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The Genome-linked Protein and 5' End RNA Sequence of Plum Pox Potyvirus


Abstract and Figures

The infectivity of plum pox potyvirus (PPV) RNA was decreased by treatment with proteases. Ribonuclease digestion of iodinated PPV RNA yielded material which had an electrophoretic mobility corresponding to Mr 22,000. This protein presumably corresponds to the protease-sensitive structure needed for infectivity. A protein-linked RNase T1-resistant oligonucleotide, 38 nucleotides long, was sequenced and shown to correspond to the 5' terminus of the RNA by sequence comparison to the RNAs of two other potyviruses, tobacco etch virus and tobacco vein mottling virus. A 12 nucleotide block was found to be completely conserved in the RNAs of the three viruses.
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J. gen. Virol.
(1989), 70, 2785-2789.
Printed in Great Britain
Key words:
PPV/genome-linked protein/nucleotide sequence
The Genome-linked Protein and 5' End RNA Sequence of
Plum Pox Potyvirus
Centro de Biologia Molecular (CSIC-UAM), Universidad Aut6noma de Madrid, Canto Blanco,
28049 Madrid, Spain
(Accepted I June 1989)
The infectivity of plum pox potyvirus (PPV) RNA was decreased by treatment with
proteases. Ribonuclease digestion of iodinated PPV RNA yielded material which had
an electrophoretic mobility corresponding to Mr 22000. This protein presumably
corresponds to the protease-sensitive structure needed for infectivity. A protein-linked
RNase Tl-resistant oligonucleotide, 38 nucleotides long, was sequenced and shown to
correspond to the 5' terminus of the RNA by sequence comparison to the RNAs of two
other potyviruses, tobacco etch virus and tobacco vein mottling virus. A 12 nucleotide
block was found to be completely conserved in the RNAs of the three viruses.
The RNA genomes of several animal and plant viruses have a protein covalently linked to
their 5' termini (Daubert & Bruening, 1984) and genome-linked viral proteins (VPgs) have been
found attached to the RNAs of tobacco etch potyvirus (TEV) (Hari, 1981) and tobacco vein
mottling potyvirus (TVMV) (Siaw et al., 1985). In this paper we show that plum pox potyvirus
(PPV) also has a VPg and show that proteolytic enzyme treatment decreases the infectivity of
the viral RNA. Also, the existence of a terminal protein in the PPV genome has allowed us to
sequence the 5' end of the RNA directly.
PPV, Rankovic strain, was propagated in Nicotiana clevelandii and purified as described by
Lain et al. (1988). RNA was extracted from purified virions using SDS and phenol (Zimmern,
1975) and was recovered from the aqueous phase by ethanol precipitation. In the t25I-RNA
labelling experiments the RNA was not precipitated; instead, to remove the contaminating
capsid protein molecules, it was subjected to SDS-sucrose gradient centrifugation (Hellmann et
al., 1980) in a Beckman SW40 rotor for 9 h at 20 °C at 16000 r.p.m. The RNA band was then
collected and ethanol-precipitated.
Results presented in Table 1 show that digestion with proteolytic enzymes always diminished
the infectivity of PPV RNA, although some variability was observed between different
experiments. This suggested the presence of a protein structure, presumably a VPg, associated
with the genomic RNA. This result is in contrast with that obtained in similar experiments with
TEV (Hari, 1981), in which the infectivity of TEV RNA was not decreased by treatment with
proteinase K but, for undetermined reasons, considerably increased. When the influence of
proteolytic treatments on the infectivity of the RNAs of several nepoviruses was studied (Mayo
et al., 1982), the effect observed was different and characteristic for each of them. Treatment
with different proteases can cause different extents of decrease in the infectivity of the same
RNA (Mayo et al., 1982), which suggests that the peptides that remain attached to the RNA
after the proteolytic treatments may be different, and thus may contribute differently to an early
step of the viral life cycle. In the case of PPV both pronase and proteinase K digestions
decreased the infectivity of the RNA although, as in the case of raspberry ringspot virus (Mayo
et al., 1982), pronase seemed to be more efficient. No data are available on the effect of pronase
digestion on the infectivity of TEV RNA. In any case, these results might show not that intact
0000-8967 © 1989 SGM
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Table 1.
Effect of proteolytic treatments on the infectivity of PPV RNA*
Infectivity after treatment with
RNA concentration A
Expt. ~g/ml) Buffer only Pronase
1 3.5 (20)
4/7 0/7
2 45 (60) 32/4 0/4
3 70 (79) 76/10 3/10
4 80 (50) 192/4 77/4
5 100 (40) 276/6 2/6
6 80 (59) 192/4
Proteinase I~
74/4 (a)
37/4 (b)
* PPV RNA, at the concentration indicated in parentheses, was incubated for 4 h at 37 °C in the absence or
presence of pronase (Calbiochem) (0.8 mg/ml, or 0.4 mg/ml in expt. 3) or proteinase K (Merck) (0.2 mg/ml) in 10
mM-Tris-HC1 pH 7-5, 5 mM-EDTA, 150 mM-NaC1, 0"5~ SDS. Digestion with proteinase K was for 5 h (a) or 8 h
(b). Both proteases were self-digested before use, and the integrity of the viral RNA after the protease treatments
was confirmed by agarose gel electrophoresis. Treatments were stopped by phenol extraction and RNA was
recovered by ethanol precipitation, resuspended at the indicated concentrations in 50 mM-phosphate pH 7.0 and
used to inoculate
plants. Infectivity is given as the number of lesions/the number of leaves
VPg is required for the infectivity of the viral RNA, but that the peptide structure remaining
attached to RNA after the proteolytic treatment interferes with RNA infectivity.
To demonstrate the presence of the putative VPg in the PPV RNA, gradient-purified viral
RNA was iodinated using the Bolton and Hunter reagent, which is specific for proteins, as
described by Siaw
et al.
(1985). Electrophoresis of 125I-labelled RNA in 0.8~ agarose gels
indicated that the radioactivity was associated with RNA of genomic size (data not shown).
Electrophoresis in an SDS-polyacrylamide gel of 125I-labelled RNA digested with ribonuclease
A revealed a band with an apparent Mr of approximately 22000 (Fig. 1, lane 2) that was absent
from the undigested RNA (Fig. 1, lane 1). The faint band with higher mobility that also
appeared in lane 2 was absent in other experiments and comigrated with the RNase A employed
in the digestion and stained with Coomassie Brillant Blue. When the iodinated RNA was
digested with RNase T1 (Fig. 1, lane 3) the new band had a mobility lower than the one
produced after digestion with RNase A. This was expected because the 5' regions of potyvirus
RNA have few G residues. These bands disappeared upon treatment of the 125i_labelled RNA
with pronase (Fig. 1, lane 4). The putative 125I-labelled VPg-5' terminal oligonucleotides
remained in the organic phase after phenol extraction, in agreement with the proteinaceous
nature of the labelled material and a covalent linkage between it and the RNA.
Although the possession of genome-linked proteins seems to be a general characteristic of
potyvirus RNA, there are great differences in the Mr of the VPgs as estimated by SDS-
polyacrylamide gel electrophoresis. Values of 6000 and 24 000 have been reported for the VPgs
ofTEV (Hari, 1981) and TVMV (Siaw
et al.,
1985) respectively, and we have found that of PPV
VPg to be 22000. These differences among three viruses that are quite similar in genomic
structure and sequence (Allison
et al.,
1986; Domier
et al.,
1986; Maiss
et al.,
1989; Lain
et al.,
1989) are surprising. The potyvirus genome is expressed as a polyprotein that is autocatalytically
cleaved into the functional polypeptides (Dougherty & Carrington, 1988). The cleavage sites are
characterized by conserved series of amino acids, different for each potyvirus. The N terminus
of TVMV VPg has been located at one of these sites (Sbahabuddin
et al.,
1988), but no such
recognition sequence is available at its C terminus. Indeed, aberrant mobilities in
polyacrylamide gel electrophoresis of the VPgs of several viruses are well known (Daubert &
Bruening, 1984). This renders any discussion about the Mr of the potyvirus VPgs purely
speculative, and points to the need for additional sequence data on the N and C termini of the
The presence of the T 1 oligonucleotide linked to the labelled protein in the organic phase after
phenol extraction (see above) allowed its identification, purification and sequencing. If the
labelled protein is an authentic VPg, this oligonucleotide should correspond to the 5' terminus of
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1 2 3 4
Fig. 1. Analysis by SDS-PAGE and autoradiography of the protein released by RNase digestion of
zSI-labelled PPV RNA. Electrophoresis was in a discontinuous SDS gel system (Laemmli, 1970). The
separating gel was 15 ~ acrylamide, 0-25 ~ bisacrylamide, 0-1 ~ SDS. Lane 1, no treatment; lane 2, after
RNase A digestion; lane 3, after RNase T1 digestion; lane 4, after pronase digestion. The numbers at
the left refer to the Mr values of marker proteins.
PPV RNA. A sample of 20 ~tg of PPV RNA was treated with RNase T1 (55 units; Boehringer)
and alkaline phosphatase (120 units; Boehringer) in a reaction volume of 50 ~tl, essentially as
described by Fon Lee & Fowlks (1982). The incubation mixture was extracted three times with
phenol-chloroform and the resulting organic phases were combined, re-extracted with 20 mM-
Tris-HCl pH 7-5, 2 mM-EDTA (TE 2 × ) and then mixed with 2-5 volumes of ethanol. The
recovered material, presumably the VPg-linked Y-terminal T1 oligonucleotide, was 3' end-
labelled with 40 ~tCi [5 '-32P]pCp (3000 Ci/mmol; Amersham) and T4 RNA ligase (New England
Biolabs) (Fon Lee & Fowlks, 1982), ethanol-precipitated and resuspended in TE containing
0-125 ~ SDS. The products of the labelling reaction, either intact or digested with pronase, were
analysed in a 7 M-urea/20 ~o acrylamide gel (Fig. 2a). Besides a large amount of contaminating
oligonucleotides not removed by the phenol extractions, a band that was absent from the
untreated material (Fig. 2a, lane 1) appeared when it was digested with pronase (Fig. 2a, lane 2).
This band should correspond to the T 1 oligonucleotide linked to the remaining amino acids after
the proteolytic treatment. The intact protein--oligonucleotide band was not seen, presumably
because it could not enter the gel.
This procedure to obtain the putative 5'-terminal T 1 oligonucleotide was slightly modified to
get an oligonucleotide preparation with a higher specific radioactivity to enable its sequencing.
After digestion of 15 ~tg of PPV RNA with RNase T1 and alkaline phosphatase as above, the
mixture was subjected to three successive cycles of phenol extraction and ethanol precipitation
of the organic phases. After the last one, the precipitated material was resuspended in 0-125~
SDS in TE and treated with pronase, phenol-extracted again, and labelled with 40 ~tCi [5'-
32p]pCp. The 3' end-labelled putative Y-terminal oligonucleotide was purified by polyacryl-
Short communication
~ '7
+ + = u~ IIi
~ ~ < 0 I //// A
TEV 5'
Fig. 2. Identification and sequencing of the 5'-terminal T1 oligonucleotide of PPV RNA. (a) PPV
RNA digested with RNase T1 and 3' end-labelled with [5"-32p]pCp analysed in a 20~ acrylamide/7 M-
urea gel. Lane 1, no treatment; lane 2, after pronase digestion. (b) Autoradiograph of a sequencing gel
(20~ polyacrylamide/7 M-urea) showing the first 36 nucleotides of the PPV Y-terminal RNase T1
oligonucleotide labelled at its 3' end and sequenced by partial digestion with RNases. The bands
corresponding to the two last nucleotides were absent because they migrated out of the gel in this
experiment. Digestions were performed with RNase T1 (lane 1), RNase Phy M (lane 2), RNase BC
(lane 3), RNase U2 (lane 4), 50 mM-NaHCO3 for 5 min at 100 °C (lane 5) or no enzyme (lane 6). XC,
xylene cyanol marker dye; BPB, bromophenol blue. (c) Comparison of the 5" end RNA sequences of
amide gel electrophoresis and sequenced by partial digestion with the site-specific ribonucleases
T 1, Phy M, BC and U2 (P-L Biochemicals RNA sequencing enzyme kit, employed according to
the supplier's instructions) (Fig. 2b). A 38 nucleotide long sequence was obtained. There was one
uncertainty, at position 13, where a band appeared in the alkali ladder but was not present in any
RNase lane. The artefactual bands that appeared at positions 20, 33 and 34 were absent in gels
from other experiments. The sequence obtained showed significant homology with the 5'
terminus of the TEV (Allison
et al.,
1986) and TVMV (Domier
et al.,
1986) RNAs, confirming
that we had identified a VPg linked to the 5' end of the PPV RNA. As the linkage between VPg
and RNA was not expected to be cleaved by RNases and the largest bands appeared at the same
Short communication 2789
level in the alkali ladder and RNase Phy M lanes (Fig. 2b) it can be inferred that the sequence up
to the first 5' ribonucleotide had been obtained and that the bond between the latter and the VPg
was not cleaved by the mild alkali conditions employed. There is a 12 nucleotide block
completely conserved among the three potyvirus RNAs compared (Fig. 2c). This sequence
conservation at the 5' end of the RNAs is in contrast with the diversity of their 3' non-coding
regions (Lain et al., 1988), suggesting that this nucleotide block could play an important role in a
step of the virus life cycle in which either only the 5' end is involved, such as encapsidation or
translation, or where both ends participate but in different ways, such as replication.
Note. After submission of this paper the complete nucleotide sequence of the RNA of a non-aphid-transmissible
strain of plum pox virus (PPV-NAT) was published (Maiss et al., 1989). Its Y-terminal sequence is identical to that
reported here for the PPV Rankovic strain,
We thank Dr M. Salas for useful discussions and laboratory facilities. We are also grateful to the Departamento
de Quimica Agricola of the Universidad Aut6noma de Madrid for greenhouse space. This investigation was aided
by grants from Comision Interministerial de Ciencia y Tecnologia (BI088-0257), Fondo de Investigaciones
Sanitarias and Fundaci6n Ram6n Areces. J.L.R. and S.L. received fellowships from Plan de Formaci6n del
Personal Investigador and Fondo de Investigaciones Sanitarias, respectively.
ALLISON, R., JOHNSTON, R. E. & DOUGHERTY, W. G. (1986). The nucleotide sequence of the coding region of tobacco
etch virus genomic RNA: evidence for the synthesis of a single polyprotein. Virology 154, 9-20.
DAUBERT, S. D. & BRUENING, G. (1984). Detection of genome-linked proteins of plant and animal viruses. Methods in
Virology 8, 347-379.
M. F. E., LOMONOSSOFF, G. P., SHAW, J. G. & RHOADS, R. E. (1986). The nucleotide sequence of tobacco vein
mottling virus RNA. Nucleic Acids Research 14, 5417-5430.
DOUGHERTY, W. G. & CARRINGTON, J. C. (1988). Expression and function of potyviral gene products.
Annual Review
of Phytopathology 26, 123-143.
FON LEE, Y. & FOWLKS, E. R. (1982). Rapid in vitro labeling procedures for two-dimensional gel fingerprinting.
Analytical Biochemistry 119, 224-235.
ham, V. (1981). The RNA of tobacco etch virus: further characterization and detection of protein linked to RNA.
Virology 112, 391-399.
HELLMANN, G. M., SHAW, J. G., LESNAW, J. A., CHU, L. Y., PIRONE, T. P. & RHOA-DS, R. E. (1980). Cell-free
translation of
tobacco vein mottling virus RNA. Virology 106, 207-216.
LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature, London 227, 680-685.
(1988). Nucleotide sequence of the 3' terminal region of plum
pox potyvirus RNA. Virus Research 10, 325-342.
GARCI.A, J. A. (1989). The complete nucleotide sequence of plum pox potyvirus RNA.
Virus Research 13, 157-172.
(1989). The complete nucleotide sequence of plum pox virus RNA. Journal of General Virology 70, 513-524.
(1982). Specificity and properties of the genome-linked proteins of
nepoviruses. Journal of General Virology 59, 149-162.
SnArlAmrDDIN, M., SHAW, J. G. & RnOADS, R. E. (1988). Mapping of the tobacco vein mottling virus VPg cistron.
Virology 163, 635-637.
(1985). Identification of a protein
covalently linked to the 5' terminus of tobacco vein mottling virus RNA. Virology 142, 134-143.
D. (1975).
The 5' end group of tobacco mosaic virus RNA is m[7]G[5']pppGp. Nucleic Acids Research 2,
(Received 20 March 1989)
... PPV virions are long, flexuous and rod-shaped, around 750 nm in length and 12.5-20 nm in width, formed by a single coat protein (CP) of about 36 kDa arranged helicoidally around one molecule of single-stranded RNA (ssRNA) of a positive polarity (Brunt et al., 1996;Büchen-Osmond, 2004). The genomic RNA of PPV has a protein (Vpg) linked to its 5'-end (Riechmann et al., 1989) and a long poly (A) tail heterogeneous in size at its 3'end (Lain et al., 1988). PPV has been well characterized molecularly in recent years (Olmos et al., 2006;James & Glasa, 2006). ...
... It is commonly spread by aphids feeding on infected plants and then transferring PPV to uninfected plants [53]. The potyvirus genome consists of a ssRNA of ~ 10 kb with a viral protein genome-linked (VPg) at its 5′ end and a poly(A) tail at its 3′ end encapsidated by a single type of capsid protein (CP) subunit [54]. The N-terminal part of PPV CP was chosen as a site for the expression of foreign antigenic peptides, because it is exposed on the virion surface and is highly immunogenic. ...
Full-text available
Plant virus-based nanoparticles (PVNs) are self-assembled capsid proteins of plant viruses, and can be virus-like nanoparticles (VLPs) or virus nanoparticles (VNPs). Plant viruses showing helical capsid symmetry are used as a versatile platform for the presentation of multiple copies of well-arrayed immunogenic antigens of various disease pathogens. Helical PVNs are non-infectious, biocompatible, and naturally immunogenic, and thus, they are suitable antigen carriers for vaccine production and can trigger humoral and/or cellular immune responses. Furthermore, recombinant PVNs as vaccines and adjuvants can be expressed in prokaryotic and eukaryotic systems, and plant expression systems can be used to produce cost-effective antigenic peptides on the surfaces of recombinant helical PVNs. This review discusses various recombinant helical PVNs based on different plant viral capsid shells that have been developed as prophylactic and/or therapeutic vaccines against bacterial, viral, and protozoal diseases, and cancer.
... As regiões amino e carboxi-terminal estão voltadas para o exterior da partícula viral, e são responsáveis pelas propriedades antigênicas da proteína e, consequentemente, da partícula viral (Shukla et al., 1991). O RNA dos potyvírus é covalentemente ligado a uma proteína de origem viral ("genome-linked viral protein", VPg) em sua extremidade 5' (Riechmann et al., 1989) e apresenta uma cauda poliadenilada de origem viral em sua extremidade 3' (Allison et al., 1986). O RNA genômico apresenta duas fases abertas de leitura (open reading frames, ORFs) sobrepostas localizadas entre duas regiões não codificadoras (5'NTR e 3'NTR). ...
... Prieto et al., (2001) also reported that infected zucchini samples showed yellow mosaic and severe leaf blistering. Also, the experimental results are in harmony with that found by Lisa et al., (1981); Siaw et al., (1985); Riechmann et al.,(1989); Murphy et al., (1990); Desbiez and Lecoq (1997); Svoboda and Polák (2002) and Verma et al., (2006) The electron micrograph of negatively stained partially purified virus preparations shows the presence of flexuous filamentous virus particles with dimensions 750 X 13 nm (Fig. 2). Similar results wereobtained by Abdel-Ghaffar et al., (1998) and Mahmoud et al. (2004). ...
Full-text available
Squash is considered as one of the important vegetable crops worldwide including Egypt. Zucchini yellow mosaic potyvirus (ZYMV) is reported to be the most series viruses infecting cucurbits. In this study, an Egyptian isolate of ZYMV infecting squash plant (Cucurbita pepo cv. Eskandarani) was identified based on its biological, serological, and molecular properties. The isolate appeared severe mosaic, vein banding and deformation the infected squash plant under open field and greenhouse conditions. Positive reactions with polyclonal antibodies specific to ZYMV were obtained when samples were subjected to direct antigen coated (DAC)-enzyme-linked immunosorbent assay (ELISA). The electron microscopy of purified virus prepared from ZYMV-infected squash plants, showed the presence of filamentous virus-like particles measuring 750X13 nm. The viral isolate was confirmed to be belonging to Potyviruses group through producing cylindrical inclusions (pinwheels, scroll, and laminated aggregates) in the cytoplasm of cells infected with ZYMV. At the level of molecular characterization, the cylindrical inclusions (CI) protein and nuclear inclusions (NIb) genes of the ZYMV-EG isolate comprised 1902, and 1551 nucleotides, and encoding 634 and 517 amino acids protein, respectivelyand their similarities to some overseas isolates were addressed. The two genes appeared 100% homology compared to ZYMV TW-TN3 strain (AF127929).
... [52][53][54] The viral protein genome-linked (VPg) is covalently attached at its 5 0 end and a poly(A)-tail terminates at its 3 0 end. [55][56][57] This genome contains untranslated or noncoding regions (UTR) at each of its ends that surround a single-ORF encoding a polyprotein B3000 residues long. After translation, the polyprotein is proteolytically processed by three Potyvirus-encoded proteases P1, Helper component proteinase (HC-Pro) and Nuclear inclusion proteinase (NIa-Pro) into ten mature proteins. ...
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Within proteins, intrinsically disordered regions (IDRs) are devoid of stable secondary and tertiary structures under physiological conditions and rather exist as dynamic ensembles of inter-converting conformers. Although ubiquitous in all domains of life, the intrinsic disorder content is highly variable in viral genomes. Over the years, functional annotations of disordered regions at the scale of the whole proteome have been conducted for several animal viruses. But to date, similar studies applied to plant viruses are still missing. Based on disorder prediction tools combined with annotation programs and evolutionary studies, we analyzed the intrinsic disorder content in Potyvirus, using a 10-species dataset representative of this genus diversity. In this paper, we revealed that: (i) the Potyvirus proteome displays high disorder content, (ii) disorder is conserved during Potyvirus evolution, suggesting a functional advantage of IDRs, (iii) IDRs evolve faster than ordered regions, and (iv) IDRs may be associated with major biological functions required for the Potyvirus cycle. Notably, the proteins P1, Coat protein (CP) and Viral genome-linked protein (VPg) display a high content of conserved disorder, enriched in specific motifs mimicking eukaryotic functional modules and suggesting strategies of host machinery hijacking. In these three proteins, IDRs are particularly conserved despite their high amino acid polymorphism, indicating a link to adaptive processes. Through this comprehensive study, we further investigate the biological relevance of intrinsic disorder in Potyvirus biology and we propose a functional annotation of potyviral proteome IDRs.
The tobacco etch potyvirus (TEV) polyprotein is proteolytically professed by three viral proteinases (NIa, HC-Pro, and P1), While the NIa and HC-Pro proteinases each provide multiple functions essential for viral infectivity, the role of the P1 proteinase beyond its autoproteolytic activity is understood poorly, To determine if P1 is necessary for genome amplification and/or virus movement from cell to cell, a mutant lacking the entire P1 coding region (Delta P1 mutant) was produced with a modified TEV strain (TEV-GUS) expressing beta-glucuronidase (GUS) as a reporter, and its replication and movement phenotypes were assayed in tobacco protoplasts and plants, The Delta P1 mutant accumulated in protoplasts to approximately 2 to 3% the level of parental TEV-GUS, indicating that the P1 protein may contribute to but is not strictly required for viral RNA amplification. The Delta P1 mutant was capable of cell-to-cell and systemic (leaf-to-leaf) movement in plants but at reduced rates compared with parental virus, This is in contrast to the S256A mutant, which encodes a processing-defective P1 proteinase and which was nonviable in plants. Both Delta P1 and S256A mutants were complemented by P1 proteinase expressed in a transgenic host, In transgenic protoplasts, genome amplification of the Delta P1 mutant relative to parental virus was stimulated five- to sixfold, In transgenic plants, the level of accumulation of the Delta P1 mutant was stimulated, although the rate of cell-to-cell movement was the same as in nontransgenic plants. Also, the S256A mutant was capable of replication and systemic infection in P1-expressing transgenic plants. These data suggest that, in addition to providing essential processing activity, the P1 proteinase functions in trans to stimulate genome amplification.
The potyviruses have long been favored subjects for study by plant virologists. They constitute the most numerous of the three dozen or so groups of plant viruses, and collectively are responsible for more damage to the world’s crop plants than is caused by the viruses of most of if not all the other groups. This was rather dramatically illustrated by a recently conducted international election of “favorite” filamentous plant viruses among several eminent virologists in which the potyviruses emerged with a clear “victory” (Milne, 1988). The ecological and epidemiological aspects of diseases caused by potyviruses, with their many fascinating but varied and complex considerations, have also prompted the major efforts in research that have been directed over many years to this group of viruses.
Plum pox virus (PPV), the cause of the Sharka disease, is considered to be the most serious virus of stone fruits (Prunus spp.), which is transmitted in a non-persistent manner by several aphid species and vegetative production materials. Following its first determination in plums in Bulgaria, the virus has progressively spread to European continent and the Mediterranean Basin. It has also been sporadically detected in America (Argentina, Chile, Canada and USA) and in Asia (China, India, Iran, Kazakhstan and Pakistan). The diagnosis of the virus can be achieved by indicator plants, serological (i.e., ELISA) and molecular techniques (i.e., RT-PCR, IC-RT-PCR, microarray). However, trees infected with PPV cannot be treated. The practical prevention of the disease involves the production of healthy plants for planting, control of aphid vectors and destruction of infected trees in orchards. Several methods are used for obtaining initial virus-free propagation materials, such as thermotherapy, chemotherapy, electrotherapy, and cryotherapy. Breeding for resistance is the only way to control Sharka efficiently. However, at present very limited tolerant or resistant varieties of Prunus species are exist in the nature. The pathogen-derived resistance has been experimented to control the disease. In view of the crucial importance of Sharka disease, characteristics of the virus and the disease, detection techniques together with the potential applications and benefits of both classical and biotechnological treatments were discussed in this chapter.
Multiplication is the basic biological function of all organisms and is dependent upon replication of genomes. This is also true of plant viruses even though they possess a minimum of essential genetic information. Phylogenetically, replication-associated genes constitute the core elements of RNA virus genomes while other gene modules are considered as accessory elements. However, the biochemical studies on plant virus RNA replication were in their infancy even in 1999-2000. The picture has improved much since structural and sequence requirements of viral RNA replication, and synthesis are beginning to be understood, primarily because of the genetic, molecular, biochemical, and enzymatic studies conducted during the last six years. Certain virus-encoded essential proteins, nucleotide sequence motifs, and RNA secondary structures are central to virus RNA replication, which has a number of stages. Each stage is a complex phenomenon requiring specific factors and conditions. All this has generated much new information so that replication of plus-sense RNA plant viruses has now emerged as a rapidly developing field. However a lot of distance still has to be covered and traversing this distance could prove difficult because no one organised corpus of knowledge is available. Hopefully, this book fills the niche and generates understanding of multiplication of plus-sense RNA plant viruses, especially at molecular level. Nearly all the information on various aspects of plant virus multiplication has been collected, collated, and organized in eleven chapters spanning 332 pages so that this book comprehensively covers all facets of multiplication of plus-sense RNA plant viruses. No such book has been published so far.
A brief account is given on the situation of Sharka disease in the Mediterranean countries. The recent developments in the domain of virus characterization, diagnosis, epidemiology, genetic resistance and control measures are briefly reviewed. The necessity to intensify and extend the territorial monitoring activity and the use of certified plant propagating material of stone fruits is strongly recommended.
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SUMMARY The infectivity of the RNA of six nepoviruses was decreased or abolished by proteinase K treatment, whereas that of the RNA of cowpea mosaic virus (comovirus group) or tomato bushy stunt virus was unaffected. The extent of the decrease in infectivity was characteristic for each nepovirus and was independent of the plant species used as virus source or as assay host. The infectivity of raspberry ringspot virus (RRV) RNA was less affected than that of the other nepoviruses but treatment with Pronase decreased infectivity more than treatment with proteinase K. Proteinase K treatment also abolished the infectivity for tobacco mesophyll protoplasts of RNA of tobacco ringspot virus (TRSV) and tomato black ring virus (TBRV). Tests on virus RNA, labelled with ~25I by the chloramine T method, provided evidence that three nepoviruses and Echtes Ackerbohnenmosaik-Virus (EAMV; comovirus group) have genome-linked proteins (VPg). Pronase treatment rendered about half (RNA of strawberry latent ringspot virus; SLRV), or nearly all (RNA of the other nepoviruses and EAMV), of the '25I soluble in 70 % ethanol. Treatment of nepovirus RNA with ribonuclease P 1 yielded a product with an estimated mol. wt. of 4000 __+ 900. Mobilities in polyacrylamide gels of VPg from the RNA of different viruses differed slightly (SLRV > TBRV > TRSV > RRV). TRSV VPg yielded one ~25I-labelled tryptic peptide whereas the genome-linked proteins of RRV and TBRV both yielded two major products, of which one was resistant to further digestion and the other was converted, apparently via intermediates, to a second more stable product. No difference was detected between the tryptic peptides obtained from VPg of different strains of RRV, or of TBRV, or between those obtained from RNA-1, RNA-2 or RNA-3 (satellite RNA) of TBRV. Nepovirus VPg is therefore virus-specific. It seems to be coded on RNA-1 and probably has multiple functions.
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The nucleotide sequence of the RNA of tobacco vein mottling virus, a member of the potyvirus group, was determined. The RNA was found to be 9471 residues in length, excluding a 3′-terminal poly(A) tail. The first three AUG codons from the 5′-terminus were followed by in-frame termination codons. The fourth, at position 206, was the beginning of an open reading frame of 9015 residues which could encode a polyprotein of 340 kDa. No other long open reading frames were present in the sequence or its complement. This AUG was present in the sequence AGGCCAUG, which is similar to the consensus initiation sequence shared by most eukaryotic mRNAs. The chemically-determined amino acid compositions of the helper component and coat proteins were similar to those predicted from the nucleotide sequence. Amino acid sequencing of coat protein from which an amino-terminal peptide had been removed allowed exact location of the coat protein cistron. A consensus sequence of V-(R or K)-F-Q was found on the N-terminal sides of proposed cleavage sites for proteolytic processing of the polyprotein.
This chapter explains the detection of genome-linked proteins of plant and animal viruses. The search for a VPg becomes an effort to demonstrate that a given protein is associated with the virion nucleic acid, that the association is covalent, and that the association is sequence specific and does not have an explanation that is unrelated to genome expression. Strong, but not sequence-specific, binding of protein to virion nucleic acids is common. The nonspecific association may become covalent. The sensitivity of the infectivity of isolated virus nucleic acid to proteinase treatment has been an indicator of the presence of a VPg. However, such sensitivity alone is insufficient evidence of a VPg. For example, several RNA and DNA bacteriophages have proteins that enter the cell along with the virion nucleic acid during the normal infection initiation process. These proteins are not covalently attached to the nucleic acid but can stimulate the infectivity of the isolated nucleic acid by several orders of magnitude. A positive evidence of a VPg requires highly purified virion nucleic acid and a sensitive test for protein.
The complete nucleotide sequence of the tobacco etch virus (TEV) RNA genome has been determined excepting only the nucleotide(s) present at the extreme 5' terminus. The assembled TEV genomic sequence is 9496 nucleotides in length followed by a polyadenylated tract ranging from 20 to 140 residues. A computer search of the sequence reveals the following. A 5' untranslated region, rich in adenosine and uridine, is present between nucleotides 1 and 144. A putative initiation codon, at nucleotides 145-147, marks the beginning of a large open-reading frame (ORF) which ends with an opal (UGA) termination codon at positions 9307-9309. A 186-nucleotide untranslated region is present between the termination codon of the ORF and the beginning of the 3' polyadenylated region. The predicted translation product of this ORF is a 3054 amino acid polyprotein with a mol wt of 345,943. A function for the large (54,000 Mr) nuclear inclusion protein is suggested by a comparison of the deduced amino acid sequence with a protein data bank. This protein displays biochemical similarities to other viral RNA-dependent, RNA polymerases.
The location and order of cistrons in the RNA of the potyvirus tobacco vein mottling virus (TVMV) were investigated. Hybrid-arrested translation, using cloned single-stranded DNA probes complementary to various regions of the viral RNA, was performed and the resulting translation products were analyzed by electrophoresis. The pattern of polypeptides produced with each probe was different from that of control reactions containing RNA alone. Immunoprecipitation of reaction products with antisera to five potyviral proteins revealed that, in some cases, portions of cistrons were masked by the DNA probe resulting in the precipitation of altered polypeptides. In other cases, the entire cistron was affected, resulting in the total loss of immunoreactive material. By correlating the location of each DNA probe with the resulting pattern of translation products, it was possible to construct a map of known cistrons and potential coding regions for additional cistrons in the genomic RNA. DNA probes representing several regions of the viral RNA were expressed in E. coli. Immunoprecipitation of cell proteins revealed the presence of TVMV-related polypeptides; the results were consistent with the cistron order determined by hybrid-arrested translation experiments. Our proposed cistron order and the sizes in kilodaltons of the corresponding polypeptides are: 5'-25 kDa unidentified protein-53 kDa helper component protein-50 kDa unidentified protein-70 kDa cylindrical inclusion protein-52 kDa nuclear inclusion protein-56 kDa nuclear inclusion protein-32 kDa coat protein-3'.
The complete nucleotide sequence of the RNA of an aphid non-transmissible plum pox virus (PPV-NAT) isolate has been determined from five overlapping cDNA clones. cDNA prepared by primer extension was used to determine the 5' terminus. The assembled RNA is 9741 nucleotides in length, excluding a 3' terminal poly(A) sequence. One large open reading frame starts at nucleotide positions 36 to 38 and is terminated with an UAG codon at positions 9522 to 9524. The putative start codon is located at positions 147 to 149. The encoded polyprotein has a predicted Mr of 353.8K. Comparison of cistrons from tobacco vein mottling virus and tobacco etch virus with those predicted for PPV-NAT indicated a similar genome organization. A highly conserved sequence of 12 nucleotides was found in the 5' non-coding region of these three potyviruses. The potential polyadenylation signal from yeast (UAUGU) was found in the 3' non-coding region of PPV-NAT and several other members of the potyvirus group.
The location of the cistron encoding the genome-linked protein (VPg) in the potyvirus tobacco vein mottling virus (TVMV) was investigated. Precipitation of 125I-labeled VPg with anti-tobacco etch virus 49K nuclear inclusion protein antiserum (which reacts with the NIa nuclear inclusion protein of TVMV) indicated that the TVMV VPg is immunologically related to NIa. Lysyl residues were found to be present at positions 2, 11, and 16 of the amino-terminal region of the VPg. A search of the TVMV polyprotein sequence for this distribution of lysyl residues revealed a unique location beginning at amino acid residue 1801, the proposed amino-terminus of the NIa protein.
Rapid in vitro labeling procedures for two-dimensional gel fingerprinting The RNA of tobacco etch virus: further characterization and detection of protein linked to RNA
  • Fon Lee
  • Y Fowlks
  • E R Ham
FON LEE, Y. & FOWLKS, E. R. (1982). Rapid in vitro labeling procedures for two-dimensional gel fingerprinting. Analytical Biochemistry 119, 224-235. ham, V. (1981). The RNA of tobacco etch virus: further characterization and detection of protein linked to RNA. Virology 112, 391-399.
The 5' end group of tobacco mosaic virus RNA is m[7]G[5']pppGp
ZIMMERN, D. (1975). The 5' end group of tobacco mosaic virus RNA is m[7]G[5']pppGp. Nucleic Acids Research 2, 1189-1201. (Received 20 March 1989)