Download Sequence analysis of a faba bean necrotic yellows virus DNA

Journal of General Virology (1995), 76, 475-479. Printed in Great Britain
475
Sequence analysis of a faba bean necrotic yellows virus DNA component
containing a putative replicase gene
Lina Katul,* Edgar Maiss and H. J o s e f Vetten
Biologische Bundesanstalt fiir Land- und Forstwirtschaft, Institut fiir Biochemie und Pflanzenvirologie,
Messeweg 11-12, 38104 Braunschweig, Germany
Faba bean necrotic yellows virus (FBNYV) has a circular
ssDNA genome possibly consisting of several components of about 1 kb each. The complete nucleotide
sequence of one component of FBNYV (FBNYV DNA
1) containing a putative replicase gene is presented. This
component consists of 1002 nucleotides and, in the
virion orientation, contains one large open reading
frame (ORF1) potentially encoding a 32.3 kDa replicase
with the NTP-binding motif GGEGKS. No obvious
functions could be assigned to two smaller ORFs (7"4
and 9-3 kDa) occurring in the complementary often-
Introduction
Faba bean necrotic yellows is an economically important
disease of crop and pasture legumes in west Asia and
north Africa. Isometric virus-like particles are closely
associated with the disease and have been regarded as the
putative causal agent although their infectivity has not
yet been shown. The putative causal agent is not
transmitted mechanically, but by aphids in a persistent
manner. The virus-like particles measure 18 nm in
diameter, contain a capsid protein of about 22 kDa and
circular ssDNA of about 1 kb and are called faba bean
necrotic yellows virus (FBNYV) (Katul et al., 1993).
Based on these criteria FBNYV is very similar to banana
bunchy top virus (BBTV) (Thomas & Dietzgen, 1991;
Harding et al., 1991, 1993), coconut foliar decay virus
(CFDV) (Randles & Hanold, 1989; Rohde et al., 1990),
milk vetch dwarf virus (MDV) (Sano et al., 1993) and
subterranean clover stunt virus (SCSV) (Chu & Helms,
1988). In addition, MDV and SCSV particularly resemble FBNYV in not only having the same aphid vector
species and a similar host range, but also in being
* Author for correspondence. Fax +49 531 299 3006. e-mail
[email protected]
The nucleotidesequence reported here will appear in the EMBL,
GenBankand DDBJnucleotidesequencedatabasesunderthe accession
number X80879.
0001-2821 © 1995 SGM
tation. Amino acid sequence comparisons of the putative
replicase of FBNYV with that of other similar ssDNA
viruses yielded higher homologies to subterranean clover
stunt virus than to banana bunchy top and coconut foliar
decay viruses. A potential stem-loop structure and a
TATA box were identified within the noncoding region.
Two oligonucleotides derived from FBNYV DNA 1
were used for direct sequencing of the virion ssDNA to
determine its virion polarity and for amplifying part of
this component by immunocapture PCR in extracts
from FBNYV-infected plants.
serologically related (Katul et al., 1993). Recent studies
on the genome of BBTV (Harding et al., 1993; Burns et
al., 1994; Yeh et al., 1994), CFDV (Rohde et al., 1990)
and SCSV (Boevink et al., 1993; Chu et al., 1993; Surin
et al., 1993) confirmed the tentative grouping of these
viruses since their circular ssDNA genomes share similar
organization and appreciable sequence homologies.
Apart from CFDV, the genome of which has not been
further analysed, BBTV and SCSV were shown to have
a multipartite genome, consisting of at least six and seven
circular components, respectively (Burns et al., 1993;
Boevink et al., t993). This clearly differentiates them
from geminiviruses, the only other group of plant viruses
with a circular ssDNA genome (Lazarowitz, 1987).
Based on the results from our previous cloning and
hybridization experiments with FBNYV DNA (Katul et
al., 1993) and by analogy with BBTV and SCSV, we
expect the genome of FBNYV to be made up of several
covalently closed circular ssDNA components of about
1 kb each. In this paper we present the sequence of the
first FBNYV DNA component and compare it with the
corresponding genome components of similar viruses.
We also describe the use of the polymerase chain
reaction (PCR) for the amplification and detection of
viral DNA from plant extracts.
Extraction of the nucleic acid from purified virus
preparations and cloning of the DNA into the phagemid
vectors pGEM-3Zf(+) (Promega) and pT7T3 19U
(Pharmacia LKB) was as previously described (Katul et
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476
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Fig. 1. Complete nucleotide sequence of the
FBNYV DNA 1 component and the amino acid
sequence of ORF 1, encoding a putative replicase.
The stem-loop structure is in bold and underlined,
the TATA box (TATAAA) and the poly(A)
signal (AATAAG) are in bold and double
underlined, and the potential NTP-binding motif
(GGEGKS) is in bold and shaded. The positions
of primer 1 (I~
I~) and the reverse complement of primer 2 ('II
.q) are indicated
by arrows over the corresponding nucleotide
sequence.
K
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C~GCGATGGAGATGATG~G~TAGATGCTTTGC~GTACGAAATATAGATCTGTAGAT
Q A M E M M K N R C F A S T K Y R S V D
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CTTTGTTGT~TAAAAATGTTCATTTAGTTGTTTTTGCC~CGTGGCATATGACCCCACA
L C C N K N V H L V V F A N V A Y D P T
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/~,~TGAGGATAGGATTGT~TTA~C~TTGTTG~TT 1002
K I S E D R I V I I N C
al., 1993). The dideoxynucleotide chain termination
sequencing method (Sanger et al., 1977) was conducted
using the T7 sequencing kit (Pharmacia LKB) on ssDNA
templates generated with the helper phage M13K07.
Sequences were analysed using the program package of
the Genetics Computer G r o u p (Devereux et al., 1984).
Sequences of at least 20 templates from independent
clones overlapped partially or completely with no
mismatches. When connected, they formed a covalently
closed circle of 1002 nucleotides, thereafter called
F B N Y V D N A 1 (Fig. 1). Moreover, several fragments of
about 1 kb (from agarose gel analysis) were found to
have 1002 bp when completely sequenced and were
therefore considered to contain the full length of this
circular component.
Sequence analysis of this component revealed three
open reading frames (ORFs) potentially encoding proteins larger than 5 k D a (Fig. 2). One large O R F (ORF1)
in the virion orientation (polarity determination described
below) spans from nucleotides 163 to 996 (278 amino
acids) and codes for a protein of 32.3 kDa. There are two
other smaller O R F s in the complementary orientation
(ORF2 from nucleotides 354 to 160, 65 amino acids,
7.4 k D a ; ORF3 from nucleotides 949 to 719, 77 amino
BgllI
SacI
NruI
HindlII
BarnHI
Fig. 2. Genome organization of the FBNYV DNA 1 component,
containing a putative replicase gene. ORFs are shown in the virion
orientation (ORF1, shaded arrow) or the complementary orientation
(ORF2 and ORF3, open arrows). The stem-loop structure is
represented by a black rectangle. Cleavage sites of selected restriction
enzymes are indicated.
acids, 9.3 kDa). ORF1 is preceded by a T A T A box
(TATAAA) 33 nucleotides upstream from the A T G start
codon and contains a polyadenylation [poly(A)] signal
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(a)
A
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et al., 1993) gave sequence identities of 58.5 %, 47.7 %,
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BBTV-C1 (Hardinget al., 1993)
BBTV-C2 (Yehet al., 1994)
GEMINI (Fariaet al., 1994)
CFDV (Rohdeet al., 1990)
FBNYV
Fig. 3. (a) Putative stem-loop structure of the FBNYV DNA 1
component; (b) nucleotidesequence alignmentof the potential loop
region of several ssDNA viruses. The geminivirus sequence is a
consensus sequence from 15 geminiviruses.X = A, T, C or none;
(TA) = TA, TT or AA.
(AATAAG) which falls 34 nucleotides upstream from
the TGA stop codon (Fig. 1). Although the consensus
sequence (AATAAA) for the poly(A) is also present, it
falls 53 nucleotides further upstream. We chose to
consider the further downstream variant, AATAAG, as
the possible poly(A) signal based on the findings of
Rothnie et al. (1994). They suggested that, in contrast to
poly(A) signals in vertebrate systems, a high degree
of sequence variation of the AATAAA motif can be
tolerated by plants, and that all single base variants of
this consensus sequence were actually recognized and
directed efficient processing at the cauliflower mosaic
virus poly(A) site. Moreover, ORF1 potentially encodes
a protein with replicase activity since it contains the
NTP-binding motif GXGKS [G(GE)GKS] which has
been shown to be present in the replicases of several
viruses (Gorbalenya et al., 1990) and may therefore
encode a protein with replicase activity. Boevink et al.
(1993) showed that the SCSV genome has two distinct,
possibly co-existing or interchangeable components
(SCSV2 and SCSV6) which potentially code for a
replicase. Whether a similar phenomenon exists for
FBNYV isolates is not known yet. Further work aimed
at studying the expression level of these potential ORFs
and their function will be required.
Alignments of the amino acid sequence of the putative
replicase of FBNYV DNA 1 with those of SCSV2,
SCSV6 (Boevink et al., 1993, and personal communication), CFDV (Rohde et al., 1990) and BBTV (Harding
35-7 % and 35.0 %, respectively, when using the program
Align (Lipman & Pearson, 1985). This sequence comparison supports our earlier observations that FBNYV is
more similar to SCSV than to BBTV and CFDV (Katul
et al., 1993). Sequence data for MDV, which appeared to
be serologically closely related to FBNYV (Katul et al.,
1993), have not been reported yet. For ORF2 and ORF3,
the two smaller ORFs, no striking amino acid sequence
similarities to any of the ORFs of BBTV and CFDV
were revealed. Similarly, no significant amino acid or
nucleotide sequence similarities were found in the
GenBank database and hence no obvious function could
be assigned to them.
Further sequence analysis of the FBNYV DNA 1
component revealed a potential stem-loop structure
outside the coding region consisting of a 10 bp stem
(5' CCAAGGCGGG 3' and 5' CCCGCCTTGG 3') and
an 11 nucleotide loop (5' TATAGTATTAC 3') (Fig.
3a). Similar to geminiviruses, the potential stem-loop
structure consists of a GC-rich base-paired stem and an
AT-rich loop (Lazarowitz, 1987). Lazarowitz et al. (1992)
and Fontes et al. (1994) have pointed out that the
stem-loop structure is highly conserved among the
components of each geminivirus and have suggested that
it is necessary for DNA replication and may be part of
the viral replication origin. Partial comparisons of the
noncoding region of FBNYV DNA 1 with that of BBTV
(Harding et al., 1993; Yeh et al., 1994), CFDV (Rohde et
at., 1990) and several geminiviruses (Faria et at., 1994)
confirmed earlier findings that the potential loop structure is a highly conserved region among these viruses
(Fig. 3b). Moreover, the noncoding region was also
found to contain sequence stretches common among the
different genome components of BBTV (Burns et al.,
1993, 1994) and SCSV (Chu et al., 1993; Boevink et al.,
1993, and personal communication). Yeh et al. (1994)
referred to another domain of 12 nucleotides
(5' ATTTAAATTATG 3') which is present within the
coding region of BBTV component 1 (Harding et al.,
1993) and of CFDV DNA (Rohde et al., 1990), as well as
within the noncoding region of BBTV component 2 (Yeh
et al., 1994). This domain was not found in the FBNYV
DNA 1 component. However, long stretches of high
levels of amino acid identity were observed in the large
ORFs of FBNYV DNA 1 and SCSV2, but were chiefly
restricted to their C-terminal halves (data not shown).
To determine the polarity of the virion ssDNA, two
19-mer oligonucleotides (primer 1, 5' AATTAAATATGGCTTGTTC 3'; primer 2, 5' AAACAAATTCAACAATTGA 3') (Fig. 1) derived from the FBNYV DNA 1
were synthesized and used separately for direct
sequencing of the noncloned viral ssDNA. Only primer
2 primed with the viral ssDNA producing a readable
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478
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M
1
2
3
bp
2840
-
-
1159-805--
516 --
264 -Fig. 4. Agarose gel analysis of the PCR products. Primers 1 and 2 were
used in an IC-PCR experiment to amplify a 852 bp fragment from
FBNYV-infected pea tissue (lane 2) and from cloned viral DNA (lane
3), but not from noninoculated pea tissue (lane 1). Lane M contains
phage 2 dsDNA cut with PstI for use as a size marker. The molecular
sizes are indicated.
oligonucleotide sequence which perfectly matched the
sequence obtained from the cloned fragments. Primer 1
produced no signals on the autoradiogram indicating
that it did not prime with the viral ssDNA. This allowed
us to conclude that the orientation of the virion ssDNA
is complementary to primer 2 and that it is present in the
virions as presented in Fig. 1.
Moreover, this primer pair was used in an immunocapture PCR (IC-PCR) experiment (Wetzel et al., 1992)
which was conducted as follows : using the conventional
ELISA buffers, virus particles from plant extracts were
trapped in 1.5 ml tubes coated with 200 lal of antiFBNYV IgG (2 lag/ml). Viral DNA was released by a
proteinase K treatment at 0.5 mg/ml final concentration
in the presence of 1% SDS for 30 min at 37 °C, extracted
with an equal volume of phenol, washed with chloroform
and precipitated twice with ethanol. The final pellet was
dried and redissolved in 50 ~tl 10 mM-Tris-HC1, 0-1 mMEDTA, pH 8-0. For the PCR reaction, 5 lal of this
solution was added to a 45 ~tl reaction mix containing
0.5 U Replitherm polymerase (Biozym) and run for 40
cycles (1.5 min, 95 °C; 1.5 min, 50 °C; 2 min, 72 °C) with
an initial denaturation for 7 min at 95 °C. An aliquot of
the final product (5 ~tl) was analysed in a 1% agarose gel
stained with ethidium bromide. The expected fragment
of 852 bp was amplified fi'om pea samples infected with
FBNYV, but not from noninoculated plants (Fig. 4),
which confirmed that the PCR product was virus related.
A full-length insert of the FBNYV DNA 1 component
isolated by H i n d l I I digest from a plasmid a n d subsequently religated was used as a c o n t r o l a n d p r o d u c e d
a f r a g m e n t of the same size.
O u r studies provided further evidence for earlier
conclusions ( K a t u l et al., 1993) that F B N Y V is a virus
distinct f r o m the other s s D N A viruses described so far.
The g e n o m e o r g a n i z a t i o n described here for the F B N Y V
D N A 1 c o m p o n e n t matches well with the published
work o n BBTV ( H a r d i n g et al., 1993; Burns et aL, 1993,
1994; Yeh et aL, 1994), C F D V (Rohde et al., 1990) a n d
SCSV (Boevink et al., 1993; C h u et al., 1993; Surin et al.,
1993). All these viruses have a covalently closed circular
s s D N A g e n o m e consisting o f several c o m p o n e n t s of
a b o u t 1 k b with a partially conserved n o n c o d i n g region
c o n t a i n i n g a potential s t e m - l o o p structure, a T A T A box
a n d poly(A) signals. T h e y also have a D N A c o m p o n e n t
with one m a j o r O R F potentially coding for a 32.333.5 k D a protein, possibly a replicase, with the N T P b i n d i n g m o t i f G X G K S . This seems to be the expected
general p a t t e r n of the g e n o m e of these viruses a n d a
further c o n f i r m a t i o n for their belonging together in one
new t a x o n o m i c g r o u p o f p l a n t viruses.
We thank Ms Petra Boevink for sharing SCSV sequences prior to
publication. This research was funded by the Gesellschafl ffir
Technische Zusammenarbeit (GTZ).
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(Received 8 August 1994; Accepted 4 October 1994)
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