Virology 454-455 (2014) 1–10

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Virology
journal homepage: www.elsevier.com/locate/yviro

Presence of poly(A) and poly(A)-rich tails in a positive-strand RNA

virus known to lack 3' poly(A) tails
Weimin Li n,1, Yongqiang Zhang 1, Chao Zhang 1, Xinwu Pei, Zhixing Wang, Shirong Jia
Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing, China

art ic l e i nf o a b s t r a c t

Article history: Here we show that Tobacco mosaic virus (TMV), a positive-strand RNA virus known to end with 3' tRNA-
Received 13 November 2013 like structures, does possess a small fraction of gRNA bearing polyadenylate tails. Particularly, many tails
Returned to author for revisions are at sites corresponding to the 3' end of near full length gRNA, and are composed of poly(A)-rich
8 December 2013
sequences containing the other nucleotides in addition to adenosine, resembling the degradation-
Accepted 1 February 2014
Available online 20 February 2014
stimulating poly(A) tails observed in all biological kingdoms. Further investigations demonstrate that
these polyadenylated RNA species are not enriched in chloroplasts. Silencing of cpPNPase, a chloroplast-
Keywords: localized polynucleotide polymerase known to not only polymerize the poly(A)-rich tails but act as a 3' to
Tobacco mosaic virus 5' exoribonuclease, does not change the profile of polyadenylate tails associated with TMV RNA.
Poly(A) tails
Nevertheless, because similar tails were also detected in other phylogenetically distinct positive-strand
RNA polyadenylation
RNA viruses lacking poly(A) tails, such kind of polyadenylation may reflect a common but as-yet-
unknown interface between hosts and viruses.
& 2014 Elsevier Inc. All rights reserved.

Introduction The polyadenylation-stimulated RNA degradation mechanism,

RNA polyadenylation involves addition of the homopolymeric
poly(A) tails composed exclusively of adenosines, or sometimes
heteropolymeric poly(A)-rich stretches containing the other three
nucleotides as well, to 3' end of RNA substrates (Edmonds, 2002;
Slomovic et al., 2006, 2008a, 2010). This post-transcriptional
modification is a common event present in almost all organisms,
but shows somewhat paradoxical roles. Stable poly(A) tails found
on mature 3' end of most nucleus-encoded mRNAs in eukaryotes
play roles in nucleocytoplasmic export and translation initiation,
in addition to their more known role in stabilizing transcripts
(Edmonds, 2002; Moore and Proudfoot, 2009). In bacteria, archaea
and organelles, however, polyadenylation occurs mainly on the
degradation intermediates of both coding and non-coding RNAs,
thereby generating transient poly(A) or poly(A)-rich stretches as
‘landing pads’ to recruit 3'-5' exoribonucleases for further degra-
dation (Slomovic et al., 2006a, 2008b; Condon, 2007; Regnier and
Hajnsdorf, 2009; Schuster and Stern, 2009). Hence, the transient
polyadenylate tails have been considered a tell-tale sign of the
presence of polyadenylation-stimulated RNA degradation pathway
(Slomovic et al., 2006a, 2008b).
n
Corresponding author. Tel.: þ 86 10 82106107; fax: þ 86 10 82106102.
E-mail address: liweimin01@caas.cn (W. Li).
1
W.L., Y.Z. and C.Z. contributed equally to this work.
however, is not limited to the bacterial and bacterial-like genetic
systems, non-abundant truncated nuclear transcripts having
destabilizing poly(A) or poly(A)-rich tails were also detected in
nuclei or cytoplasm of eukaryotes such as budding yeast (Kuai
et al., 2004, 2005; Wyers et al., 2005; LaCava et al., 2005;
Vanácová et al., 2005), fission yeast (Win et al., 2006), fruit fly
(Nakamura et al., 2008), trypanosome (Cristodero and Clayton,
2007), Arabidopsis thaliana (Lange et al., 2008; Zakrzewska-Placzek
et al., 2010) and human cells (Slomovic et al., 2006b, 2010). The
fact that the poly(A)-stimulated RNA degradation occurs through-
out the prokaryotes and eukaryotes reflects that stimulating
RNA degradation is an ancestral role of polyadenylation (Slomovic
et al., 2008a, 2008b; Houseley and Tollervey, 2009). Thus far,
this evolutionarily conserved mechanism has been shown to
play critical roles in rapidly removing ‘unwanted RNAs’, such
as maturation byproducts, cryptic transcripts as well as the over-
expressed, mutated, incorrectly folded or misprocessed RNAs,
thereby maintaining the RNA quality of genome expression
(Slomovic et al., 2010; Houseley and Tollervey, 2009; Lange et al.,
2009; Lange and Gagliardi, 2011).
Viruses typically are not considered to be organisms, but are
small infectious entities that can replicate only within the living
cells of organisms. It is so far clear that RNA of many eukaryotic
viruses, ranging from DNA to RNA viruses, has 3' poly(A) tails (King
et al., 2012), which, however, are synthesized via not merely

http://dx.doi.org/10.1016/j.virol.2014.02.002
0042-6822 & 2014 Elsevier Inc. All rights reserved.
2 W. Li et al. / Virology 454-455 (2014) 1–10
a post-transcriptional manner, also the direct or reiteritive tran-
scription of a poly(U) stretch in the template strand (Sanfaçon
et al., 1991; Weichs an der Glon et al., 1993; Silver and Pagano,
1997; Poon et al., 1999; Chen et al., 2005; Steil et al., 2010; Bier
et al., 2011; Ogram and Flanegan, 2011). Regardless of the synth-
esis mechanism used, the viral poly(A) tails have long been
considered as a player in RNA stability and translation, mimicking
roles of the stable poly(A) tails in eukaryotic mRNA (Dreher, 1999;
Barr and Fearns, 2010). No evidence had come to light that the
poly(A) tails function in destabilizing the viral RNA until recently.
In HeLa cells infected with vaccinia virus (VV), a double-stranded
DNA virus of the poxvirus family, types of non-abundant truncated
mRNA molecules of VV containing poly(A) or poly(A)-rich tails
were detected, resembling the polyadenylated degradation inter-
mediates of the poly(A)-stimulated RNA decay pathway (Slomovic
et al., 2010). This indicates that the VV mRNA is subjected to the
poly(A)-stimulated degradation pathway. However, due to the sole
piece of evidence as of today, it remains an open question whether
the transient polyadenylation is widespread in viruses.
Positive-strand RNA viruses constitute the largest group of
viruses, whose genome serves directly as mRNA for protein synthesis
(Ahlquist et al., 2003; King et al., 2012). It is known, however, that
many viruses within this group, do not have poly(A) tails but evolve
3'-termini as tRNA-like structure (TLS) or non-TLS heteropolymeric
sequence (Het) instead (Dreher, 1999). In contrast to the hitherto
widely accepted view, here we show that Tobacco mosaic virus (TMV),
a positive-strand RNA virus believed to terminate with TLSs (Creager
et al., 1999; Scholthof et al., 2011), does possess a certain number of
gRNA molecules that are polyadenylated at sites corresponding to
both full length and near full-length viral gRNA. Particularly, in
addition to some poly(A) tails, most of the polyadenylate tails are
of poly(A)-rich stretch resembling the degradation-stimulating poly
(A)-rich tails previously observed in all biological kingdoms (Lisitsky
et al., 1996; Mohanty and Kushner, 2000, 2010; Bralley and Jones,
2002; Campos-Guillén et al., 2005; Slomovic et al., 2006a, 2008b,
2010; Zimmer et al., 2009; Germain et al., 2011). In addition, similar
types of polyadenylate tails were also found in Odontoglossum ring-
spot virus (ORSV), Cucumber green mottle mosaic virus (CGMMV),
Cucumber mosaic virus (CMV), Tobacco rattle virus (TRV), Turnip
crinkle virus (TCV) and Tobacco necrosis virus (TNV), six more
positive-strand RNA viruses known to terminate with either TLSs
or Hets. This, to our knowledge, is the first discovery of poly(A) and
poly(A)-rich tails in positive-strand RNA viruses known to lack
poly(A) tails, and suggest a so-far-unknown virus-host interacting
but probably with respect to the polyadenylation-assisted RNA
degradation.
Results
RNA-Seq of TMV-inoculated Chenopodium amaranticolor suggested
TMV RNA bearing poly(A) tails
Using RNA sequencing (RNA-Seq), we recently constructed a
foliar transcriptome database containing 112,453 unigenes based
on the poly(A) þ RNA extracted from the Chenopodium amaranti-
color leaves that include healthy leaves, as well as TMV- and CMV-
inoculated leaves (Zhang et al., 2012). Whilst the unigenes were
searched against the NCBI NR database, we surprisingly noticed
that one assembled sequence (Unigene 24636), which has a length
of 6232 bp, matches nearly the complete genome of TMV U1
(#NC_001367). In contrast, none of the unigenes matched any of
the tripartite genomes of CMV. The immediate application of this
transcriptome data to analyze the digital gene expression (DGE)
profiles of the TMV-inoculated C. amaranticolor leaves identified
numbers of tags mapped to Unigene24636. In concert with the
course of virus multiplication in host plants, the number of these
tags increased sharply from 86 at 6 hours post inoculation (hpi) to
2776 at 28 hpi.
The presence of TMV RNA in the transcriptome and DGE
profiles implied that the TMV RNA might bear poly(A) tails. If so,
this will greatly challenge the traditional view that the TMV RNA
terminate with TLSs rather than poly(A) tails (Creager et al., 1999;
Scholthof et al., 2011). However, an alternative possibility we could
not rule out was that the poly(A) þ RNA isolated from the TMV-
inoculated leaves might have some contamination with the TMV
RNA probably due to its nonspecific interaction with oligo(dT)
magnetic beads. Further experiments were therefore carried out
here below to clarify if TMV RNA has 3' poly(A) tails indeed.
Characterization of the polyadenylate tails associated with TMV RNA
The approach of oligo(dT) primed RT-PCR was pursued to
this end (Fig. 1A). Total RNA extracted from the TMV-inoculated
leaves of C. amaranticolor, Arabidopsis thaliana Col-0 and Nicotiana
benthamiana were individually reverse transcribed with an
anchored oligo(dT) primer PT18, and then PCR amplified with
two nested primer pairs P1/TMV-5372-94 and P2/TMV-6023-44
(Fig. 1A and Table S1). The resulting PCR products were subcloned
and sequenced. Taking this approach, we indeed isolated the TMV
RNA molecules bearing ploy(A) or poly(A)-rich tails in each of the
aforementioned plant species infected with TMV (Fig. 1B and Fig.
S1). By contrast, no such TMV RNA molecules were identified in a
control experiment, in which 1 μg RNA mixture containing 0.1 μg
in vitro TMV RNA transcripts lacking poly(A) tails and 0.9 μg total
RNA from uninfected N. benthamiana leaves was analyzed with the
same approach (data not shown), ensuring that the polyadenylate
tails of TMV RNA was not of amplification artifacts. Taken together,
the data conclusively demonstrated that TMV RNA had 3' poly
(A) tails. Moreover, the presence of the polyadenylated TMV
RNA in various hosts belonging to evolutionarily distant plant
families indicated that the viral RNA polyadenylation is not host-
dependent but occurs widely in hosts of TMV.
In an effort to decipher nature of the poly(A) and poly(A)-rich
tails associated with TMV RNA, we totally analyzed 65 poly-
adenylated viral RNA molecules that were cloned from the
TMV-inoculated N. benthamiana as described above. In addition
to 15 RNA molecules bearing poly(A) tails comprised exclusively
of adenosines, a large number of RNA had poly(A)-rich tails,
a mixture of predominantly adenosines (88.68%), guanosines
(5.66%), uridines (5.53%) and cytosines (1.57%). Notably, these
poly(A)-rich tails with heterogeneity confined to their 5' ends,
but terminated in homogenous stretches of adenosines (Fig. 1B),
possibly due to the 3' bias of oligo(dT)-dependent detection.
The homopolymeric poly(A) tails ranged in size from 12 nt to
37 nt, while the length of the poly(A)-rich tails varied significantly
from 14 nt to 78 nt. However, considering that the oligo(dT)-
dependent RT-PCR does not allow determination of the maximum
length of the poly(A) and poly(A)-rich tails as previously discussed
(Lisitsky et al., 1996; Bralley and Jones, 2002; Slomovic et al., 2010),
the real tails associated with TMV RNA should be longer than those we
detected here. In addition, the adenosines within the poly(A)-rich tails,
in contrast to the pure adenosines of the poly(A) tails, were most often
clustered with other residues interspersed. Length of the contiguous
adenosines in poly(A)-rich tails varied, with 1620-nt stretches
occurring most frequently, while almost all stretches of the clustered
non-adenosine residues was not longer than four residues (Fig. 1B).
These features are similar with those of the previously observed poly
(A)-rich tails in Escherichia coli (Mohanty and Kushner, 2000), spinach,
Arabidopsis and Chlamydomonas chloroplasts (Lisitsky et al., 1996;
Zimmer et al., 2009), Streptomyces coelicolor (Bralley and Jones, 2002),
Bacillus subtillis (Campos-Guillén et al., 2005) and human cells
 W. Li et al. / Virology 454-455 (2014) 1–10 3

Fig. 1. Oligo(dT) primed RT-PCR detection of the polyadenylated TMV RNA. (A) Schematic diagram of the primers in TMV gRNA (Table S1). (B) Nature of polyadenylate tails
associated with TMV RNA in N. benthamiana. The 3' end of TMV gRNA is schematically presented. The poly(A) and poly(A)-rich tails are isolated from the TMV-inoculated
leaves at 4 dpi by means of oligo(dT) RT-PCR, and are presented below and above TMV gRNA, respectively. Vertical dashed lines with numbers indicate the exact positions of
the polyadenylate tails on TMV gRNA, and nucleotide compositions of the tails are shown.
4 W. Li et al. / Virology 454-455 (2014) 1–10
(Slomovic et al., 2006b, 2010), suggesting a high preference for
adenosine during polymerization of these polynucleotide tails.
To determine the sites of the homo- and hetero-tails addition,
we further characterized the junctions of these collected TMV
RNA-poly(A) sequences (Fig. 1B). A total of 10 RNA molecules
contain the mature 3' end of TMV gRNA. The rest, however,
correspond to an incomplete TMV gRNA, and their junctions were
located at variable positions, which were 1–337 nt away from the
3'-terminus of TMV gRNA. In addition, the 3' terminal nucleobases
of TMV RNA linked with poly(A) or poly(A)-rich tail were mostly
adenosines and uridines ( 470%), suggesting 3' end specificity for
the polyadenylate tails addition.
A small fraction of TMV gRNA species is polyadenylated
The presence of the ployadenylate tails in 3' end of TMV RNA
indicated a type of TMV RNA species yet unknown. Additional
experiments were therefore performed to characterize these novel
RNA molecules. The poly(A) þ RNA containing the polyadenylated
TMV RNA was purified from total RNA of the TMV-infected N.
benthamiana with the oligo(dT) column (Qiagen, USA), and was
analyzed by northern blot with two sets of probes (Fig. 2A). As
shown in Fig. 2B (lane 2), a probe targeted toward 5' end of TMV
gRNA detected only one band, which was comparable in size to
TMV gRNA, while its intensity was only a minor percentage ( 2%)
of that of the TMV gRNA. Clearly, this band should represent the
viral RNA bearing ploy(A) or poly(A)-rich tails, suggesting that at
least part of the ployadenylated RNA species originate from TMV
gRNA. In agreement with this hypothesis, a TMV 3'-UTR-specific
probe generated also a band correlate with TMV gRNA (Fig. 2B,
lane 4), presenting a hybridization pattern similar to that from the
5' end probe. To be sure that the detected bands do represent the
polyadenylate TMV RNA that specifically isolated by the oligo(dT)
column, a RNA sample containing 3 μg in vitro TMV RNA tran-
scripts without poly(A) tails and 27 μg total RNA from uninfected
Fig. 2. Characterization of the polyadenylated TMV RNA in TMV-inoculated
N. benthamiana. (A) Schematic diagram of two different probes used in northern
blot analysis. (B) Detection of the polyadenylated viral RNA in the TMV-inoculated
N. benthamiana leaves (4 dpi) by using poly(dT) selection-coupled northern blot.
Poly(A) þ RNA enriched from 15 μg total RNA (lanes 2 and 4), along with 3 μg total
RNA (lanes 1 and 3), was hybridized with a 5'-end probe complementary to nts
1–380 of TMV gRNA and a TMV 3'UTR-specific probe, respectively. Bands corre-
sponding to gRNA and cp-sgRNA are indicated, respectively.
N. benthamiana leaves were also analyzed with poly(dT) selection-
coupled northern hybridization. No band was detected with the 5'
end probe or the 3'-UTR-specific probe (Fig. S2, lanes 2 and 5).
It was noteworthy that, in spite of co-3'-termini of TMV gRNA
and sgRNA, the 3' end probe detected only a band corresponding to
the size of gRNA but not sgRNA (Fig. 2B, lane 4). This showed that
TMV sgRNA, unlike its gRNA, was absent of the polyadenylate tail
addition, suggesting selective polyadenylation between these two
kinds of viral RNA species. In particular, the sgRNA species, regarding
that they could not be purified with the oligo(dT) column, could
serve as an internal control to ensure the specific enrichment of the
polyadenylated viral RNA by using this approach.
Collectively, the poly(dT) selection-coupled northern hybridi-
zation revealed the possible size as well as the relative level
of the polyadenylated TMV RNA in TMV-inoculated leaves of
N. benthamiana. The data, along with the above findings that the
poly(A) or poly(A)-rich tails were added at the 3' end of TMV
gRNA, indicated that many, if not all, polyadenylated TMV RNA
molecules were of the full length and near-full-length TMV gRNA
bearing polyadenylate tails.
TMV virions contain a trace amount of the polyadenylated viral RNA
It is known that, for TMV U1, only its gRNA is able to be
encapsidated in virus particles (Butler, 1999). To determine if the
polyadenylated TMV gRNA is encapsidated, we tested the viral
RNA prepared from TMV virions by means of oligo(dT) primed
RT-PCR used above. The TMV RNA species bearing the poly(A) or poly
(A)-rich tails were successfully identified (Fig. 3A). Particularly, like
the poly(A) tails isolated from total RNA of the TMV-infected plants
(Fig. 1B), all but one tail isolated here were associated with 3'
truncated TMV RNA (Fig. 3A). This clearly showed presence of the
polyadenylated TMV RNA in virions. Hence, northern blot analysis
was employed to evaluate the relative level of the packaged poly-
adenylated TMV RNA. A total of 15 μg packaged TMV RNA was
subjected to the oligo(dT) column to enrich the polyadenylated viral
RNA. However, no any signal relevant to TMV gRNA was detected in
the enriched RNA (Fig. 3B).
Taken together, the lines of evidence demonstrated that TMV
virions contain but only a trace amount of the polyadenylated
viral RNA, indicating that this type of RNA was less likely to be
encapsidated. It is known that the complete genome of TMV was
initially deduced from the viral RNA prepared from the purified
virions (Guilley et al., 1979; Goelet et al., 1982; Ohno et al., 1984),
few number of polyadenylated TMV RNA in virions might account
for the unawareness of the polyadenylate tails in TMV genome
during the past years.
Chloroplast PNPase has a monir role in the TMV RNA polyadenylation
Nature of poly(A)-rich tails associated with TMV RNA was
reminiscent of the previous studies in which poly(A)-rich tails
are identified to be polymerized by bacterial and chloroplast
polynucleotide phosphorylase (PNPase) or Archaeal exosome
(Slomovic et al., 2008a; Lange et al., 2009; Mohanty and
Kushner, 2010). Considering that the exosome in eukaryotes is
active in RNA degradation but absent of polymerization activity
(Slomovic et al., 2008a, 2010), we asked if chloroplast PNPase
(cpPNPase) of higher plants was the candidate for polymerization
of poly(A)-rich tails observed here.
It is known that cpPNPase is a chloroplast-localized poly
(A) polymerase (Hayes et al., 1996; Li et al., 1998). Thus, if the poly
(A)-rich tails associated with TMV gRNA were indeed generated by
cpPNPase, TMV gRNA from chloroplasts would be enriched greatly in
poly(A)-rich viral RNA compared with the viral RNA in the population
from total RNA. To test this possibility, RNA was extracted from the
 W. Li et al. / Virology 454-455 (2014) 1–10
Fig. 3. TMV virions contain a trace amount of the polyadenylated viral RNA.
(A) Polyadenylate tails of TMV RNA isolated from virions. Vertical dashed lines with
numbers indicate the exact positions of the tails on the 3' end of TMV gRNA, and
nucleotide compositions of the tails are shown. (B) Northern blot analysis of the
polyadenylated TMV RNA in virions. Poly(A) þ RNA enriched from 15 μg TMV RNA of
virions (lanes 2 and 4), along with 0.3 μg TMV RNA (lanes 1 and 3), was hybridized
with the 5'-end probe specific for nts 1–380 of the TMV gRNA and the TMV 3'UTR-
specific probe, respectively. Bands corresponding to TMV gRNA are indicated.
intact chloroplasts, which were purified from the TMV-inoculated N.
benthamiana leaves with Percoll gradients and treated with RNase A
to remove the cytoplasmic RNA. As reported previously (Schoelz and
Zaitlin, 1989), only TMV gRNA, but not sgRNA, is present in chlor-
oplasts. In particular, since TMV sgRNA is abundant in cytoplasm but
absent in chloroplasts, this type of RNA could be used as an internal
control to assess the purity of the chloroplast RNA. Accordingly, the
chloroplast RNA prepared here was tested by northern blot analysis
using the TMV 3'-UTR-specific probe. Consistent with the previous
observation (Schoelz and Zaitlin, 1989), only TMV gRNA was present
in the chloroplast RNA, and sgRNA was virtually undetectable (Fig. 4A,
lane 3). Next, poly(dT) selection-coupled northern blot analysis was
performed to evaluate the relative level of the polyadenylated TMV
gRNA in chloroplasts. Up to 60 μg chloroplast RNA was purified by the
oligo(dT) column, but no signal relevant to TMV gRNA was detected in
the purified RNA (Fig. 4A, lane 4). This showed that the polyadeny-
lated TMV gRNA was not enriched in chloroplasts as anticipated.
The putative role of cpPNPase in the TMV RNA polyadenylation
was further determined by using the TRV-based VIGS (Liu et al.,
2002). A mixture of Agrobacterium culture containing the pTRV2-
cpPNPase and pTRV1 was infiltrated into the leaves of N. benthamiana
plants. At 10–14 days post Agro-infiltration, the plants exhibited
yellowing or chlorosis in the upper leaves (Fig. 4B), a phenotype
similar with that of the Arabidopsis PNP-mutants (Germain et al.,
2011; Sauret-Güeto et al., 2006). RT-PCR quantification confirmed the
significant reduction in cpPNPase mRNA levels in the yellowing leaves
(Fig. S3). These leaves were inoculated with TMV and collected
at 4 days post inoculation (dpi) to analyze the TMV RNA poly-
adenylation.
5
Referring to both poly(A) and poly(A)-rich tails associated with
TMV gRNA in wt N. benthamiana, we anticipated that, if cpPNPase
was indeed responsible for generating the poly(A)-rich tails of
TMV, cpPNPase silencing would leave only the poly(A) tails or
cause a significant shift in favor of the poly(A) tails, as observed in
chloroplasts of Arabidopsis PNP- mutants (Germain et al., 2011).
However, by using oligo(dT) primed RT-PCR, a number of poly(A)-rich
tails rather than only poly(A) tails were detected in N. benthamiana
with cpPNPase silencing, and no increase was observed in the
percentage of poly(A) tails within the total number of the cloned
polyadenylated TMV RNA molecules (Fig. 4C).
Also of note was that cpPNPase is a reversible enzyme
(Slomovic et al., 2008a). Besides its role in 5' to 3' RNA polymer-
ization, this enzyme functions in the processive 3' to 5' phosphor-
olysis as well. Depletion or knockout of the Arabidopsis cpPNPase
can greatly enhance the accumulation of the polyadenylated
transcripts in chloroplasts (Walter et al., 2002; Marchive et al.,
2009). Herein, however, silencing of the N. benthamiana cpPNPase
directed no change in the proportion of polyadenylated TMV RNA
with respect to TMV gRNA (Fig. 4D).
Taken together, these data conclusively demonstrated that
chloroplasts were not the site for the TMV RNA polyadenylation,
and cpPNPase played, at best, the minor roles in either polymer-
ization of the poly(A)-rich tails or degradation of the tails asso-
ciated with TMV gRNA.
Polyadenylation occurs widely in positive-strand RNA viruses known
to lack poly(A) tails
TMV belongs to the genus Tobamovirus, whose members all
possess TLSs but not poly(A) tails in 3' end of the viral gRNA (King
et al., 2012). To define if, in addition to TMV, the other members
in genus Tobamovirus also undergo the polyadenylation, two
additional tobamoviruses, ORSV and CGMMV, were investigated
herein. By using oligo(dT) primed RT-PCR with the gene-specific
primers (Table S1), we successfully identified the viral RNA bearing
poly(A) or poly(A)-rich tails from total RNA extracted from ORSV-
or CGMMV-infected N. benthamiana (Fig. 5A and B). This disclosed
that, like TMV, both ORSV and CGMMV bear poly(A) or poly(A)-
rich tails at or close to 3' end of the viral RNA.
The presence of polyadenylate tails in tobamoviruses prompted
us to investigate if the polyadenylation occurs in other genera of
RNA viruses known to lack poly(A) tails. Examination was there-
fore extended to CMV (genus Cucumovirus), TRV (genus
Tobravirus), TCV (genus Carmovirus) and TNV (genus Necrovirus),
four more phylogenetically distinct positive-strand RNA viruses
known to terminate with TLS or Het. With the same approaches,
total RNA from the N. benthamiana plants infected with each of
aforementioned viruses was analyzed using the corresponding
primers (Table S1). The results showed that all these viruses do
have the poly(A) and poly(A)-rich tails (Fig. 5C–F). Notably, TRV
RNA was inclined toward homopolymeric adenylation, a large
proportion of the tails associated with TRV RNA1 and RNA2 are
exclusively composed of adenosines (Fig. 5D). For CMV, no poly-
adenylated viral RNA was identified by using the transcriptome
method as described in the first section. However, such molecules
were undoubtedly detected by means of oligo(dT) primed RT-PCR
(Fig. 5C). These data suggested that CMV could generate the
polyadenylated viral RNA in host plants, but the level was probably
below the detection limit of RNA-Seq.
The polyadenylate tails in CMV and TRV, as detected in
tobamoviruses, were either located at or close to 3' end of gRNA.
Particularly, a total of 10 tails associated with CMV gRNA3 were all
at the mature 3' end, and most of them present a consensus
sequence U3An (Fig. 5C). By contrast, the isolated tails of TCV and
TNV were at least 185 nt and 31 nt upstream of the 3' terminus of
6 W. Li et al. / Virology 454-455 (2014) 1–10

Fig. 4. Functional analysis of the N. benthamiana cpPNPase in TMV gRNA polyadenylation. (A) Northern blot analysis of the polyadenylated TMV gRNA in chloroplasts. Poly
(A) þ RNA purified from a total of 60 μg chloroplast RNA (lane 4), along with 3 μg chloroplast RNA (lane 3), was blotted with a TMV 3'UTR-specific probe. (B) Phenotypes of
N. benthamiana infiltrated with Agrobacterium containing the empty pTRV2 vector (TRV2) or pTRV2-PNPase (TRV2-PNP). The photograph was taken at 14 days post
agroinfiltration and the bar represents 2 cm. (C) Polyadenylate tails of TMV RNA isolated from the N. benthamiana plants with cpPNPase silencing. Vertical dashed lines with
numbers indicate the exact positions of the tails on the 3' end of TMV gRNA, and nucleotide compositions of the tails are shown. (D) Northern blot analysis of the
polyadenylated TMV RNA in the N. benthamiana plants with cpPNPase silencing. Poly(A) þ RNA enriched from 15 μg total RNA (lanes 2 and 4), along with 3 μg total RNA
(lanes 1 and 3), was blotted with a TMV 3'UTR-specific probe. Bands corresponding to gRNA and cp-sgRNA are indicated.

the vial gRNA, respectively (Fig. 5E and F), suggesting that there
should be no or an extremely low level of polyadenylate tails
associated with the mature 3' end of TCV or TNV gRNA.
It is known that CMV and TRV, like tobamoviruses, have
3'-UTRs terminated with TLSs, while TCV and TNV possess 3' Hets.
Thus, the presence of polyadenylate tails in viruses with different
terminal structures indicated the widespread polyadenylation in
positive-strand RNA viruses previously not reported to have 3' poly
(A) tails.
Discussion
TMV, as the type member of genus Tobamovirus, is the first
known virus and the first completely sequenced RNA virus,
and has a leading role in virology research to the present time
(Creager et al., 1999; Scholthof et al., 2011). Since 1982, the
complete genome of the TMV common strain was deciphered
(Guilley et al., 1979), numbers of distinct TMV strains have been
sequenced over the past 30 years. From then to now, it has been
firmly established that the genome of this positive-strand RNA
virus terminates with TLSs but not poly(A) tails (Creager et al.,
1999; Barr and Fearns, 2010). However, in this work, we deter-
mined that, in a wide range of TMV-infected host species, at least
a low level of the TMV gRNA molecules are polyadenylated. Both
homo- and heteropolymeric poly(A) tails were detected in the full-
length and near full length gRNA species.
Interestingly, such polyadenylate tails were also found in other six
positive-strand RNA viruses from distinct virus families and genera
known to lack poly(A) tails. As is well accepted that the 3' poly
(A) structure is a unique feature of many, but not all positive-strand
RNA viruses. Thus, these unexpected poly(A) and poly(A)-rich tails
detected in this study indicate that occurrence of polyadenylation in
viruses is much more common than we currently appreciate, and
may represent an as-yet-unknown interface between virus and host.
The tails isolated here, in light of their specific nature, strictly
resemble the transient polyadenylate tails, an indication of the
presence of the polyadenylation-assisted RNA degradation path-
way, one ancient RNA surveillance mechanism (Slomovic et al.,
2006a, 2008b; Mohanty and Kushner, 2010). Given that a virus
begins to flood a host cell with its own RNA, the viral transcripts,
regarding their inherent molecular features, are apt to be perceived as
 W. Li et al. / Virology 454-455 (2014) 1–10 7

Fig. 5. Polyadenylate tails are present in other positive-strand RNA viruses known to lack poly(A) tails. The polyadenylate tails of viral RNA were isolated from the N.
benthamiana leaves inoculated with ORSV, CGMMV, CMV, TRV, TCV or TNV at 4 dpi by oligo(dT) primed RT-PCR (A–F). Vertical dashed lines with numbers indicate the exact
positions of the polyadenylate tails on the 3' end of the viral RNA, and nucleotide compositions of the tails are shown.

the ‘unwanted RNAs’ by the cellular RNA surveillance mechanisms
(Dickson and Wilusz, 2011; Moon et al., 2012). It is, therefore, rational
that the viral RNA is subjected to the polyadenylation-stimulated
degradation pathway. Generally, the process of the polyadenylation-
stimulated RNA decay comprises three sequential steps: endonucleo-
lytic cleavage, addition of polyadenylate tails to the cleavage products,
and exonucleolytic degradation (Slomovic et al., 2006a, 2008a). Thus,
the non-abundant viral RNAs bearing polyadenylate tails detected in
this study should represent the polyadenylated degradation inter-
mediates of this RNA decay pathway. Recently, a double-strand DNA
virus has been linked with this conserved RNA decay mechanism,
non-abundant, fragmented viral mRNA species bearing poly(A) or poly
(A)-rich tails were detected in human cells infected with this
virus (Slomovic et al., 2010). The discovery of polyadenylate tails
in two different virus orders implied a wide occurrence of the
polyadenylation-assisted RNA degradation in viruses.
As far as is known, of the degradation-stimulating poly(A) tails
observed to date, the homo-poly(A) tails are usually generated by
non-conical poly(A) polymerase, while the poly(A)-rich tails are
produced by bacterial and chloroplast PNPase, as well as Archaeal
exosome (Slomovic et al., 2008a). Here, in plant cells, the observation
of both homo- and heteropolymeric poly(A) tails associated with the
viral RNA opened the question concerning the identity of the poly-
merizing enzyme, in particular the one(s) for heteropolymeric tails.
In the case of TMV, only its gRNA but not sgRNA was
polyadenylated. This, along with a previous observation that only
8 W. Li et al. / Virology 454-455 (2014) 1–10
TMV gRNA but not sgRNA is present in the chloroplasts (Schoelz
and Zaitlin, 1989), suggested that the poly(A)-rich tails of TMV
gRNA was added by cpPNPase in chloroplasts. However, our
further investigations showed that few polyadenylated TMV gRNA
molecules were detected in chloroplasts, and silencing of cpPNPase
in N. benthamiana directed no change in the poly(A)-rich tails
associated with TMV gRNA. The data clearly did not support the
postulate above, but implied that cytoplasm of plant cells might be
the site where TMV gRNA was polyadenylated regarding that TMV
does not enter the nucleus during its life cycle (Más and Beachy,
1999; Buck, 1999). This is reminiscent of a recent study, in which
non-abundant, truncated RNA species with poly(A) and poly(A)-
rich tails were detected in cytoplasm of human cells, but neither
hPNPase nor exosome is a candidate for synthesis of the hetero-
polymeric tails (Slomovic et al., 2010). Taken together, the cyto-
plasm of eukaryotes should contain an unknown enzyme or
complex toward the poly(A)-rich tails synthesis. Regarding the
selective susceptibility of TMV gRNA and sgRNA to polyadenyla-
tion, this unknown polyadenylating enzyme or complex recog-
nized its substrates possibly by dependency upon structure and/or
nucleotides at the 5' end of gRNA. The effect of the 5' end of RNA
on polyadenylation has been reported previously, an in vitro study
shows that the unpaired nucleotides at the 5' end of RNA govern
both the rate and length of the 3' poly(A) tails added by Escherichia
coli poly(A) polymerase I (Feng and Cohen, 2000).
In conclusion, we demonstrated here that a small fraction of TMV
gRNA species, as well as RNA of several other positive-strand RNA
viruses known to lack poly(A) tails, become tailed presumably in
cytoplasm of plant cells. The viral RNA species with poly(A) or poly
(A)-rich tails addition do bear a close resemblance to the degradation
intermediates of the polyadenylation-assisted RNA degradation path-
way witnessed in prokaryotes, organelles and eukaryotes, but much
remains to be done to decipher the biogenesis as well as biological
relevance of these unexpected polyadenylate tails.
Materials and methods
Viruses and plant inoculation
TMV, ORSV, CGMMV, CMV, TRV, TCV and TNV were propagated
in 5- to 6-week-old N. tabacum plants, respectively. Infectious sap
was prepared with 1 g systemically infected leaves macerated
in 5 mL inoculation buffer (50 mM KH2PO4, pH 7.0, 1% Celite),
and was mechanically inoculated in leaves of C. amaranticolor,
N. benthamiana and A. thaliana Col-0. For total RNA extraction,
inoculated leaves of these three plant species were collected
at 3 dpi, 4 dpi, and 10 dpi, respectively. The healthy and virus-
infected plants were cultured at 25 1C in a growth chamber with
16 h light/8 h dark cycle.
Transcriptome analysis and digital gene expression tags analysis
Transcriptome analysis and digital gene expression tags analy-
sis of the C. amaranticolor leaves were carried out previously as
described (Zhang et al., 2012).
Oligo(dT) primed RT-PCR
The 3' polyadenylate tails associated with the viral RNA were
cloned with oligo(dT) primed RT-PCR according to the instructions
of the GeneRacerTM Kit (Invitrogen, USA). Briefly, 1 μg total RNA
was reverse transcribed by M-MLV reverse transcriptase (Promega,
USA) with anchored oligo(dT)18, and the resulting first strand
cDNA was used as template for nested PCR amplification with the
corresponding primer pairs (Table S1).
Virus particles purification
TMV-U1 was inoculated in leaves of  6-week-old N. benthamiana,
and 100 g of inoculated leaves were harvested at 4 dpi. Virus particles
were isolated and fractionated on linear sucrose gradients essentially
as described (Beachy and Zaitlin, 1977). The fine purified virus
particles were suspended in 1ml of 0.01 M phosphate buffer at pH
7.0, and were treated with 1mg RNase A at 25 1C for 1 h to clear the
residual RNAs. RNA was extracted from virions by using the TRIzol
reagent (Invitrogen, USA).
Chloroplasts isolation
Chloroplasts were isolated following the procedure as described
(Schoelz and Zaitlin, 1989). The TMV-inoculated N. benthamiana
(2 dpi) was first placed in the dark to reduce the amount of starch
in leaves. Intact chloroplasts were isolated 2 days after incubation by
using a 40%:85% (vol/vol) Percoll step gradient and treated with RNase
A to reduce the contamination of cytoplasmic RNA to a minimum. The
chloroplast preparations were stored at  70 1C for RNA extraction.
Virus-induced NbcpPNPase silencing
A 2441-bp fragment representing the partial cDNA of
N. benthamiana chloroplast PNPase was amplified by using a
degenerated primer pair PNP-1-21/PNP-2421-01 (Table S1) designed
according to the Solanaceae homologous sequences, and was
submitted to Genbank (#KF318459). Accordingly, a 741-bp frag-
ment corresponding to nts 1153–2256 of the partial NbcpPNPase
was amplified by using a gene-specific primer pair PNP-1553-72/
PNP-2256-45 (Table S1), and digested with EcoR I and Sma I and
ligated into pTRV2. The resulting pTRV2-PNPase and the empty
pTRV2 vector were then transformed into Agrobacterium strain
GV2260, respectively. Virus-induced gene silencing assays were
performed as described (Liu et al., 2002). Agrobacterium containing
pTRV2-PNPase or pTRV2 was infiltrated into the lower leaves of
3-week-old N. benthamiana plants. Two weeks after infiltration,
the mRNA expression level of NbcpPNPase in upper leaves of plants
was measured using semi-quantitative RT-PCR with a primer pair
PNP-28-47/PNP-574-55 (Table S1), as previously described (Burton
et al., 2000). The silenced and healthy upper leaves were imme-
diately inoculated with TMV-U1, and the inoculated leaves were
collected at 4 dpi for total RNA extraction.
Poly(dT) selection and northern blot analysis
Poly(dT) selection was performed by using the oligo(dT) column
(Oligotex mRNA mini Kit, Qiagen) according to the manufacturer's
recommendations. The purified poly(A) þ RNA, along with total RNA,
was electrophoresed through a 1.2% agarose-formaldehyde gel. Blots
were hybridized with a 5'-end probe complementary to nts 1–380
of the TMV gRNA and/or a 3'-end probe specific for the TMV 3'UTR
(nts 6191–6395). Intensities of the bands were quantified using
ImageJ (Version 1.45S).
Acknowledgments
We thank Dr. William Dawson (University of Florida, USA)
for providing the cDNA clone of TMV U1, and Dr. Yule Liu
(Tsinghua University, PR China) for supplying the TRV-based VIGS
vectors and Agrobacterium strain GV2260. We are also grateful to
Dr. Nongnong Shi (Hangzhou Normal University, PR China)
for providing ORSV, Dr. Yongjiang Zhang (Chinese Academy of
Inspection and Quarantine, PR China) for CGMMV, Dr. Dawei Li
(China Agricultural University, PR China) for TCV and TNV, and
 W. Li et al. / Virology 454-455 (2014) 1–10
Dr. Xiaorong Tao (Nanjing Agricultural University, PR China) for
CMV. This work was supported by the National Natural Science
Foundation of China (31370181), the Special Fund for Agro-
scientific Research in the Public Interest (201303028), the Funda-
mental Research Funds for Central Public Welfare Research
Institutes (2013ZL046), and the National Major Special Project
of China on New Varieties Cultivation for Transgenic Organisms
(2011ZX08001-002, 2011ZX08004-004).
Appendix A. Supplementary materials
Supplementary data associated with this article can be found in
the online version at http://dx.doi.org/10.1016/j.virol.2014.02.002.
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