J. gen. Virol. (1989), 70, 963468.
Printed in Great Britain
963
Key words: TRV/infectious transcripts/tobravirus
Infectious RNA Produced by in vitro Transcription of a Full-length
Tobacco Rattle Virus RNA-I cDNA
By W I L L I A M D. O. H A M I L T O N *
AND D A V I D C. B A U L C O M B E
A F R C Institute o f Plant Science Research (Cambridge Laboratory), Marls Lane, Trumpington,
Cambridge CB2 2JB, U.K.
(Accepted 19 December 1988)
SUMMARY
A near full-length c D N A clone of tobacco rattle virus (TRV) RNA-1 was
constructed by joining together nine overlapping c D N A clones using restriction sites in
the regions of overlap. At the 5' end of the cDNA, oligonucleotide mutagenesis was
used to insert nucleotides which were missing from the cDNA placing the construct
immediately on the 3' side of the Pr promoter of phage ). to create pTR7116. Extraneous
non-viral nucleotides had been deleted from the 3' end of the TRV c D N A to create a
unique Sinai site in pTR7116 in which the nucleotides C C C were provided by the viral
cDNA, and G G G by the vector. As a result, pTR7116 could be linearized with SmaI
and transcribed in vitro to yield R N A molecules with 5' and 3' termini identical to
those of natural TRV RNA-1. These transcripts were infectious when inoculated onto
leaves of tobacco and produced the subgenomic RNA species typical of an infection
with TRV RNA-1.
The tobravirus, tobacco rattle virus (TRV) has a bipartite RNA genome but is unusual in that
it can cause infections in the absence of a gene for the capsid protein which is carried on the
smaller component (RNA-2) of the genome (Harrison & Robinson, 1986). These infections,
called NM-type infections, occur when RNA-2 is lost; those caused by the complete TRV
genome are called M-type infections.
NM infections cause symptoms on the host plant which may be more severe than those of the
corresponding M-type infection and involve both replication of the viral R N A and its slow
spread from cell to cell (Cadman & Harrison, 1959; Harrison & Robinson, 1978). In the
anticipation that features of NM infections will be common to those of infections caused by
other plant RNA viruses in their non-encapsidated phase, we have initiated a detailed molecular
analysis of the larger RNA (RNA-1) of TRV to understand its ability to cause disease in plants.
The analysis so far, based on the analysis of interviral homologies in the nucleotide sequence
of RNA-1, has suggested particular functional significance for several coding and non-coding
regions. For example, in a gene encoding a 194K protein there are three domains that show
similarity to plant viruses outside the tobravirus group (Hamilton et al., 1987). Two of these
domains have been assigned functions related to R N A replicase activity: one domain is thought
to be involved in nucleotide binding and the other in R N A recognition. Both of these domains
are found in genes of a diverse group of eukaryotic viruses, sometimes referred to as the Sindbis
group (Goldbach, 1986).
There is also similarity between the 29K protein encoded by TRV RNA-1 and the 30K
protein of tobacco mosaic virus (TMV) (Boccara et al., 1986). This region of similarity in the
29K protein includes a domain which, in the TMV 30K protein, is known to be involved with
the cell-to-cell movement of the virus (Ohno et al., 1983; Zimmern & Hunter, 1983).
Other features of the TRV RNA-1 which have been revealed by nucleotide sequence analysis
and which may have functional significance include a tRNA-like structure at the 3' end (Van
0000-8683 O 1989 SGM
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964
(a)
II
II
I
I
II I I
I
II
II
¥
II
]
25B----I
1 kb
17A
4A
'!
I
I
t
I
I
I 13A
--t
543B
1
31B ----~
25A
t----
24B
pTR18
pTR713
]--- ---q
I
pTR722 -----1
pTR741
I
pTR7621
I
I
]_____---q
:pTR752
?-_-
[ _ _
I
1II
II
(b)
pBPM1 + I
II
...... ~ ' 1 - - 1
......
pTRMA61
pTRP]
I~---
41
21
...... ~ \ \ \ \ \ ]
pTRP22
pTR771
F----- pTRP33
pTR785
L......
-----{
5i
~\ \ \ \ \ \ N
6i~
N~q,\\\\\\l
pTR791
H
7 ~
+II
~pTR7102
8 ~~
pTR7116
+ III
,,-¢~\\\\\~
134K
194K
V / / / / / / / / / / / / I / / / / / ~ I
TRV RNA-1
16K
V / / i F-7-1
29K
Fig. 1. Construction of the full-length TRV RNA-1 clone. (a) The eight overlapping cDNAs are shown
relative to one another together with a map of the restriction enzyme sites used in the construction
(excluding polylinker sites). The cDNAs were cloned into the PstI site of either pUC 19 or pBR322 after
G-C tailing. All intermediate clones leading to the formation of pTR7621 involved the ligation of at
least two fragments with pUC19. The first clone, pTR18, contained the KpnI/XhoI fragment from
cDNA 25B and the XholIPstI (polylinker) fragment from 13A. The cDNA 543B was found to extend
the sequence further towards the viral 3' end and was therefore added to pTRI8. This resulted in clone
pTR752 which contained the KpnI/AvalI fragment from pTRI8 and the AvalI/PstI (polylinker)
fragment from 543B. At the 5' end, pTR713 was formed by ligation of a HindlII/AccI fragment from
31B and an AccI/SstI fragment from 4A. A partial AccI digest was used to isolate both fragments.
PTR722 was produced by ligation of these fragments: PstI (polylinker)/BglI] fragment from 24B, a
BgllI/AccI (partial) fragment from 25A and an AccI (partial)/SstI fragment from pTR713, pTR741
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965
Belkum et al., 1987) and repeated sequences at the 5' end, which are also present in R N A - 2
(Hamilton et al., 1987).
A functional assay is required to test the significance of the various features of T R V RNA-1.
Recently, two approaches have proved useful as functional assays of plant virus genomes. One
involves the expression of viral genes from the genome of a transgenic plant to complement
mutations in the viral genome (Deom et al., 1987). The second approach involves the creation of
mutations in the viral genome at the D N A level. The mutated viral sequence is then inoculated
onto plants either as D N A or after in vitro transcription for viruses with R N A genomes (Meshi et
al., t987; Saito et al., 1987; K n o r r & Dawson, 1988; Meshi et al., 1988). We are following the
second approach and in this communication we describe the construction of a full-length clone
of T R V RNA-1 and its transcription in vitro into infectious RNA.
The promoter used for in vitro transcription of the T R V RNA-1 c D N A was derived from
plasmid pPM1 (Ahlquist & Janda, 1984). This promoter, rather than the T3, T7 or SP6
promoters, was selected because the final constructs would allow the transcription of an R N A
molecule with a 5' A residue identical to that of natural T R V RNA-1. Transcripts obtained
using the other promoters would contain a 5' G residue derived from the promoter sequence
itself which extends into the transcribed region.
Attempts to synthesize full length c D N A of T R V RNA-1 were not successful, so the fulllength c D N A was reconstructed from a series of shorter overlapping clones. These clones were
derived from purified T R V (strain SYM) R N A as described previously (Boccara et al., 1986)
and have been sequenced over the entire length of the viral R N A (Hamilton et al., 1987).
Construction of the full-length c D N A is shown in Fig. 1. Firstly isolated fragments were ligated
in a stepwise m a n n e r to form a near full-length c D N A (pTR7621), a clone which was missing 45
bases at the 5' terminus and 96 at the 3' terminus compared with the viral sequence. Then a
contained the pTR722 SphI (polylinker)/SstI and 17A Sstl/KpnI fragments. Finally pTR7621 was
constructed using the pTR741 SphI (polylinker)/Kpnl and pTR752 KpnI/HindlII (polylinker)
fragments. (b) (1) Clone pBPM1 was constructed by subcloning into Bluescribe+ a PstI/EcoRI
fragment containing the Pmpromoter isolated from plasmid pPM 1 (Ahlquist & Janda, 1984).The 5"446
base fragment from pTR7621, resulting from Sinai (polylinker)/EcoRI digestion, was cloned into pBP1
to produce pTRP1. (2) pTRP1 was mutagenized using an 89-mer nucleotide (A. Northrop,
Microchemical Facility, I.A.P. Babraham, U.K.) to remove the G-C tail and polylinker sequences and
insert the missing 5' bases determined previously (Hamilton et al., 1987). The clone was transformed
into JM 101 which was subsequently superinfected with phage VCS-M13 (Stratagene) and ssDNA was
prepared. The oligonucleotide contained 23 bases partly complementary to the P~ promoter. The
variant nucleotides converted the three 3'-terminal bases (part of the introduced SmaI site) back to the
original 2 Pr sequence. The oligonucleotide also contained 21 bases complementary to the 5' end of
cDNA 24B. The oligonucleotidewas treated with kinase and together with normal M13 forward 17-mer
sequencing primer was annealed to the ssDNA template (Zoller & Smith, 1987). Primer extension was
carried out using both ligase and Klenow enzymes for approximately 5 h at 16 °C. The DNA was then
transformed into JM101 and resultant colonies screened by colony hybridization using the 89-mer as a
probe. Of the colonies, 16~ hybridized stably to the 89-mer and one clone (pTRP22) was confirmed by
sequencing. (3) A fragment from pTRP22 containing the Pr promoter and the complete 5' terminus of
TRV RNA-1 was cloned into pUCI9 to remove various flanking restriction sites. This was carried out
by digesting pTRP22 with PstI, filling in with Klenow polymerase and releasing the fragment with
EcoRI. The fragment was then cloned between the SmaI and EcoRI sites of pUC19 to create pTRP33.
(4) The cDNA pTRMA61 extends the viral sequences contained in pTR7116 to the genuine viral 3'
end. Deletion of the poly(A) tail and creation of the Smal site in pTRMA61 was carried out by Bal 31
deletion from the polylinker SstI site. After Bal 31 digestion the insert was released with PstI and cloned
into pUC19. Colonies were screened for the presence of a SmaI site and positive ones further screened
by sequencing. (5) The insert from one clone was isolated using KpnI/HindlII digestion and cloned into
Bluescript SK +. (6) The insert fragment from pTRP33 was released by EcoRI/BamHI digestion and
cloned into pTR785 to produce pTR791. (7) The fragment (II) from pTR7621 was then cloned into
pTR791 to form pTR7102. (8) The final step involved cloning the pTR7621 SalI/StuI fragment (III)
into pTR7102 to form the complete construct pTR7116. Sequences are indicated as follows: open
boxes, TRV sequence; hatched boxes, Pm/Pr promoter sequence; straight lines, pUC19; dotted lines,
Bluescribe+ ; sawtooth lines, Bluescript SK +. Heavy lines at box ends indicate G-C tails. The gene
organization of TRV RNA-1 is shown at the bottom.
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966
Short communication
plasmid was constructed which contained the Pm promoter derived from the plasmid pPM1
(Ahlquist & Janda, 1984) and the 5' 446 bases of pTR7621. A synthetic oligonucleotide was used
to insert the missing 5' bases, delete the non-viral sequence between the promoter and the TRV
sequence, and change the three 3' bases of the Pm promoter back to the original 2 Pr promoter
sequence (see Fig. 1 legend). A c D N A containing the viral T-terminal sequences was treated
with Bal 31 to remove the homopolymer tail acquired during the cloning procedure. The 3'terminal vira~ sequence ends in -CCCoH (Minson & Darby, 1973). Deletion of the homopolymer
tail and subsequent ligation into a SmaI-cut vector created a unique SmaI site. Use of Sinai
would thus allow the production of transcripts with 3' termini identical to the viral RNA. The
completed 5' and 3' ends were joined into one construct and the remaining TRV RNA-1 c D N A
sequence inserted. The last part required a two-step ligation as it was not possible to insert the
remaining part of the c D N A as one fragment. All junction points were verified by sequencing
subclones containing the junction regions as previously described (Hamilton et al., 1987).
The final outcome of this construction was a plasmid, pTR7116, which could be transcribed in
vitro to produce R N A molecules which are identical at both the 5' and 3' termini to the natural
viral RNA-1.
Transcription reactions were based on those described by Ahlquist & Janda (1984). R N A
polymerase was obtained from four independent companies; reactions using lots from New
England Biolabs and Boehringer Mannheim contained full-length transcripts, although it also
became apparent that different batches from these companies gave different amounts. As the
viral R N A is most probably capped (AbouHaidar & Hirth, 1977), the cap analogue
mTG(5')ppp(5')A (Pharmacia) was also introduced into the transcription reaction. The standard
reaction contained 2 ~tg of SmaI-digested pTR7116, 25 mM-Tris-HCl (pH 8.0), 5 mM-MgC12,
150 mM-NaCI, 1 mM-dithiothreitol, 200 ~tM-rGTP, -rCTP and -rUTP, 50 ~tM-rATP, 0-5 mM of
the cap analogue, 30 units RNAsin (Anglian Biotech.) and 2 units RNA polymerase in a total
volume of 20 ktl. The reaction was carried out at 37 °C for 30 min. Two ~tl of 500 ~tM-rATP was
then added and the reaciion continued at 37 °C for a further 30 rain. Thirty ~tl of 50 mM-EDTA
was added to stop the reaction. Approximately 1.2 ktg of R N A transcripts was produced from
each reaction and estimations based on Northern blot analysis and densitometer scans of
autoradiograms indicated that between 40 and 5 0 ~ of them were full-length transcripts.
Young Nicotiana tabacum vat. Samsun N N plants were inoculated at the four to five leaf stage
by manually inoculating the transcription reactions, diluted to 20 raM-sodium phosphate
(pH 7.0) and mixed with bentonite (5 mg/ml), onto carborundum-dusted leaves (Lister &
Hadidi, 1971) which were then rinsed with water not more than 15 min post-inoculation. Plants
were scored for visual symptoms and total R N A was extracted using the proteinase K-phenol
method (Baulcombe & Buffard, 1983) and analysed by Northern blotting.
The Northern blots of viral R N A extracted from inoculated leaves showed that there were no
detectable differences at the R N A level between natural NM infections and the infections
produced by pTR7116 RNA (Fig. 2). The genomic RNA-1 was detected as were the two
subgenomic R N A species, la and lb, which encode the 29K and 16K proteins, respectively
(Robinson et al., 1983; Boccara et aL, 1986). Much less RNA- 1, - l a and - 1b were found in leaves
inoculated with transcripts produced in the absence of the cap analogue. No viral R N A was
found in leaves inoculated with linearized, non-transcribed pTR7116 and these leaves were
indistinguishable from healthy leaves. Leaves inoculated with uncapped transcripts showed
very small lesions 3 days after inoculation which became large necrotic areas after a further 3
days. However, consistent with the Northern blot analysis, inoculation with the capped
transcripts produced more and larger necrotic lesions after 3 days. After 6 days the leaves died.
On some plants, the necrotic symptoms moved down the petiole of the inoculated leaves and
could be seen eventually as a necrotic band extending up the main stem, ultimately causing the
whole plant to die. This spread accounts for the detection of TRV R N A in the 'systemically'
infected leaves of plants (Fig. 2, lane 7). The lesions formed by inoculation with uncapped
transcripts did not spread to the same extent (Fig. 2, lane 6). These symptoms were similar to
those of natural TRV NM-type infections in the production of necrotic lesions and the ability of
the infection to spread from cell to cell (Harrison & Robinson, 1981). However, inocula of
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967
S h o r t communication
1
2
3
4
5
6
7
8
9
2a
la
lb
Fig. 2. Northern blot of RNA from pTR7116-infected leaves. Lane t, an aliquot of capped RNA
transcripts from pTR7116 that was used for inoculation (equivalent to 0.1 Ixg of the template DNA).
Other lanes represent RNA prepared from healthy, buffer-inoculated plants (lane 2), from leaves
inoculated (lane 3) or systemically infected (lane 4) with linearized plasmid, from leaves inoculated with
transcripts (lane 5) or systemically infected by transcripts produced without (lane 6) or with (lane 7) cap
analogue, and from NM-infected (lane 8) and M-infected (lane 9) plants. The genomic TRV RNA-1 (1)
and TRV RNA-2 (2) are indicated together with the associated subgenomic species (la, lb and 2a). In
addition the full-length pTRT116 transport is arrowed. Approximately 7 ~tg of total RNA was run in
each lane and the blot was probed with a randomly primed BstEII/SmaI fragment from pTR7116,
corresponding to the Y-terminal 1.44 kb ofTRV RNA-1. RNA was prepared 14 days post-inoculation.
transcripts induced lesions that were more uniform and more intensely necrotic than leaves
inoculated with total RNA prepared from NM (strain SYM)-infected plants. Induction of these
slightly different symptoms may have resulted from one of the cDNAs used in the construction
of pTR7116 representing a natural variant RNA species or from sequence differences
introduced during the cloning procedure. On N. clevelandii the symptoms induced by the
pTR7116 transcripts and NM RNA were very similar except that the pTR7116 infection was
slightly more vigorous.
We have described in this communication the construction of a full-length clone of TRV
RNA-1 from multiple cDNA fragments and have demonstrated the infectivity of in vitro derived
transcripts. This validates the biological activity of the previously reported sequence (Hamilton
et al., 1987). Clone pTR7116 will therefore be extremely useful in the analysis of the basic
characteristics of NM-type infections and will form the basis for future mutagenesis and gene
replacement experiments.
This work was supported by the Rockefeller Foundation.
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