Download A single silent substitution in the genome of Apple stem grooving

Journal of General Virology (2003), 84, 2579–2583
Short
Communication
DOI 10.1099/vir.0.19179-0
A single silent substitution in the genome of Apple
stem grooving virus causes symptom attenuation
Hisae Hirata, Xiaoyun Lu, Yasuyuki Yamaji, Satoshi Kagiwada,
Masashi Ugaki and Shigetou Namba
Laboratory of Bioresource Technology, Graduate School of Frontier Sciences, The University of
Tokyo, 202 Bioscience Bldg, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8562, Japan
Correspondence
Shigetou Namba
[email protected]
Received 21 February 2003
Accepted 29 April 2003
Among randomly mutagenized clones derived from an infectious cDNA copy of genomic RNA of
Apple stem grooving virus (ASGV), we previously identified a clone, pRM21, whose in vitro
transcript (ASGV-RM21) does not induce any symptoms characteristic of the original (wild-type)
cDNA clone (ASGV-wt) in several host plants. Interestingly, ASGV-RM21 has only a single,
translationally silent nucleotide substitution, U to C, at nucleotide 4646 of the viral genome
within open reading frame (ORF) 1. Here, we characterize and verify this unprecedented
silent-mutation-induced attenuation of symptoms in infected plants. Northern and Western blot
analyses showed that less ASGV-RM21 accumulates in host plants than ASGV-wt. In addition, two
more silent substitutions, U to A and U to G, constructed by site-directed mutagenesis at the
same nucleotide (4646), also induced attenuated symptoms. This is the first report that a single
silent substitution attenuates virus-infection symptoms and implicates a novel determinant of
disease symptom severity.
Apple stem grooving virus (ASGV) is the type species of
the genus Capillovirus, a group of plant viruses with a
monopartite genome consisting of a 6?5 kb, single-stranded,
plus-sense RNA that is 59-capped and 39-polyadenylated.
ASGV RNA contains two overlapping open reading
frames: ORF1 (6?3 kb) and ORF2 (1?0 kb), which encode
proteins of molecular mass 241 and 36 kDa, respectively
(Yoshikawa et al., 1992; Ohira et al., 1995) (Fig. 1). The
larger ORF1 encodes an apparently chimeric polyprotein
containing at least two conserved regions: the replicase
(Rep) region contains several domains (methyltransferase,
papain-like protease, NTP-binding helicase and RNAdependent RNA polymerase domains) characteristic of
viral Rep, and the coat protein (CP) region is located at
the C-terminal end and contains motifs typical of CP
(Yoshikawa et al., 1992; Ohira et al., 1994, 1995). ORF2
encodes a protein with conserved motifs for both movement proteins (MP) and viral proteases (Yoshikawa et al.,
1992; Ohira et al., 1995). The expression strategies of these
ORFs are not yet well understood. By using an antiserum
against purified ASGV particles, the expected 241 kDa
polyprotein for ORF1 was not detected in a plant infected
with ASGV; only a small protein thought to be the CP was
detected (Yoshikawa & Takahashi, 1992). These results
suggest that CP is expressed from subgenomic RNA rather
than polyprotein processing of the 241 kDa protein. The
mechanism of expression of ORF1 thus remains to be
elucidated. Construction of a full-length cDNA clone,
pITCL, of ASGV lily strain (formerly Citrus tatter leaf virus
lily strain: Nishio et al., 1989; Yoshikawa et al., 1993; Ohira
0001-9179 G 2003 SGM
et al., 1995) that produces infectious viral RNA transcripts
(ASGV-wt) in vitro has been described (Ohira et al., 1995),
allowing mutagenesis of the ASGV genome.
We randomly mutagenized pITCL using a DNA repairdeficient mutator Escherichia coli strain (Greener et al.,
Fig. 1. Gene organization of ASGV genomic RNA. Open
boxes represent ORFs. Encoded proteins are indicated as follows: Rep, replicase; CP, coat protein. The exact coding
regions for Rep and CP are not known. Shaded boxes represent conserved domains for replicase: Mt, methyltransferase
domain; P-Pro, papain-like protease domain; Hel, NTP-binding
helicase domain; Pol, RNA-dependent RNA polymerase domain.
Some of the genomic RNA is shown and a triplet codon for
glycine is underlined. The third letter U is nucleotide 4646,
which was mutagenized into three other possible nucleotides
(shown beneath) to produce translationally silent mutations.
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H. Hirata and others
1997). An in vitro transcript (ASGV-RM21) of one of the
resultant clones, pRM21, was found to induce almost no
symptoms in inoculated plants (Chenopodium quinoa) and
unexpectedly had only one silent nucleotide substitution
within ORF1 (Lu et al., 2001). The silent mutation was U
to C (T to C for cDNA) at nucleotide 4646 of the ASGV
genome, which is located downstream from the RNAdependent RNA polymerase domain of the replicase region
within ORF1 (Fig. 1). Here, we extensively analyse this
clone, pRM21, and several site-directed mutagenized clones.
Infectivity assays of each ASGV cDNA clone were performed as follows. RNA transcribed in vitro was immediately used to inoculate C. quinoa seedlings at the five- to
ten-leaf stage after adjusting the concentration of the
transcribed RNA to 1 mg ml21, as described by Ohira et al.
(1995). Plants were tested for virus infection by RT-PCR
with an AMV RNA PCR kit (Takara) using total RNA
extracted by Isogen (Nippon Gene) and ASGV-specific
primers. The amplified cDNA of infecting viruses was
detected by agarose gel electrophoresis and sequenced with
a DNA sequencer following the manufacturer’s protocol
(Applied Biosystems). RNA transcribed in vitro from
pITCL (ASGV-wt) induced symptoms characteristic of
ASGV. These consist of severe chlorotic and necrotic local
lesions on inoculated C. quinoa leaves, seen about 1 week
post-inoculation (p.i.) (Fig. 2a) and chlorotic mottling and
malformation on upper leaves (Fig. 2b). In contrast, ASGVRM21, which has a silent nucleotide substitution within
ORF1, usually induced no symptoms (data not shown) or
occasionally very mild symptoms, with only a few, small
chlorotic spots on inoculated leaves (Fig. 2c) and no symptoms on upper leaves (Fig. 2d). Dwarfing was observed
in the plants infected with ASGV-wt, but no dwarfing
occurred in those infected with ASGV-RM21 (Fig. 2e).
RT-PCR analysis confirmed the presence of ASGV-RM21
progeny in the asymptomatic upper leaves (data not shown).
The attenuated symptoms caused by ASGV-RM21 were
observed also in additionally tested plants including
Nicotiana glutinosa and Vigna sesquipedalis cv. KurodaneSanjaku. In N. glutinosa plants, ASGV-wt induced mosaic,
chlorotic spots or faint ring spots with etched surface on
the systemically infected leaves (Fig. 2f), while ASGV-RM21
infected asymptomatically (Fig. 2g). In V. sesquipedalis,
ASGV-wt produced necrotic spots on the upper leaves
(Fig. 2h), but ASGV-RM21 showed no infectivity (Fig. 2i).
The stability of the ASGV-RM21 mutation was tested after
four serial passages in C. quinoa plants using crude leaf
extracts as an inoculum. Inoculated plants invariably
produced attenuated symptoms identical to those induced
by the original ASGV-RM21 (data not shown). The complete nucleotide sequences of the progeny viruses were
determined using 26 internal primers. No nucleotide
alteration during these passages was detected, indicating
that the single nucleotide mutation of ASGV-RM21 is
stable. In order to confirm that the single U to C substitution at nucleotide 4646 is responsible for the attenuated
symptoms with ASGV-RM21, the C at nucleotide 4646 of
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pRM21 was mutagenized back to the wild-type T nucleotide using a QuikChange site-directed mutagenesis kit
(Stratagene). As expected, transcripts of the resultant
construct produced identical symptoms to ASGV-wt (data
not shown). Similarly, when the T at nucleotide 4646 of
pITCL was mutagenized into C, transcripts of the resultant
construct produced no symptoms, as in the case of pRM21
(data not shown).
To find whether the attenuated symptoms with ASGVRM21 were accompanied by an altered amount of virus,
the accumulation of viral CP and RNA was analysed by
Western and Northern blot analyses, respectively, in plants
inoculated with ASGV-wt or -RM21. Fresh plant tissue
(0?5 g) of either inoculated or upper leaves was collected
from each plant, and the amounts of ASGV CP were
compared by SDS-polyacrylamide gel electrophoresis and
Western blot analysis (Sambrook & Russell, 2001a). The
blotted CP was detected using anti-ASGV antibody and an
ECF Western Blotting Reagent Pack (rabbit) (Amersham).
In both inoculated and upper leaves, less of the viral CP
expressed by ASGV-RM21 progeny accumulated as compared with ASGV-wt progeny (Fig. 3a). Northern blot
analysis was performed by fractionating total RNA extracted
from infected plants, as described by Verwoerd et al. (1989),
in 1 % agarose/formaldehyde gels (Sambrook & Russell,
2001b). Blotted viral RNA was detected with an alkaline
phosphatase-labelled DNA probe (cDNA of ORF2 region)
prepared by PCR and with a chemiluminescent ECL
substrate following the manufacturer’s protocol (Amersham)
and analysed by LAS 1000 (Fujifilm) and Image Gauge
software version 3.45 (Fujifilm). ASGV-RM21 progenies produced less 6?5 kb genomic RNA than ASGV-wt progenies
(Fig. 3b). In addition to the 6?5 kb genomic RNA, 2?0 and
1?0 kb bands were also detected. Using probes that covered
the entire ORF2 or only the 59 half of ORF2, we concluded
that the 2?0 and 1?0 kb bands were subgenomic RNA for
ORF2 and CP, respectively (data not shown). This finding
is in agreement with Magome et al. (1997), who reported
2?0 and 1?0 kbp dsRNA species in ASGV-infected plants.
To determine the effect of the nucleotide species at
nucleotide 4646 in the ASGV genome, the T at nucleotide
4646 of the original pITCL was mutagenized into two
other possible nucleotides, A and G, and the pathogenicity
of the resultant constructs (pT4646A and pT4646G, respectively) was analysed. The transcripts from pT4646A
(ASGV-U4646A) induced almost no symptoms, as
observed for ASGV-RM21 (Fig. 2j and 2k). The transcripts
from pT4646G (ASGV-U4646G) induced clearly attenuated
symptoms in infected C. quinoa plants (Fig. 2l and 2m).
Northern (Fig. 3c) and Western (data not shown) blot
analyses indicated that both of these mutants accumulated
less in host plants than ASGV-wt. These results indicate
that any change of nucleotide 4646 U in the ASGV genome
results in reduced virus accumulation and attenuated
pathogenic phenotypes, although such changes are translated silently in the ORF1 polypeptide.
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Journal of General Virology 84
Asymptomatic mutant of Apple stem grooving virus
Fig. 2. Comparison of the pathology of the original ASGV-wt (a, b, f and h) and mutants, ASGV-RM21 (c, d, g and i) in
Chenopodium quinoa (a, b, c and d), Nicotiana glutinosa (f and g; transmitted light image of the reverse side) and Vigna
sesquipedalis (h and i) at 14 days p.i. (e) Growth of C. quinoa plants (21 days p.i.) inoculated with water (mock) (left), ASGVwt (right) or -RM21 (middle). (j)–(m) Symptoms of the other mutants, ASGV-U4646A (j; inoculated-, and k; upper-leaf), or
ASGV-U4646G (l; inoculated-, and m; upper-leaf) on C. quinoa.
The relationship between viral genetic information and
the symptoms induced by the virus in host plants has long
attracted much attention. Reduced virus replication or
http://vir.sgmjournals.org
reduced cell-to-cell or long-distance movement, or combinations of these factors, may cause attenuated symptoms.
Amino acid alterations in viral replicase causing attenuated
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2581
H. Hirata and others
symptom development (Shintaku & Palukaitis, 1992;
Banerjee et al., 1995; Rao & Grantham, 1995; Fujita et al.,
1996; Moreno et al., 1997; Andersen & Johansen, 1998;
Szilassy et al., 1999; Takeshita et al., 2001; de Assis Filho
et al., 2002). It has also been implied that symptom
development is a complex process involving interactions
between viral genes or their products and host plant factors.
The viral RNA 39- and 59-noncoding sequences can also
affect the expression of disease symptoms (Rodrı́guezCerezo et al., 1991; Zhang et al., 1994; van der Vossen et al.,
1996). In this study, RM21 and the other two substitution
mutations at nucleotide 4646 of the ASGV genome, which
are responsible for the attenuated pathogenic phenotype,
are translationally silent. To our knowledge, this is the first
example of silent mutations in the coding region of a plant
viral sequence affecting viral symptoms.
Fig. 3. (a) Western immunoblot detection of ASGV CP from
inoculated or upper systemic leaves collected from plants 1, 4,
6, 9 or 13 days p.i. with ASGV-wt (WT) or -RM21 (RM21).
Bars at the side of the figure indicate the migration distance of
the standard ASGV CP. (b, c) Northern blot analysis for ASGV
viral RNA species with a probe specific for ASGV ORF2 using
total RNA extracted from plants (b, 11 days p.i.; c, 21 days
p.i.) inoculated with water (mock) (H), ASGV-wt (WT), -RM21
(RM21), -U4646A (U4646A) or -U4646G (U4646G). Arrows
mark the ASGV viral RNA species detected. The lower panels
in (b) and (c) show ribosomal RNA (rRNA) stained with ethidium bromide as a loading control.
symptoms have been well documented (Nishiguchi et al.,
1985; Holt et al., 1990; Lewandowski & Dawson, 1993;
Hagiwara et al., 2002). In addition, it has been reported
that alteration of the amino acids in CP or MP modifies
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As for the mechanism of the reduced accumulation of
the viruses with a mutation in nucleotide 4646, one can
argue that the nucleotide is located in the promoter region
of the subgenomic RNA for MP, since the mutation is
located upstream from the coding region (ORF2) of the
putative MP. Although the promoter for MP subgenomic
RNA has not yet identified, a homologous sequence, 59GCTNGAN(2)TTNGAAAN(2)TTNGGN(2)AN(14)AATG-39,
is located upstream from both the MP and CP genes, and
might be part of the subgenomic promoter of both genes
(Magome et al., 1997). Nucleotide 4646 is 104 and 141 bases
upstream from the homologous sequence and the initiation codon of MP, respectively. If the MP subgenomic
promoter is somehow affected by the mutation, then the
transcription of MP subgenomic RNA should be affected.
Therefore, we quantified the amount of genomic, MP
subgenomic, and CP subgenomic RNA species using the
data shown in Fig. 3(b) (RNA signal intensity in inoculated
leaves). The relative abundance (genomic RNA=100) of
genomic RNA:MP subgenomic RNA:CP subgenomic RNA
was 100 : 12 : 18 for ASGV-wt and 100 : 13 : 20 for ASGVRM21, meaning that the relative abundance of MP
subgenomic RNA compared to that of genomic RNA is
not specifically reduced in ASGV-RM21, although the
absolute abundance of all three RNA species is reduced
equally. This suggests that the mutation at nucleotide 4646
does not affect MP subgenomic RNA transcription.
Another possible explanation of the reduced accumulation
of RM21 is that nucleotide 4646 might be involved in the
expression or activity of viral replicase, thereby affecting
virus replication. A stable stem–loop structure was predicted near nucleotide 4646 of the ASGV-wt genomic
RNA using the program MFOLD (Zuker & Jacobson,
1998), and altered stem–loop structures were predicted
for genomes of ASGV mutants having any of three
substitutions at nucleotide 4646 (data not shown). The
mechanism of symptom attenuation caused by a single
nucleotide substitution which is translationally silent
might be related to the alteration of the secondary
structure of the genome.
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Journal of General Virology 84
Asymptomatic mutant of Apple stem grooving virus
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