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

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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
2785
The Genome-linked Protein and 5' End RNA Sequence of
Plum Pox Potyvirus
By JOS]~ L. RIECHMANN,* SONIA LAIN AND JUAN A. GARCIA
Centro de Biologia Molecular (CSIC-UAM), Universidad Aut6noma de Madrid, Canto Blanco,
28049 Madrid, Spain
(Accepted I June 1989)
SUMMARY
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
2786
Short communication
Table 1.
Effect of proteolytic treatments on the infectivity of PPV RNA*
Infectivity after treatment with
RNA concentration A
t
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
Chenopodiumfoetidum
plants. Infectivity is given as the number of lesions/the number of leaves
inoculated.
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
VPgs.
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
Short communication
1 2 3 4
2787
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-
2788
Short communication
(a)
(c)
12
!
XC~
BPB
~ '7
(b)
A
'A
+ + = u~ IIi
U
~ ~ < 0 I //// A
XC~
BPB
123456
PPV
5"
TEV 5'
TVMV 5'
AI'~AUA~AI~AXUCAACACAACA~ACAA~AUUUUAUG...3 '
~AAA~AACA'A'~UC~CAACACAACA~AUAC~AACAAAC
.3'
~AAU~AA~AA~CAACACAACA~UAUA~GAACCAG
.3'
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
PPV, TEV and TVMV.
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.
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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.
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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.
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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.
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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'.
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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.
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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)