Journal of General Virology (1992), 73, 1297-1301.
1297
Printed in Great Britain
Characterization of the discontinuities in rice tungro bacilliform
virus DNA
Yiming Bao and Roger Hull*
Department of Virus Research, John Innes Institute, John Innes Centre for Plant Science Research, Colney Lane,
Norwich NR4 7UH, U.K.
The dsDNA of rice tungro bacilliform virus (RTBV)
has two discontinuities, one on each strand, each in a
specific position as found in other pararetroviruses.
The 5' end of discontinuity 1 was mapped to nucleotide
1 of the published RTBV DNA sequence which
suggests that tRNA~ ~t serves as a primer for negative
strand DNA synthesis. This 5' terminus contains up to
two ribonucleotides and the 3' terminus overlaps it by
five to 25 nucleotides. The discontinuity 2 (D2) did not
map to a purine-rich region as has been found in other
similar viruses. Both the 5' and 3' termini of D2 were
heterogeneous in position giving structures varying
from a gap of 10 nucleotides to an overlap of 103
nucleotides.
Rice tungro bacilliform virus (RTBV) is one of the two
viruses in the complex causing rice tungro, the most
severe viral disease of rice in South East Asia (Hibino et
al., 1978). RTBV has a circular dsDNA genome, and
from the sequence of an 8002 bp viral genome clone, Hay
et al. (1991) suggested that it is a plant pararetrovirus
(Hull & Will, 1989; Temin, 1989) similar in its structure
and D N A replication to caulimoviruses (see Covey,
1985; Mason et al., 1987). It is classified with the
badnaviruses, along with Commelina yellow mottle virus
(CoYMV) (Medberry et al., 1990) and cacao swollen
shoot virus (Lot et al., 1991). These plant pararetroviruses have two to three discontinuities located at
specific sites, one in the negative strand and one or more
in the other strand. These discontinuities are believed to
be priming sites for D N A replication (Covey, 1985;
Mason et al., 1987; Medberry et al., 1990). The sequence
around the non-coding negative strand discontinuity,
taken as the zero point on the map of these viral
genomes, has complementarity to the 3' end of tRNAiM~t
whereas the positive strand discontinuities are rich in
purines (Franck et al., 1980; Hull et al., 1986; Richins et
al., 1987; Verver et al., 1987; Medberry et al., 1990; Luo
et al., 1990). The structure of the D N A around the three
discontinuities of cauliflower mosaic virus (CaMV) has
been shown to comprise a fixed 5' terminus with some
ribonucleotides attached. The 3' terminus overlapped the
5' terminus by a variable amount of five to 35 nucleotides
(Richards et al., 1981). In strand separation experiments,
Jones et al. (1991) showed that there are two discontinuities in the RTBV genome, one (D1) in the negative
strand at the zero point of the restriction map and the
other (D2) in the positive strand at around 0.58 map
units. In this paper, we describe the structure of these
two discontinuities.
RTBV (Philippines isolate) was propagated in rice
(Oryza sativa) cv. TN1 after agroinoculation (Dasgupta
et al., 1991). Virus particles and virion D N A were
isolated as described by Jones et al. (1991). Cloned
RTBV D N A pJIIS2 (Hay et al., 1991) was sequenced
using [~-35S]dATP and a Sequenase Kit (United States
Biochemical) as recommended by the manufacturer, or
by a simplified method (Hsiao, 1991). Oligonucleotides
used for DNA sequencing and primer extension experiments were: V36 (5774 GGCTTGTATCCATCC 5760),
V104 (4383 A T A T G A A T G C A A A T G A G G 4366),
V105 (5192 T G C G G C T G A A G A C T A A T C 5175), V285
(7911 A G T C G A C G G A T G A G G T C A 7929), V305 (110
AATTCAAGTTTTTCGTAA 127), V361 (7937 GCACTAGTAGGTAACTAA 7954) and V403 (4111 GACGAAACTTTTGGAATA 4128) (the nucleotide numbers refer to the sequence of Hay et al., 1991) (Fig. 1).
Restriction fragments of virion D N A were recovered
from low melting point agarose, or by the liquid nitrogen
freeze-squeeze method (Gaastra et al., 1984).
The recovered fragments were dephosphorylated and
[y-32p]ATP 5' end-labelled using polynucleotide kinase
as recommended by the manufacturer (Boehringer
Mannheim).
Oligonucleotides V361 and V 104 were used for primer
extension to map the 5' ends of DI and D2. These
primers use the positive and negative strand of RTBV
D N A as the template, respectively, and stop extending
at the discontinuity position. Primer extension was
0001-0711 © 1992 SGM
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1298
Short communication
HinclI
EcoRI
(a)
A
BamHI
÷
C
G
T
1-5'
5'
/
RTBV
8002 bp
5'
I
I~
EcoRI
T
C
G
T
T
G
C
T
C
T<
T
T
T
C
A
A
T
C
C
C
C
C
C
(b)
T
T
C
A
G
C
13
A
G
A
C
T
A
T
G
G<
T* <
A<
A
A
G
A
T
C
C
1-3'A
C
G
T
C
G<
(3<
T<
C
T
T
T
T
G
G
G
G
G
G
A
e
c
(3
.
HinclI
-- 3 ' - T C G T T G C T C T T T T C A A T C C C C C C A C G G A T C T T t A c
Fig. 1. The position of oligonucleotides (V36, V104, V105, V285,
V305, V361 a n d V403) and restriction endonuclease cutting sites used
to map the discontinuities (Q) of RTBV virion DNA. The orientations
of the oligonucleotides and the plus and minus strand of RTBV are
indicated by arrows.
performed as described by Medberry et al. (1990) except
that an excess of primers was used and the virion D N A
was denatured by heating in a boiling water bath for
5 min and then cooling immediately in ice. Plasmid
pJIIS2 was sequenced using the same primer as in primer
extension and the sequencing reactions were run in
parallel to the primer extension samples on 6 ~ polyacrylamide sequencing gels.
In the mapping of the 5' position of D1, three adjacent
stop positions were detected (Fig. 2a), the strongest
being at nucleotide 1 (Hay et al., 1991). The proportion of
each band was estimated by measuring the peak areas
given by densitometer scanning of the autoradiographs
to be 32~, 4 5 ~ and 23~o for bands corresponding to
nucleotides 8002, 1 and 2 respectively. This is similar to
the 5' terminus of discontinuity 1 found in CaMV
(Franck et al., 1980; Richards et al., 1981) and CoYMV
(Medberry et al., 1990) and supports the suggestion that
tRNAiMet serves as the primer for negative strand D N A
synthesis in these plant pararetroviruses (Mason et al.,
1987).
After some initial anomalous observations (see below)
using primer V104, the 5' terminus of D2 was mapped to
six different nucleotides, namely 4263, 4289, 4291, 4303,
4317 and 4318 (Fig. 3a) in the positive strand. The result
was of interest since the multiple nature of the 5'
3'
+
t ..............
5'
ggAtCTTTACCATAGTCT-5'
5"-AGCAACGAGAAAAGTTAGGGGGGTGCCTAGAAATGGTATCAGA-3'
7970
i0
Fig. 2. Mapping the 5' and 3' ends of RTBV D1. (a) Primer extension
to map the 5' end of virion D N A using oligonucleotide V361 (lane 1-5').
The other four lanes are from dideoxynucleotide sequencing on cloned
R T B V DNA using V361 as primer. To the right is the sequence with
the positions of the extension stops marked by arrowheads, the
strongest being also asterisked. (b) Mapping of the 3' ends of virion
D N A by comparing the migration of end-labelled EcoRI fragment
(lane 1-3') with a sequencing gel. The position of the 3' ends are
indicated on the sequence as in (a). (c) The structure of D1. The
positions on the sequence of the first and last nucleotides are indicated.
The lower case letters indicate the faint bands in lanes 1-5' and 1-3".
terminus differs from that found in CaMV and CoYMV,
and there was no purine-rich region in this part of the
RTBV sequence.
It has been shown that the 5' end at the discontinuities
of CaMV virion D N A possess RNA tracts (Richards et
al., 1981). To test for RNA tracts in RTBV DNA, virion
D N A was treated with 0.2 M-NaOH and incubated at
65 °C for 15 min before primer extension. There was no
difference between the use of alkali and denaturation by
the heating and cooling method (data not shown). This
suggested that there were no extensive RNA tracts at the
5' terminus of D2 in RTBV virion DNA. The situation
with regard to D1 is discussed later.
To determine the 3' termini of D1 and D2, RTBV
D N A was digested with EcoRI and HinclI respectively
and DNA fragments of nucleotides 2134 to 123 (5991 bp
for D1) and 4108 to 7912 (3804 bp for D2) were recovered
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Short communication
(a)
T
G
C
C
C
T<
T
A
T
A
T
G
G
G
T
T
G
T
C
G
G
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A
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T
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G
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C<
C
T
O
x_g
(c)
+
3 " -ACTCCTTAAAACAATATCCAAAGTCCTTACAAACTTTTATTACCGCCCGCA
3" t . . . . . . . . . . . . .
a .......... AAA-TTt t a ...... c .....
5" - T G A G G A A T T T T G T T A T A G G T T T C A G G A A T G T T T G A A A A T A A T G G C G G G C G T
4370
4320
GgT .............
G ........... a-t .........................
.............
CGCGTTTGAGACTAAATTCAAGTACAGACCGACAACCCATATAAGGG
CCACCATGCTTTCGCGCAAACTCTGATTTAAGTTCATGTCTGGCTGTTGGGTATATTCCC-
4319
A
5"
- 5"
3 '
4260
Fig. 3. Mappingthe 5' and 3' endsof RTBVD2 as in Fig. 2. (a) Primer
extension to map the 5' end using oligonucleotideV104 (lane 2-5').
(b) End-labelledHinclI fragment(lane 2-3') used to map the 3' ends.
(c) The structureof D2.
from agarose gels. These fragments were dephosphorylated and 5' end-labelled in the presence of 25 ~ DMSO
according to Franck et al. (1980). The samples were
incubated in a boiling water bath for 2 min just before
loading onto a 6 ~ acrylamide gel. To determine the size
of the end-labelled fragments, pJIIS2 was sequenced
using oligonucleotides V305 and V403 as primers. These
primers were designed to hybridize to the 5' end of the
restriction fragment containing the 3' end of each
discontinuity.
1299
The 3' termini of D1 and D2 were mapped to
nucleotides 7980, 7995 to 7998 in the negative strand and
4306, 4325, 4332 to 4336, 4338 to 4340, 4351 and 4365 in
the positive strand respectively (Fig. 2b and 3b). (There
might be one nucleotide change in the above data
because the fragments from RTBV were end-labelled
whereas the primers for the sequencing ladder were not.)
This showed an overlap of five to 25 nucleotides between
the 5' and 3' ends of D1. Thus, the structure of D1 closely
resembles that of the negative strand discontinuity (G 1)
of CaMV (Richards et al., 1981). At D2 the separation of
the 5' and 3' ends varied from a gap of 10 nucleotides to
an overlap of 103 nucleotides. The structure differs from
the positive strand priming site of CaMV in that there is
a gap in, at least, a portion of the molecules. The
pararetrovirus hepatitis B virus (HBV) has a gap of up to
700 nucleotides in the positive strand (Delius et al.,
1983).
Initial experiments on priming crude virion D N A
templates with the oligonucleotide V36 indicated that
the 5' end of D2 mapped to nucleotides 5681 to 5683,
which is close to the purine-rich region identified by Hay
et al. (1991). However, no 3' terminus of RTBV D N A
could be found in this region. When further oligonucleotides hybridizing to different positions were used for
primer extension, they all gave extension stop fragments
corresponding to 91 to 93 nucleotides (Fig. 4). Even when
no primer was added, the 91 to 93 nucleotide extension
stop fragments were found. This suggested that the crude
DNA preparation contained an endogenous primer. To
test this possibility, virion D N A was further purified by
agarose gel electrophoresis. It did not prove possible to
recover intact virion D N A from either low melting point
agarose gel or ordinary agarose by the liquid nitrogen
freeze-squeeze method. Virion DNA was therefore
linearized by BamHI digestion and recovered from the
gel by the liquid nitrogen freeze-squeeze method. When
this D N A was used as the template with primers V36,
V104 and without primers, the 91 to 93 nucleotide
fragments were not found (data not shown) indicating
that there was an endogenous primer. If the crude D N A
preparation was treated by alkali, the non-specific
fragment was one to six nucleotides shorter indicating
that the endogenous primer contained terminal ribonucleotides (data not shown). The nature of this endogenous primer has yet to be determined.
As noted above, D1 has the same basic structure as GI
of CaMV. The majority of the 5' ends were nucleotide 1
of the sequence which corresponds to the 3' nucleotide of
the tRNA primer, a situation similar to that in CoYMV.
If the molecule which actually primes the synthesis of the
negative strand was the authentic tRNA with the 3'terminal CCA, we would expect the 5' nucleotide of the
negative strand D N A to be at the - 1 or 8002 position.
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1300
Short communication
A
C G
T
1
2
3
4
5
T* <
A*<
A
A
G
A
T
C
C
G
T
G
G
G
G
G
G
A
T
T
G
A
A
A
A
G
A
Fig. 4. Primer extension using a crude virion DNA preparation as the
template and different oligonucleotides as the primer. Lanes I to 5
represent oligonucleotides V285, V36, V104, V105 and no oligonucleotide added respectively.The nucleotide coordinate of the asterisked T
is 1 in the RTBV sequence.
Only a small portion of negative strand molecules
appeared to prime at this site. There are two possible
answers to this problem. Either the 3' end of t R N A was
incomplete or a specific R N a s e H cleavage leaves the
terminal r C A from the t R N A attached to the D N A
strand as suggested by Whitcomb et al. (1990) for human
immunodeficiency virus type 1. C C A at the 3' terminus
of eukaryotic t R N A is added as individual nucleotides
by t R N A nucleotidyltransferase to t R N A precursor
(Deutscher, 1983). Thus, there might be some t R N A
molecules having C C or even C at their 3" end, and such
t R N A might be selected to serve as primers for D N A
synthesis. When alkaline treatment before primer
extension was performed, the percentages for primer
extension stop bands corresponding to nucleotides 8002,
1 and 2 are 2 2 ~ , 50~o and 2 8 ~ respectively. Thus, there
are some changes in proportions of the three stop sites
when compared with heat denaturation, which indicates
that some o f the 5' termini contained one or two
ribonucleotides. This suggests that both possibilities
about the 3' end of t R N A existed. The 3' end of D1
overlaps the 5' end indicating that the reverse transcriptase strand displaces the 5' end by a certain amount.
The structure of D2 displays various unusual features
when compared with the positive strand p r i m i n g sites of
other retroviruses and pararetroviruses. With the retroviruses and the pararetroviruses, the priming site is
specific and, except for HBV, is associated with a purinerich region (Mason et al., 1987; Franck et al., 1980; Hull
et al., 1986; Richins et al., 1987; Verver et al., 1987;
Medberry et al., 1990; Luo et al., 1990). The 5' end of
R T B V D2 ranged across 55 nucleotides though some
were preferred over others. There was no obvious purinerich region, and the 5' end did not fine m a p to a purinerich region which had been previously identified (Hay et
al., 1991). It is generally considered that the priming
molecule for positive strand synthesis is R N A remaining
after digestion of the reverse transcription template by
viral R N a s e H (Varmus et al., 1982; Mason et al., 1987).
Some specificity between the individual viral enzymes
and the priming sequence has been suggested (Luo et al.,
1990). I f this is so, the specificity of R T B V R N a s e H m a y
differ from other retroviral and plant pararetroviral
enzymes. Comparison of polypeptide sequences encoding the core consensus sequence of R N a s e H of RTBV
and other plant pararetroviruses and retroviruses (Hay et
al., 1991) revealed four amino acids in R T B V which
differed from those common to similar viruses. This may
account for R T B V R N a s e H having a different specific
activity. Another possibility is that R T B V positive
strand synthesis is not primed by R N A fragments left
behind by R N a s e H, but by host R N A sequences.
We thank Mr M. Harvey for synthesizing oligonucleotides and for
preparing Fig. 1, Mr A. Lucy for synthesizing oligonucleotides, and
Professor J. W. Davies, Drs J. Hay, I. Dasgupta and G. Dahal, Mr G.
Lee, S. Zhang and Z. Fan for helpful comments during the preparation
of this manuscript. Y.B. was funded from a grant from the Rockefeller
Foundation. Tungro is held under MAFF Plant Health licence no.
PHF48A/116.
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(Received 29 October 1991; Accepted 2 January 1992)
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