Download Cloning and sequencing of the S RNA from a Bulgarian isolate of

Journal of General Virology (1991), 72, 461-464. Printedin Great Britain
461
Cloning and sequencing of the S RNA from a Bulgarian isolate of tomato
spotted wilt virus
E. Maiss, 1. L. Ivanova, 2 E. Breyel 3 and G. Adam 3
1Biologischen Bundesanstalt f~r Land- und Forstwirtschaft, Institut fur Biochemie, Braunschweig, Germany, ZInstitute of
Genetic Engineering, Agricultural Academy, Bulgaria and 3DSM-Deutsche Sammlung yon Mikroorganismen und
Zellkultur GmbH, Abteilung Pflanzenviren, Braunschweig, Germany
Libraries of cloned cDNA were prepared from complete genomic RNA and isolated S RNA of the
Bulgarian L3 isolate of tomato spotted wilt virus
(TSWV-L3). Northern blotting of TSWV genomic
RNA detected clones specific for the L, M and S RNAs
in the library from complete RNA. S RNA-specific
clones selected from both libraries covered approximately 2-8 kb (about 95 %) of the S RNA. Sequencing
of these clones showed TSWV-L3 S RNA to be
ambisense. It contains two open reading frames
(ORFs); one of 1401 nucleotides located on the viral
RNA encodes an Mr 52400 (52K) protein, and the
other of 774 nucleotides on the complementary strand
encodes an Mr 28 900 (29K) protein. Expression of the
29K ORF in bacteria and immunological analysis of
the fusion protein synthesized confirmed that the 29K
protein is the N protein of TSWV-L3. Comparison
with the published sequence for the S RNA of a
Brazilian TSWV isolate, CNPH1, revealed almost
complete identity in the amino acid sequences for the
29K protein, but several clustered amino acid exchanges in the putative 52K protein. In addition, the
separating non-translated intergenic region of the S
RNA of the Bulgarian isolate is 81 nucleotides longer
than that of CNPH1.
Tomato spotted wilt virus (TSWV) has gained in both
economic importance during the last 10 years (Cho et al.,
1989) and academic interest since it seems to represent a
group of plant viruses that should be considered a genus
in a family of otherwise vertebrate-infecting viruses, the
Bunyaviridae (Milne & Francki, 1984; de Haan et al.,
1989a, 1990; Elliott, 1990). This proposed classification,
hitherto based solely on morphological and protein
chemical data (Milne & Francki, 1984), has recently
been substantiated by nucleic acid sequence data for one
of its genomic RNA species and by elucidation of the
genome arrangement of this RNA and the replication
strategy of the virus (de Haan et al., 1989a, 1990;
Kormelink et al., 1990).
The variability among different TSWV isolates has
been estimated by differences in symptom expression
(Best, 1954) and confirmed by serological comparisons
using either polyclonal (Wang & Gonsalves, 1990) or
monoclonal antibodies (Adam et al., 1990; de .Avila et al.,
1990).
In order to compare the nucleotide and amino acid
sequences of TSWV isolates from different geographical
locations, we have started to sequence the S RNA from
the Bulgarian isolate TSWV-L3 that is described in more
detail by Adam et al. (1990). The TSWV S RNA
sequence obtained for a Brazilian isolate, TSWV
CNPH1 (de Haan et al., 1990), was used for comparison.
In this paper we report the partial nucleotide sequence
covering two open reading frames (ORFs), of the S RNA
from the Bulgarian L3 isolate and present a comparison
of the two available sequences.
The virus isolate TSWV-L3 and its purification and
core preparation are described in detail by Adam et al.
(1990). RNA from purified cores was isolated according
to Maiss et al. (1988). Complementary DNA of total
genomic and gel-purified S RNA was synthesized
(Gubler & Hoffman, 1983), blunt-end ligated into
HinclI-cut pT7T3 19U phagemid vector (Pharmacia),
and subsequently used to transform Escherichia coli strain
N M 522 (Hanahan, 1983). Denatured genomic RNAs of
TSWV were separated in 1~ agarose gels (Carmichael &
McMaster, 1980), blotted onto nitrocellulose and hybridized with random primed 32p-labelled cDNA clones
(Feinberg & Vogelstein, 1983a, b).
Single-stranded DNA templates of original clones, or
of clones after exonuclease III treatment (Henikoff,
1984), were produced (Vieira & Messing, 1987) and
sequenced using both the universal and the T3 promoter-
0000-9798 © 1991 SGM
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1
121
241
361
48l
601
721
841
961
1081
1201
1321
1441
1561
1681
1801
1921
2041
2161
2281
2301
rAGAAAArcA~AArAcr~rAATAAGAAcA~AGrAc~AArAAc~ArA~rcrrcAA~rGrrrAr~AarcGArcArrcA~A~AA~AGcTr~AGr~rGGGGAr~AA~rGcAr~rGGrAAA~c120
~rrG~A~A~r~Ac~GGA~A~GAAc~GGrA~rGGT~c~c~Ac~AGr~AAAcc~AGc~G~A~c~GAr~AAGAA~cAAAAG~AGc~G~A~Ac~G~AA~AGGGAA~
240
~ccc~GT~AAGAAGA~GAGA~Tc~cTcT~AGcATaTGTATATcccTATTTTTGATGATATT~A~TAGcA~AATA~TcAT~Ac~cTG~Tc~GGcAcTATcTG~TTGc~cAAATAcA~360
~AA~Ac~AA~AGTGAAAcAT~AA~TcA~TTGAAAG~T~TG~cTc~GA~AGcT~cA~T~A~TGGAT~AccATaAA~AaAT~GATAT~AAAGAc~aA~TccAG~AAGAAAA
480
AGAcATAAx~c~cAATGAcAGATAcAT~GAAG~TG~AAA~AAA~Gc1cT~TGTcTTGTG~cAAAGA~ATAccTATAAGATcaA~AcG~Gc~A~AA~AAGc~GGGcAAA~GAATG~
600
~TTATccccTAAcAGGAATGTccATGAA~GGc~GTAcAG~TTc~AGccAAGTT~cAA~cAA~TTGAAAGcAAcAAcAGAAcTGTAAATTcTcITGcAGTGAAA~cTcT~cTcATGTcA~c
720
AGAAAAcAAcA~cATGccTAA~Tc~AGG~rTTTGrcAAAGcr~ccAcrGAT~TcAT~cAAA~GAGccr~TGGcTAAGGaTrccAAAGGTTc~GAAGcAG~c~ccATTcAGAAAT~
840
GTTcAAGGT~GcAGGA~A~GAAAcAAA~AAAAcA~r~ATTTATcTATTGrT~acATTccAAAccA~AAcAGTG~TGAGAcAac~TTAAAcA~T~cTGTTA~TTGcAAGcATcAGcTccc960
AAT~cG~AAATG~AAA~cTcc~TTGAAT~ATcAAT~AT~T~r~cTGA~TTGAA~GAGccTTAcAAcATTG~cA~AT~c~A~ATcc~cAGAGGATTG~TcATGcTcTGcrT~AAAc
1080
T~AcAcAT~TT~TGcA~AA~TTcTT~cAA~AAcT~AcAAGAA~ATGT~ATcAT~TAcA~TTT~AAcAAcTA~AGcTAA~Tcc~AAA~TTA~ATTTAGa~GAAA~AA~cTTAAA~TA
1200
cAG~AAGA~A~c~AAAA~GAAATAT~IccTrIcAAAAAcAcTT~AAT~IcrTccATc~AAcAcAcAAAcTAT~rc~IAcTTGGAcAGcATcc~AATcccTTcc~AAGATA~Ac~
1320
T~c~AG~GA~AAAwAA~ATTT~Tc~AcAA~T~T~TcA~TTGcAAAAT~TTTG~TAAAGcTTGATTTAA~GATcAAAA~AAA~A~TAAGA~cG~AAAcAcATGcT~cA~
1440
AT~AAAA~t~`~rTGc~GTG~ccG~T~TT~TAAr~ATGT~ATG~rTAT~r~T~cTT~A~TTArAArTA~T~TcTG~TTTGT~A~Tc~rTAA~TT~c~TTGTTTAA~AGAAAcc 1560
A~AAAAcAAAAAATAAAATAAAAATAAAAA~cAAAAAT~AAAcAAAA~TcAAAAAA~AAAcAAAAATAAAAAAT~AAATAAAAcAAcAAAAAAA~TAAAAAAcAAAAAAccAAAAAA6A
1680
TcccGAAAGG~AcAATTTTGGccAAA~TT~G~T~TTTT~TTT~GTTT~TTTTTT~TTTTTATTTT~ATT~ATTTATTTATTTTA~TTTATTTTT~A~TTTTTA~TTTT~TT~T
1800
TATTTTATa~T~TT~GTTGT~TTT~TATT~TGTTTGTT~ATTAAGcAcAAcAcAcAGAAA~cAAAcTTTAATTAAAcAcAcTTATTT~AAATTTATTAAGcAcAAcAcAcAGAAAGcAA
1920
AcTTTA~TTAAAcA~AcTTATTT~A~AT~AAcAcAcTAAGcAAGcAc~A~cAA~AAAGATAAAGAAAGcTT~ATATATITG~AGAcTTT~ccATAATT~AAcTTAcAAcTGcTT~-----~G
2040
cAA~TTcT~A~T~TT~ccT~TTTTTAAc~ccAAAcA~TcATAGAAcTTGTTAAGAGTTTcAcTGTAATGTT~cA~AGcAATA~TccT~AGcATTAGGATTGcTGGAGcTGAG~A2160
rAG~AG~A~A~c~T~rccTT~c~T~AccTGAr~rrcA~rcA~cAAA~Gc~T~c~TcAGcAcAG~cAAAcTTT~rAAG~cT~GG~G~cA~A~TT~GGTcAATcc2280
cGAaGTcT~TGTA~TTGcATc~TGArATA~AGc~AAGAcAA~Ac~GATcA~cT~AAAGcTATcAAcTGAA~AA~AAGAGG~AAG~TAccTcccAGcATTATG~cAA~ccTcA~A~Ac~
2400
~GcATcATcAAaAGGTAATccATAa~c~GAATcA~AG~G~GGGAAGcAATcTTAGATT~A~A~ATIGAGArTcrcAGAATTccc~G~ccTcTAcAAGccTGAccc~GATc~Gc
2520
2421
2541
2661
~ATcAAaccTTcTGAAGGTcATGTcA~T~GcTcc~ATccTGTcTaAAG~TTTcTTTAT~TAAIITTAccAAAA~TAAAATcAcT~T~TT~AATAAccTTcAT~ATAc~cT~AcGATIcT2640
~AG~AA~rcAG~cA~GAAATAA~G~T~A~cTT~TTGA~Gr~GA~r~r~rAGA~AAAAAGrc~GAA~rGAATGcrA~AGA~GA~r~rcAAA~r~AAGG~c~c 2760
CTTGTGTCAACAAAGCAACAATGCTTTCCTTAGTGAGCTTAACCTTAGA~GATGATTGTAGAAGTTGTTATATGC
...........................................
2837
Fig. 1. Partial (approx. 95~) sequence of TSWV-L3 S RNA. The starts (hatched box) and stops (underlined) of the two open reading
frames are indicated.
specific primer according to Sanger et al. (1977).
Sequence data were processed by the Sequence Analysis
Software Package of the Genetics Computer Group
(University of Wisconsin; Devereux et al., 1984).
When the clones, which were obtained from complete
viral RNA preparations, were hybridized with endlabelled viral RNA, approximately I00 clones with
inserts ranging between 0.3 and 2.2 kb in size were
obtained. Further screening by hybridization against
Northern-blotted viral RNAs revealed that this library
contained clones against the L, M and S RNAs. The
clone pTSWV-L3/335, which reacted specifically with
the S RNA, was used to detect further S RNA-specific
clones in the library of cDNA clones prepared from
purified S RNA. According to an established physical
map our S R N A clones covered approximately 95 ~ of
the complete RNA and had a restriction enzyme pattern
similar to that of the TSWV isolate C N P H 1 (de Haan et
al., 1989b).
Sequence determinations were carried out mainly
from the two clones pTSWV-L3/335 and pTSWVL3/308. The assembled sequence of 2837 nucleotides is
shown in Fig. 1. Comparison with the terminal
sequences of the TSWV isolate CNPH1 revealed a 100~
overlap at the 5'-terminal end, starting at position 42 (de
Haan et al., 1989b, 1990). Screening of our cDNA
libraries indicated that the extreme 5' and 3' ends were
not represented.
Two ORFs were present on the S R N A of TSWV-L3
(Fig. 1), the larger one on the viral R N A strand and the
smaller one on the complementary strand. The ORFs
were separated by a non-coding region of 586 nucleotides. Computer analysis suggested that this region could
form a highly stable secondary structure by internal base
pairing of the thymidine-rich (41.5~) stretch and the
inverted complementary adenosine-rich (37-5 ~) region.
The genome organization was identical to that described
for the CNPH1 isolate of TSWV (de Haan et al., 1990).
Comparison of the nucleic acid sequences from L3 and
CPNH1, using the program BESTFIT, revealed 9 4 ~
and 6 2 ~ identity, respectively when tested with and
without alignment of the two sequences. In order to
reveal variabilities between the two ORFs of TSWV-L3
and C N P H I we have analysed and compared the
corresponding homologous nucleic and amino acid
sequences.
The 1401 nucleotide ORF on the L3 virus R N A
contained the information for a protein with 467 amino
acids, and an Mr of 52400. Comparison of the respective
nucleic acid sequences from CNPH1 and L3 with
BESTFIT resulted in 9 6 ~ similarity or 6 7 ~ identity.
When the amino acid sequences of the two putative
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proteins encoded by the homologous ORFs were compared and aligned, it became obvious that the changes at
the nucleic acid level also led to substantial differences
between the two proteins; the TSWV-L3 sequence had
an insertion of four amino acids (residue 234) and a
deletion of one amino acid (residue 342). In addition,
several amino acid exchanges were observed, some of
which were clustered in two regions, positions 54 to 71
and positions 335 to 355 (Fig. 2). However, the 92~0
similarity calculated for the two amino acid sequences of
the suggested NSs proteins of the CPNH1 and the L3
isolate indicated that the two proteins are related. The
number and location of possible glycosylation sites
(NXT/S; Doolittle, 1986; Fig. 2) were identical to those
described for CNPH1 (de Haan et al., 1990), suggesting
that some of them might be used. No homologies could
be observed with proteins in the GenBank (Release 60-0)
and the EMBL Data Bank (Release 19.0).
The second ORF, located on the complementary S
RNA strand, was almost identical to that of the CNPH 1
isolate. Both sequences were 774 nucleotides long and
could code for a protein of 258 amino acids with an Mr of
28900. No glycosylation sites were detected. A nucleic
acid sequence comparison revealed 97~ similarity and
65~ identity, whereas the amino acid sequences were
almost completely identical, except for four amino acid
exchanges in our sequence at the following positions: 42,
lie to Leu; 88, Gly to Ala; 230, Val to Ile; 255, Ala to Thr.
Only the last exchange resulted in a change to a
dissimilar amino acid.
We assumed that the protein encoded by this ORF was
the N protein of TSWV-L3, for which an apparent Mr of
28000 was estimated by SDS-PAGE (Adam et al., 1990).
To prove this, we inserted pTSWV-L3/308 into the
expression vector pEX2 to give pEXcpTSWV-L3 This
construct was expressed in E. coli pop 2136 cells (Maiss et
al., 1990) and the resulting fusion protein was analysed
after SDS-PAGE, transfer to nitrocellulose (Adam et al.,
1987) and incubation with TSWV N-specific antibodies
(Loewe Biochemicals). The antibodies specifically
stained a protein band of Mr 79000 which is in good
agreement with the calculated value of 76000 for the
expected fusion protein (results not shown). This result
further verifies that the 29K ORF codes for the N
protein, as has been reported by de Haan et al. (1990) for
TSWV CNPH1 and emphasizes the close serological
relationship between the two TSWV isolates reported by
Adam et aL (1990).
Due to several insertions, the intergenic region of
TSWV-L3 S RNA which separated the two ORFs was 81
nucleotides longer than the corresponding part of the
CNPHI sequence. Suggested secondary structures resulting from internal base pairing in both intergenic
regions were analysed with the program FOLD, using
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'''''"''"'"'''' 464
Fig. 2. Comparison of the deduced amino acid sequences of the 52K
ORFs from TSWV-L3 (upper sequence) and TSWV-CNPH1 (lower
sequence). Vertical bars mark identical amino acids. Amino acid
exchanges are marked by no sign, a single point or a colon, indicating
an increasing order of chemical similarity. Hatched boxes indicate
putative glycosylation sites.
positions 1485 to 1986 (CNPH1) and 1452 to 2036
(TSWV-L3). Both structures had a similar stability as
indicated by minimum free energy values of -468 and
-482 kJ/mol for CNPH1 and L3, respectively. The
insertions in our sequence caused a much more complex
folding, but the same nucleotide sequence
(AC)AATTT(TGG) remained exposed in the prominent
stem loop region. The conservation of this sequence in
an identical structure suggests that this region might
have a regulatory function, as proposed by de Haan et al.
(1990).
In summary our data obtained regarding the genome
organization of the S RNA of a Bulgarian isolate of
TSWV are in accordance with the results of de Haan et
al. (1990). As a similar genome organization is observed
for the S RNAs of viruses belonging to the genera
phlebovirus and uukuvirus in the family Bunyaviridae
(Elliott, 1990), our results corroborate the grouping of
tomato spotted wilt viruses into a separate genus of the
Bunyaviridae as suggested by Peters (I990). However,
comparison of nucleic acid and amino acid sequences
revealed differences between the two isolates, especially
in the putative NSs protein ORF and in the intergenic
region, whereas the N protein sequences of both strains
were almost identical, as expected from serological
comparisons (Adam et aL, 1990). Whether the differ-
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ences detected in the NSs protein and the intergenic
region are of genetic importance remains to be
determined.
We gratefully acknowledge the skilful technical assistance of Ute
Biirstenbinder, Renate Nieliinder and Anke Brisske. Thanks are due to
Dr P. de Haan for sending us the nucleic acid sequence of TSWV
CNPH1 before publication.
References
ADAM, G., CHAGAS,C. M. & LESEMANN,D.-E. (1987). Comparison of
three plant rhabdovirus isolates by two different serological
techniques. Journal of Phytopathology 120, 31-43.
ADAM, G., LESEMANN, D.-E. & VEX'tEN, J. (1990). Monoclonal
antibodies against tomato spotted wilt virus: characterisation and
application. Annals of Applied Biology (in press).
BEST, R. J. (1954). Cross protection by strains of tomato spotted wilt
virus and a new theory to explain it. Australian Journal of Biological
Sciences 7, 415-424.
CARMICHAEL, G. G. & MCMA~TER, G. K. (1980). The analysis of
nucleic acids in gels using glyoxal and acridine orange. Methods in
Enzymology 65, 380-391.
CliO, J. J., MAU, R. F. L., GERMAN,T. L., HARTMAN,R. W., YUDIN,
L. S., GONSALVES,O. & PROVVIDENTI,R. (1989). A multidisciplinary
approach to management of tomato spotted wilt virus in Hawaii.
Plant Disease 73, 375-383.
DE[kVILA,A. C., DEO. RESENDE,R., KITAJIMA,E. W., GOLDBACH,R. &
PETERS,n. (1990). Tomato spotted wilt virus: occurrence, differentiation and detection of isolates. In Abstracts of the VIIlth International
Congress of Virology, Berlin, p. 132.
DE HAAN, P., WAGEMAKERS,L., GOLDBACH,R. & PETERS,D. (1989a).
Tomato spotted wilt virus, a new member of the Bunyaviridae? In
Genetics and Pathogenicity of Negative Strand Viruses, pp. 287-290.
Edited by D. Kolakofsky & B. W. J. Mahy. Amsterdam: Elsevier.
DE HAAN,P., WAGEMAKERS,L., PETERS,D. & GOLDBACH,R. (1989b).
Molecular cloning and terminal sequence determination of the S and
M RNAs of tomato spotted wilt virus. JournalofGeneral Virology70,
3469--3473.
DE HAHN, P., WAGEMAKERS,L., PETERS,D. & GOLDBACH,R. (1990).
The S RNA segment of tomato spotted wilt virus has an ambisense
character. Journal of General Virology 71, 1001-1007.
OEVEREUX,J., HAEBERLI,P. & S~tITmES, O. (1984). A comprehensive
set of sequence analysis programs for the VAX. Nucleic Acids
Research 12, 387-395.
DOOLrI'rLE, R. F. (1986). On URFs and OFSs. Oxford: Oxford
University Press.
E LLIOTI',R. M. (1990). Molecular biology of the Bunyaviridae. Journal
of General Virology 71, 501-522.
FEINBERG, A. P. & VOGELSTE1N, B. (1983a). A technique for
radiolabeling DNA restriction endonucIease fragments to high
specific activity. Analytical Biochemistry 132, 6-13.
FEINBERG, A. P. & VOGELSTEIN, B. (1983b). A technique for
radiolabeling DNA restriction endonuclease fragments to high
specific activity (addendum). Analytical Biochemistry 137, 266-267.
GtJBLER, U. & HOFFMAN,B. J. (1983). A simple and very efficient
method for generating eDNA libraries. Gene 25, 263-269.
HANAHAN,D. (1983). Studies on transformation of Escherichia coil with
plasmids. Journal of Molecular Biology 166, 557-580.
HENIKOEF, S. (1984). Unidirectional digestion with exonuclease III
creates targeted breakpoints for DNA sequencing. Gene 28, 351-359.
KORMELINK, K., DE HAAN, P., PETERS, D. & GOLDBACH,R. (1990).
Replication strategy of the tomato spotted wilt virus genome. In
Abstracts of the VIIlth International Congress of Virology, Berlin,
p. 129.
MAISS, E., BREYEL, E., BRISSKE,A. & CASPER, R. (1988). Molecular
cloning of DNA complementary to the RNA-genome of plum pox
virus (PPV). Journal of Phytopathology 122, 222-231.
MAISS, E., TIMPE, U. & CASPER, R. (1990). Expression of the coat
protein gene of plum pox virus in Escherichia coll. Archly 3'~r
Phytopathologie und Pflanzenschutz 26 (in press).
MmNE, R. G. & FItANCKI, R. I. B. (1984). Should tomato spotted wilt
virus be considered as a possible member of the family Bunyaviridae? lntervirology 22, 72-76.
PETERS, D. (1990). Overview of tomato spotted wilt virus. In Abstracts
of the USDA TSWV Workshop, Beltsville, Maryland, USA.
gANGER,F., NICKLEN,S. & COULSON,A. R. (1977). DNA sequencing
with chain-terminating inhibitors. Proceedings of the National
Academy of Sciences, U.S.A. 74, 5463-5467.
VIEIRA,J. & MESSING,J. (1987). Production of single-stranded plasmid
DNA. Methods in Enzymology 153, 3-11.
WANG, M. & GONSALVES,D. (1990). ELISA detection of various
tomato spotted wilt virus isolates using specific antisera to structural
proteins of the virus. Plant Disease 74, 154--158.
(Received 10 July 1990; Accepted 6 November 1990)
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