the manufacturer's recommendations, except that 6 µg plasmid template per 50 µl reaction mixture was used. A plasmid bearing the luciferase gene was used as a positive control for coupled in vitro transcription/translation reactions. In vitro translation products (45 µl) and 3 µl of diluted antiserum raised against the WSMV CI protein (Langenberg, 1993) or a synthetic peptide (LDTMASGAMKDYKIG) corresponding to the carboxy terminus of WSMV HC-Pro were added to 300 µl IP buffer (10 % glycerol, 50 mM HEPES-KOH, 100 mM potassium glutamate, 0·5 mM DTT, 6 mM magnesium acetate, 1 mM EGTA, 0·1 % NP40 and 0·5 mg ml1 BSA, pH 7·3) (Marcus et al., 1994
), and incubated (4 °C, 6 h) with gentle shaking. Protein ASepharose beads (5 mg) were added to 40 µl IP buffer; 40 µl of this slurry was added to the mixture of translation products and antiserum. Following incubation (4 °C for 4 h), the beads were washed five times with 400 µl IP buffer, resuspended in 20 µl gel loading buffer (50 mM Tris/HCl, 100 mM DTT, 2 % SDS, 0·1 % bromophenol blue, 10 % glycerol, pH 6·8), and boiled for 3 min. Boiled protein samples were analysed on 12 % SDS-PAGE followed by autoradiography.
Infectivity assays.
In vitro SP6 transcripts were generated from NotI linearized plasmid DNA templates (12·5 µg) as described previously (Choi et al., 1999) and mechanically inoculated onto leaves of 812-day-old wheat seedlings (cultivars Centurk or Arapahoe). Total RNA samples were extracted from non-inoculated, upper leaves of the primary shoot 21 days post-inoculation (p.i.). Systemic infection status of each plant was evaluated by RT-PCR using the primer CI-3 (5'-GTGGATCCTACTGGTATGACACACATGGG-3') for RT and the primers B-P1C4 (5'-GTCGGGATCCTGCGGATGATGCACTCCAAGGG-3') and P1-9-3 (5'-TTCCGAATTCCCTAGTGCTTGCAAGAATGC-3') for PCR. The resulting RT-PCR product corresponded to WSMV-Sidney-81 nt 8211461 and was detected by ethidium bromide staining after electrophoresis in 1 % agarose. Infectivity assays of WSMV transcripts bearing the GUS reporter gene were performed histochemically as described below.
Protoplast preparation and transfection.
Protoplasts were prepared from barley leaves as described by Loesch-Freis & Hall (1980). Leaves from 810-day-old barley (cv. Larker) seedlings grown in a growth chamber (16 h photoperiod, 23 °C) were sliced into
1 mm strips. Protoplasts were isolated from leaf strips by digestion (3 h, 30 °C) in enzyme mixture [1020 mg Cellulysin cellulase (Calbiochem) ml1, 1 mg Macerozyme R-10 (Yakult Pharmaceutical) ml1, 1 mg BSA ml1, 10 % mannitol, pH 5·7]. Protoplasts were resuspended at
5·5x105 ml1 in electroporation buffer (10 % mannitol, 70 mM KCl, 5 mM MES, pH 5·7). In vitro transcription products (20 µg) were mixed with 400 µl resuspended protoplasts (
2·2x105 protoplasts) just prior to electroporation (200 V, 960 µF) in a 0·2 cm electrode cuvette using the Gene Pulser apparatus (Bio-Rad). Electroporated protoplasts were washed with 10 % mannitol and incubated (36 h, 25 °C, 1000 lx continuous illumination) in 1 ml protoplast medium (Aoki & Takebe, 1969
). Following incubation, protoplasts were stored frozen at 70 °C until processed for GUS activity using the fluorometric assay described below.
GUS assays.
Histochemical detection of GUS expression was performed on small leaf pieces (11·5 cm length) of inoculated leaves 3 or 5 days p.i. (Choi et al., 2000b). Leaf pieces were fixed for 1 h by vacuum infiltration in 0·7 % formaldehyde. Fixed leaf pieces were rinsed five times (15 min each) in distilled water, then incubated (37 °C up to 12 h) in 50 mM phosphate buffer (pH 7·0) containing 2 mM 5-bromo-4-chloro-3-indoyl
-D-glucuronide (X-gluc). Following incubation with X-gluc substrate, leaf pieces were clarified first in 70 % ethanol and then in 5 % sodium hypochlorite.
Fluorometric GUS assays were performed based on the methods of Jefferson et al. (1987). Protoplasts were thawed and pelleted by centrifugation (50 g, 2 min). Pelleted protoplasts were resuspended with 500 µl protein extraction buffer (50 mM Na2HPO4, 0·1 % Triton X-100, 0·1 % Sarcosyl, 1 mM EDTA, 5mM DTT, pH 7·0). Glass beads (0·2 g, 450650 µm diameter) were added to resuspended protoplasts and the mixture was vortexed for 1 min, followed by centrifugation (10 000 g, 10 min). Aliquots of the supernatant containing 2050 µg total soluble protein were removed from each sample and adjusted to a volume of 300 µl with protein extraction buffer. Soluble protein samples were mixed with an equal volume (300 µl) of GUS assay buffer (2 mM 4-methylumbelliferyl
-D-glucuronide in protein extraction buffer) and incubated at 37 °C. Aliquots (100 µl) were removed at several time points between 0 and 120 min, measured for fluorescence (excitation at 365 nm, emission at 460 nm) using a TD-700 fluorometer (Turner Designs). Fluorescence values were converted to pmoles of methylumbelliferone (MU) min1 mg1 of soluble protein, utilizing known concentrations of MU as standards and a Bradford protein quantification (Bradford, 1976
) assay kit (Bio-Rad). To account for variation between different protoplast preparations, each GUS activity measured was converted to a percentage value of that obtained with wild-type WSMV bearing GUS (pWSMV-GUS-S1RN) 36 h after transfection of protoplasts from the same preparation. Mean values of percentage GUS activity relative to wild-type were based on 46 replicates from independent protoplast preparations.
RNA secondary structure.
Predictions of RNA secondary structure were generated using the program Alifold (Hofacker et al., 2002), using an alignment of the five completely sequenced WSMV isolates. Folding free energies of predicted stemloop structures were calculated using the methodology of Turner et al. (1988)
.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Systemic infectivity of mutants in wheat
Wild-type WSMV transcripts derived from pACYC-WSMV were infectious in wheat (Table 1) and produced characteristic systemic mosaic symptoms. Transcripts derived from pWSMV-CIM1 bearing 15 synonymous nucleotide substitutions in the CI cistron (Fig. 1
) were also infectious and induced systemic symptoms (Table 1
). In contrast, plants inoculated with transcripts of all P3 multiple synonymous substitution mutants (Fig. 1
) produced no symptoms during 30 days p.i. (Table 1
). To detect potential asymptomatic systemic infection, all inoculated plants were evaluated for the presence of WSMV-specific sequences in non-inoculated leaves by RT-PCR at 21 days p.i. All asymptomatic plants tested negative for WSMV infection, whereas all symptomatic plants tested positive for WSMV infection (data not shown). To ensure that lack of systemic infectivity was due to the introduced P3 mutations, the wild-type SphI fragment (originally subcloned for use as a mutagenesis template) was used to replace the mutant SphI fragment of pWSMV-P3M17 (Fig. 1
). This replacement restored infectivity and systemic symptom production, demonstrating that no unintended debilitating mutations had occurred in WSMV sequences outside of the SphI fragment.
|
|
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Information encoded by viral RNA genomes is not limited to codons specifying the order of amino acids incorporated into viral proteins. RNA elements direct a variety of essential functions critical to the viral life cycle and, although most act in cis, several viral RNA elements function in trans (Sit et al., 1998; Shen & Miller, 2004
). Although cis-acting RNA elements (CREs) involved in viral RNA polymerase template recognition and regulation of replication are typically associated with the non-coding regions (NTR) situated at the ends of viral RNA genomes, these elements may extend into adjacent coding regions. Other CREs may be found entirely (or nearly so) within coding regions. Species-specific CREs located in coding regions have been identified in several viruses of the Picornaviridae (Mcknight & Lemon, 1996
; Lobert et al., 1999
; Goodfellow et al., 2000
; Gerber et al., 2001
). It was shown that these CREs function as template for uridylylation of the genome-linked protein (VPg) (Rieder et al., 2000
; Yang et al., 2002
), and that the product of uridylylation, VPgpUpU, serves as a primer for the synthesis of positive-strand RNA (Goodfellow et al., 2003
; Morasco et al., 2003
; Murray & Barton, 2003
). The current model for picornavirus replication suggests that replication is coordinated by interactions among multiple cis-elements in 5'- and 3'-NTRs and coding regions (Barton et al., 2001
; Lyons et al., 2001
; Goodfellow et al., 2003
; Morasco et al., 2003
; Murray & Barton, 2003
). Based on the similarity in genome structures, WSMV and viruses of the Potyviridae belong to the picornavirus superfamily. CREs necessary for efficient amplification of the genome have been identified in the capsid protein (CP) coding region and 3'-NTR of Tobacco etch virus (TEV) (Mahajan et al., 1996
; Haldeman-Cahill et al., 1998
). Collectively, our results indicate that another CRE resides in the P3 cistron.
Systemic movement of plant viruses occurs by a two-step process: cell-to-cell movement between adjacent cells through plasmodesmata followed by long-distance movement via phloem. Mutational analyses of potyvirus genomes and ultrastructural studies of infected tissues indicated that several viral-encoded proteins are involved in movement (Dolja et al., 1994, 1995
; Klein et al., 1994
; Kasschau et al., 1997
; Rojas et al., 1997
; Carrington et al., 1998
; Lopez-Moya & Pirone, 1998
). In the case of WSMV, the P3 synonymous substitution mutants were able to initially spread from cell-to-cell. However, after several days p.i. cell-to-cell movement of the P3 mutants appeared to cease with infection foci limited to small clusters of cells. Thus, long-distance movement may be precluded indirectly by limitation of cell-to-cell movement such that infection foci fail to reach phloem-associated parenchyma. This phenotype is distinct from subliminal infections limited to single cells that result from a point mutation in the TEV CP (Dolja et al., 1995
) or from truncation of the WSMV CP (unpublished data). In many respects, the debilitated movement phenotype of WSMV P3 synonymous substitution mutants resembles that of long-distance movement deficient TEV HC-Pro mutants (Kasschau et al., 1997
) or a TEV CP mutant lacking 17 aa of the carboxy terminus (Dolja et al., 1995
).
Interestingly, the A2761 to G substitution of mutant G217 altered the predicted secondary structure (Fig. 6c), while the single substitutions in the infectious mutants G125, G313 and G41 did not (Fig. 6b, df
). At present we do not have informative data with respect to how perturbation of P3 RNA secondary structure affects WSMV replication and movement. We attempted to complement a movement-deficient P3 mutant bearing GUS by co-inoculation with a wild-type WSMV genome. No GUS activity was observed in systemically infected tissues (data not shown), suggesting that the element does not act in trans. We further examined whether the position of the P3 RNA element was important. In this experiment, we placed the wild-type P3 cistron between the NIb and CP cistrons in a genome also bearing multiple synonymous mutations in the resident P3 cistron. Infectivity was not restored (data not shown), indicating that the position of the RNA element was critical. This suggests a fundamental difference between the P3 element and poliovirus CRE, because the latter retains function independent of position (Goodfellow et al., 2000
). Because there is limited experimental evidence that encapsidation of potyviral genomes may initiate in the 5'-proximal half of genome (Wu & Shaw, 1998
), we speculated that the P3 RNA element may be required for virion assembly. To address this issue, we examined extracts from transfected protoplasts for virions by electron microscopy (data not shown). Unfortunately, the protoplast system employed here was suboptimal, as we were unable to observe virions in extracts derived from protoplasts transfected with any WSMV construct (including wild-type), precluding meaningful analysis. Clearly, additional experimentation is necessary to define the mechanism(s) by which the WSMV P3 RNA element affects replication and movement.
Although the biochemical function of P3 protein is still unclear, mutant analysis (Klein et al., 1994) and interactions between P3 and other proteins encoded by potyvirus genomes (Choi et al., 2000a
; Merits et al., 1999
; Rodríguez-Cerezo et al., 1993
) suggest an involvement of P3 in the replication process. Previous studies elegantly demonstrated that the potyvirus P3 cistron is an important determinant of pathogenicity and virulence (Sáenz et al., 2000
; Johansen et al., 2001
; Hjulsager et al., 2002
; Jenner et al., 2002
, 2003
; Suehiro et al., 2004
). In each case, the differential phenotype mapped to the P3 region was presumed to be due to differences in the P3 protein based on comparative sequence analysis or site-directed mutagenesis, in which non-synonymous mutations altered phenotype. However, based on our data, it is apparent that the primary RNA sequence of the WSMV P3 cistron plays an essential role in the viral life cycle and that it is necessary to distinguish among functions of the P3 coding region governed by RNA elements from those of the encoded protein. The secondary structure model for the region presented in Fig. 6
can be tested by additional physical and mutagenesis studies. At this time it is unknown if the genomes of divergent species of the family Potyviridae also harbour an essential CRE within the P3 coding sequence. Similar mutagenesis studies with infectious potyvirus clones would reveal whether the P3 CRE is retained among monopartite members of the family or is peculiar to the tritimovirus WSMV.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Barton, D. J., O'Donnell, B. J. & Flanegan, J. B. (2001). 5' cloverleaf in poliovirus RNA is a cis-acting replication element required for negative-strand synthesis. EMBO J 20, 14391448.
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248254.[CrossRef][Medline]
Carrington, J. C. & Dougherty, W. G. (1987). Small nuclear inclusion protein encoded by a plant potyvirus genome is a protease. J Virol 61, 25402548.
Carrington, J. C. & Herndon, K. L. (1992). Characterization of the potyviral HC-Pro autoproteolytic cleavage site. Virology 187, 308315.[CrossRef][Medline]
Carrington, J. C., Jensen, P. E. & Schaad, M. C. (1998). Genetic evidence for an essential role for potyvirus CI protein in cell-to-cell movement. Plant J 14, 393400.[CrossRef][Medline]
Choi, I.-R., French, R., Hein, G. L. & Stenger, D. C. (1999). Fully biologically active in vitro transcripts of the eriophyid mite-transmitted wheat streak mosaic tritimovirus. Phytopathology 89, 11821185.
Choi, I.-R., Stenger, D. C. & French, R. (2000a). Multiple interactions among proteins encoded by the mite-transmitted wheat streak mosaic tritimovirus. Virology 267, 185198.[CrossRef][Medline]
Choi, I.-R., Stenger, D. C., Morris, T. J. & French, R. (2000b). A plant virus vector for systemic expression of foreign genes in cereals. Plant J 23, 547555.[CrossRef][Medline]
Choi, I.-R., Hall, J. S., Henry, M., Zhang, L., Hein, G. L. & Stenger, D. C. (2001). Contributions of genetic drift and negative selection on the evolution of three strains of wheat streak mosaic virus. Arch Virol 146, 619628.[CrossRef][Medline]
Choi, I.-R., Horken, K. M., Stenger, D. C. & French, R. (2002). Mapping of the P1 proteinase cleavage site in the polyprotein of Wheat streak mosaic virus (genus Tritimovirus). J Gen Virol 83, 443450.
Dolja, V. V., Haldeman, R., Robertson, N. L., Dougherty, W. G. & Carrington, J. C. (1994). Distinct functions of capsid protein in assembly and movement of tobacco etch potyvirus in plants. EMBO J 13, 14821491.[Abstract]
Dolja, V. V., Haldeman-Cahill, R., Montgomery, A. E., Vandenbosch, K. A. & Carrington, J. C. (1995). Capsid protein determinants involved in cell-to-cell and long distance movement of tobacco etch virus. Virology 206, 10071016.[CrossRef][Medline]
Dougherty, W. G., Cary, S. M. & Parks, T. D. (1989). Molecular genetic analysis of a plant virus polyprotein cleavage site: a model. Virology 171, 356364.[CrossRef][Medline]
Gerber, K., Wimmer, E. & Paul, A. V. (2001). Biochemical and genetic studies of the initiation of human rhinovirus 2 RNA replication: identification of a cis-replicating element in the coding sequence of 2Apro. J Virol 75, 1097910990.
Goodfellow, I., Chaudhry, Y., Richardson, A., Meredith, J., Almond, J. W., Barclay, W. & Evans, D. J. (2000). Identification of a cis-acting replication element within the poliovirus coding region. J Virol 74, 45904600.
Goodfellow, I. G., Polacek, C., Andino, R. & Evans, D. J. (2003). The poliovirus 2C cis-acting replication element-mediated uridylylation of VPg is not required for synthesis of negative-sense genomes. J Gen Virol 84, 23592363.
Haldeman-Cahill, R., Daros, J.-A. & Carrington, J. C. (1998). Secondary structures in the capsid protein coding sequence and 3' nontranslated region involved in amplification of the tobacco etch virus genome. J Virol 72, 40724079.
Hjulsager, C. K., Lund, O. S. & Johansen, I. E. (2002). A new pathotype of Pea seedborne mosaic virus explained by properties of the P3-6k1- and viral genome-linked protein (VPg)-coding regions. Mol Plant Microbe Interact 15, 169171.[Medline]
Hofacker, I. L., Fekete, M. & Stadler, P. F. (2002). Secondary structure prediction for aligned RNA sequences. J Mol Biol 319, 10591066.[CrossRef][Medline]
Jefferson, R. A., Kavanagh, T. A. & Bevan, M. W. (1987). GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6, 39013907.[Abstract]
Jenner, C. E., Tomimura, K., Ohsima, K., Hughes, S. L. & Walsh, J. A. (2002). Mutations in Turnip mosaic virus P3 and cylindrical inclusion proteins are separately required to overcome two Brassica napus resistance genes. Virology 300, 5059.[CrossRef][Medline]
Jenner, C. E., Wang, X., Tomimura, K., Ohshima, K., Ponz, F. & Walsh, J. A. (2003). The dual role of the potyvirus P3 protein of Turnip mosaic virus as a symptom and avirulence determinant in brassicas. Mol Plant Microbe Interact 16, 777784.[Medline]
Johansen, E. I., Lund, O. S., Hjulsager, C. K. & Laursen, J. (2001). Recessive resistance in Pisum sativum and potyvirus pathotype resolved in a gene-for-cistron correspondence between host and virus. J Virol 75, 66096614.
Kasschau, K. D., Cronin, S. & Carrington, J. C. (1997). Genome amplification and long-distance movement functions associated with the central domain of tobacco etch potyvirus helper component-protease. Virology 228, 251262.[CrossRef][Medline]
Kekarainen, T., Mertis, A., Oruetxebarria, I., Rajamaki, M.-L. & Valkonen, J. P. T. (1999). Comparison of the complete sequences of five different isolates of Potato virus A (PVA), genus Potyvirus. Arch Virol 144, 23552366.[CrossRef][Medline]
Klein, P. G., Klein, R. R., Rodríguez-Cerezo, E., Hunt, A. G. & Shaw, J. G. (1994). Mutational analysis of the tobacco vein mottling virus genome. Virology 204, 759769.[CrossRef][Medline]
Langenberg, W. G. (1993). Structural proteins of three viruses in the Potyviridae adhere only to their homologous cylindrical inclusions in mixed infections. J Struct Biol 110, 188195.[CrossRef][Medline]
Lobert, P.-E., Escriou, N., Ruelle, J. & Michiels, T. (1999). A coding RNA sequence acts as a replication signal in cardioviruses. Proc Natl Acad Sci U S A 96, 1156011565.
Loesch-Freis, L. E. & Hall, T. C. (1980). Synthesis, accumulation and encapsidation of individual brome mosaic-virus RNA components in barley protoplasts. J Gen Virol 47, 323332.
Lopez-Moya, J. J. & Pirone, T. P. (1998). Charge changes near the N terminus of the coat protein of two potyviruses affect virus movement. J Gen Virol 79, 161165.[Abstract]
Lyons, T., Murray, K. E., Roberts, A. W. & Barton, D. J. (2001). Poliovirus 5'-terminal cloverleaf RNA is required in cis for VPg uridylylation and the initiation of negative-strand RNA synthesis. J Virol 75, 1069610708.
Mahajan, S., Dolja, V. V. & Carrington, J. C. (1996). Roles of the sequence encoding tobacco etch virus capsid protein in genome amplification: requirements for the translation process and a cis-active elements. J Virol 70, 43704379.[Abstract]
Marcus, G. A., Silverman, N., Berger, S. L., Horiuchi, J. & Guarente, J. (1994). Functional similarity and physical association between GCN5 and ADA2: putative transcriptional adaptors. EMBO J 13, 48074815.[Abstract]
McKnight, K. L. & Lemon, S. M. (1996). Capsid coding sequence is required for efficient replication of human rhinovirus 14 RNA. J Virol 70, 19411952.[Abstract]
Merits, A., Guo, D., Jarvekulg, L. & Saarma, M. (1999). Biochemical and genetic evidence for interactions between potato A potyvirus-encoded proteins P1 and P3 and proteins of the putative replication complex. Virology 263, 1522.[CrossRef][Medline]
Morasco, B. J., Sharma, N., Parilla, J. & Flanegan, J. B. (2003). Poliovirus cre(2C)-dependent synthesis of VPgpUpU is required for positive- but not negative-strand RNA synthesis. J Virol 77, 51365144.
Murray, K. E. & Barton, D. J. (2003). Poliovirus CRE-dependent VPg uridylylation is required for positive-strand RNA synthesis but not for negative-strand RNA synthesis. J Virol 77, 47394750.
Oh, C.-S. & Carrington, J. C. (1989). Identification of essential residues in potyvirus proteinase HC-Pro by site-directed mutagenesis. Virology 173, 692699.[CrossRef][Medline]
Rabenstein, F., Seifers, D. L., Schubert, J., French, R. & Stenger, D. C. (2002). Phylogenetic relationships, strain diversity, and biogeography of tritimoviruses. J Gen Virol 83, 895906.
Rieder, E., Paul, A. V., Kim, D. W., van Boom, J. H. & Wimmer, E. (2000). Genetic and biochemical studies of poliovirus cis-acting replication element cre in relation to VPg uridylylation. J Virol 74, 1037110380.
Rodríguez-Cerezo, E., Ammar, E. D., Pirone, T. P. & Shaw, J. G. (1993). Association of the non-structural P3 viral protein with cylindrical inclusions in potyvirus-infected cells. J Gen Virol 74, 19451949.[Abstract]
Rojas, M. R., Zerbini, F. M., Allison, R. F., Gilbertson, R. L. & Lucas, W. J. (1997). Capsid protein and helper component proteinase function as potyvirus cell-to-cell movement proteins. Virology 237, 283295.[CrossRef][Medline]
Sáenz, P., Cervera, M. T., Dallot, S., Quilot, L., Quilot, J.-B., Riechman, J. L. & García, J. A. (2000). Identification of a pathogenicity determinant of Plum pox virus in the sequence encoding the C-terminal region of protein P3+6K1. J Gen Virol 81, 557566.
Sharp, P. M., Tuohy, T. M. F. & Mosurski, K. R. (1986). Codon usage in yeast: cluster analysis clearly differentiates highly and lowly expressed genes. Nucleic Acids Res 14, 51255143.[Abstract]
Shen, R. Z. & Miller, W. A. (2004). Subgenomic RNA as a riboregulator: negative regulation of RNA replication by barley yellow dwarf virus subgenomic RNA 2. Virology 327, 196205.[CrossRef][Medline]
Shukla, D. D., Ward, C. W., Brunt, A. A. & Berger, P. H. (1998). Potyviridae family. AAB/CMI Descriptions of Plant Viruses, no. 366.
Sit, T. L., Vaewhongs, A. A. & Lommel, S. A. (1998). RNA-mediated transactivation of transcription from a viral RNA. Science 281, 829832.
Stenger, D. C., Hall, J. S., Choi, I.-R. & French, R. (1998). Phylogenetic relationships within the family Potyviridae: wheat streak mosaic virus and brome streak mosaic virus are not members of the genus Rymovirus. Phytopathology 88, 782787.
Suehiro, N., Natsuaki, T., Watanabe, T. & Okuda, S. (2004). An important determinant of the ability of turnip mosaic virus to infect Brassica spp. and/or Raphauns sativus is in its P3 protein. J Gen Virol 85, 20872098.
Turner, D. H., Sugimoto, N. & Freier, S. M. (1988). RNA structure prediction. Annu Rev Biophys Biophys Chem 17, 167192.[CrossRef][Medline]
Urcuqui-Inchima, S., Haenni, A.-L. & Bernardi, F. (2001). Potyvirus proteins: a wealth of functions. Virus Res 74, 157175.[CrossRef][Medline]
Wu, X. & Shaw, J. G. (1998). Evidence that assembly of a potyvirus begins near the 5' terminus of the viral RNA. J Gen Virol 79, 15251529.[Abstract]
Yang, Y., Rijnbrand, R., McKnight, K. L., Wimmer, E., Paul, A., Martin, A. & Lemon, S. M. (2002). Sequence requirement for viral RNA replication and VPg uridylylation directed by the internal cis-acting replication element (cre) of human rhinovirus type 14. J Virol 76, 74857494.
Received 4 April 2005;
accepted 4 June 2005.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |