The VPgPro protein of Turnip mosaic virus: in vitro inhibition of translation from a ribonuclease activity.

Abstract

A role for viral encoded genome-linked (VPg) proteins in translation has often been suggested because of their covalent attachment to the 5' end of the viral RNA, reminiscent of the cap structure normally present on most eukaryotic mRNAs. We tested the effect of Turnip mosaic virus (TuMV) VPgPro on translation of reporter RNAs in in vitro translation systems. The presence of VPgPro in either wheat germ extract or rabbit reticulocyte lysate systems lead to inhibition of translation. The inhibition did not appear to be mediated by the interaction of VPg with the eIF(iso)4E translation initiation factor since a VPg mutant that does not interact with eIF(iso)4E still inhibited translation. Monitoring the fate of RNAs revealed that they were degraded as a result of addition of TuMV VPgPro or of Norwalk virus (NV) VPg protein. The RNA degradation was not the result of translation being arrested and was heat labile and partially EDTA sensitive. The capacity of TuMV VPgPro and of (NV) VPg to degrade RNA suggests that these proteins have a ribonucleolytic activity which may contribute to the host RNA translation shutoff associated with many virus infections.

Full text

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The VPgPro protein of Turnip mosaic virus: In vitro inhibition of
translation from a ribonuclease activity
Sophie Cotton1, Philippe J. Dufresne1, Karine Thivierge1, Christine Ide, Marc G. Fortin⁎
Department of Plant Science, McGill University, 21,111 Lakeshore, Ste-Anne-de-Bellevue, Québec, Canada H9X 3V9
Received 16 January 2006; returned to author for revision 6 February 2006; accepted 14 March 2006
Available online 2 May 2006
Abstract
A role for viral encoded genome-linked (VPg) proteins in translation has often been suggested because of their covalent attachment to the 5′
end of the viral RNA, reminiscent of the cap structure normally present on most eukaryotic mRNAs. We tested the effect of Turnip mosaic virus
(TuMV) VPgPro on translation of reporter RNAs in in vitro translation systems. The presence of VPgPro in either wheat germ extract or rabbit
reticulocyte lysate systems lead to inhibition of translation. The inhibition did not appear to be mediated by the interaction of VPg with the eIF(iso)
4E translation initiation factor since a VPg mutant that does not interact with eIF(iso)4E still inhibited translation. Monitoring the fate of RNAs
revealed that they were degraded as a result of addition of TuMV VPgPro or of Norwalk virus (NV) VPg protein. The RNA degradation was not
the result of translation being arrested and was heat labile and partially EDTA sensitive. The capacity of TuMV VPgPro and of (NV) VPg to
degrade RNA suggests that these proteins have a ribonucleolytic activity which may contribute to the host RNA translation shutoff associated with
many virus infections.
© 2006 Elsevier Inc. All rights reserved.
Keywords: Inhibition of translation; Virus infection; Potyviruses; Picornaviruses; RNA degradation
Introduction
Turnip mosaic virus (TuMV; Potyviridae) is a member of the
picorna-like supergroup of positive-sense RNA viruses. For
many plant and animal RNA viruses, the virus-encoded VPg
protein (Viral Protein genome-linked) is covalently attached to
the 5′ terminus of their genomic RNA (van Regenmortel et al.,
2000). This protein is positioned on the viral RNA where the
m7G cap structure is normally found on cellular mRNAs; it is
not clear whether the VPg plays the same functional role as the
cap structure in translation initiation for viral RNA.
The VPg protein, and its VPgPro precursor form, are multi-
functional proteins that play important roles in the replication
cycle of potyviruses (Urcuqui-Inchima et al., 2001). The VPg
was shown to interact with translation initiation factors eIF(iso)
4E (eukaryotic initiation factor (iso)4E) and PABP (poly(A)-
binding protein) (Léonard et al., 2004; Wittmann et al., 1997).
These interactions suggest a role for the VPg in the recruitment
of translation initiation factors for viral RNA translation.
Mutations in either VPg or eIF(iso)4E result in reduced ability
of the virus to infect its host. Mutations in the eIF(iso)4E-
interacting domain of VPg lead to loss of virus infectivity
(Léonard et al., 2000), and disruption of plant eIF(iso)4E gene
prevented TuMV infection (Duprat et al., 2002; Lellis et al.,
2002). The involvement of VPg in facilitating viral RNA
translation was shown for Feline calicivirus (FCV) (Good-
fellow et al., 2005) where FCV translation is dependent on the
presence of VPg at the 5′ end of the viral genome. The VPg–
eIF4E interaction is required for virus RNA translation since
sequestration of eIF4E by 4E-BP1 inhibited translation. It was
suggested that FCV VPg acts as a ‘cap substitute’ during
translation initiation of virus mRNA.
Viruses that infect eukaryotic cells use a variety of
mechanisms for subverting the functions of the host cell.
Several viruses alter the translation machinery such that they
effectively block translation of host mRNAs. Viruses often
target translation initiation factors as a mean to increase their
own translation at the expense of that of their host. The main
strategies are either to compromise eIF4G or PABP functions by
proteolytic cleavage, to sequester eIF4E or to alter the
Virology 351 (2006) 92–100
www.elsevier.com/locate/yviro
⁎Corresponding author. Fax: +1 514 398 7897.
E-mail address: marc.fortin@mcgill.ca (M.G. Fortin).
1These authors contributed equally.
0042-6822/$ - see front matter © 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.virol.2006.03.019

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phosphorylation state of host translation factors (reviewed by
Gale et al., 2000). Another key aspect of virus infection in
plants and animals is the associated host transcription shut-
down (Aranda and Maule, 1998; Jen et al., 1980). For instance,
infection of plant cells with a potyvirus (carrying a 5′-bound
VPg) leads to transient disappearance of most cellular mRNAs
in cells supporting active viral replication (Aranda et al., 1996).
It is not clear whether the disappearance of RNAs is the
consequence of mRNA destabilization resulting from a stress
response, of host transcription inhibition, or of targeted
degradation resulting from a viral ribonuclease activity.
A more direct line of evidence implicating VPg in cellular
translation inhibition was found for Norwalk virus (NV). NV
VPg was shown to inhibit translation of a reporter RNA in
rabbit reticulocyte lysate (RRL; Daughenbaugh et al., 2003).
VPg added to cell-free translation reactions that contained either
capped RNA or RNA with an internal ribosomal entry site
(IRES) inhibited translation of these reporter RNAs in a dose-
dependent manner. Although potyviral VPgPro protein was
found to have non-specific RNA-binding properties (Daròs and
Carrington, 1997), as well as a deoxyribonuclease activity
(Anindya and Savithri, 2004), no link has been made between
those activities and an inhibition of translation or the
disappearance of mRNAs in plant cells. The mechanism by
which this occurs remains unclear.
In this report, we investigated the effect of addition of TuMV
VPgProon the translation of reporter RNA in in vitro translation
systems. Purified TuMV VPgPro inhibited translation of capped
reporter RNA, as was observed when VPg of NV was added to
an in vitro translation system. The VPgPro-eIF(iso)4E interac-
tion was likely not involved in the inhibition of translation since
a VPg mutant that does not interact with eIF(iso)4E did inhibit
translation. We observed that this inhibition of translation was
concurrent with degradation of reporter RNA in a rabbit
reticulocyte lysate. Purified total plant RNA was also degraded
when VPgPro was added; the same effect was observed with the
VPg domain alone. A similar ribonucleolytic activity was also
observed with NV VPg. The ribonucleolytic activity of VPg
proteins may contribute to the disappearance of most mRNAs
previously observed during potyvirus infection and to the
transient inhibition of translation documented for host cell
mRNAs during picornavirus infection.
Results
Expression and purification of VPg, VPgPro and eIF(iso)4E
proteins
TuMV VPgPro was expressed as a fusion protein with the S-
tag, glutathione-S-transferase (GST) and histidines fused at the
N-terminus (a fusion tag of 33 kDa). SDS-PAGE analysis of
the protein extracts obtained from soluble fractions following
GST affinity chromatography showed that protein bands
corresponding to both the VPg and VPgPro proteins were
present. This is consistent with the data of Ménard et al. (1995)
that showed that recombinant TuMV VPgPro self-cleaves into
two functional domains, the VPg and Pro domains. A
prominent 55 kDa band [VPg domain (22 kDa) + N-terminal
tag (33 kDa)] and a fainter 82 kDa band [VPgPro domain
(49 kDa) + N-terminal tag (33 kDa)] (Fig. 1) corresponding to
GST–VPg and GST–VPgPro, respectively, were found in the
preparation. Both the GST–VPg and GST–VPgPro fusions
were obtained since the GST domain was fused at the N-
terminus of the protein. The D77N mutant of VPgPro was
similarly expressed and purified (Fig. 1). The identity of the
proteins was confirmed by Western blot analysis using an anti-
GST monoclonal antibody and a polyclonal anti-VPgPro serum
(data not shown). The lower molecular weight products
resulted from degradation of the fused GST moiety. NV and
TuMV VPg proteins were also expressed in E. coli cells as N-
terminal GST fusions (a fusion tag of 26 kDa); fusion proteins
of 53 kDa (Fig. 1) [NV VPg domain (27 kDa) + N-terminal tag
(26 kDa)] and a 48 kDa [VPg domain (22 kDa) + N-terminal
tag (26 kDa)] were obtained for NV and TuMV, respectively.
Triticum aestivum eIF(iso)4E protein was expressed as an N-
terminal T7 fusion protein of 28 kDa (Fig. 1).
VPgPro inhibits in vitro translation of capped reporter RNA
A capped luciferase reporter RNA was translated either in
wheat germ extract (WGE) (Fig. 2A) or in RRL (Fig. 2B)
translation systems in the presence of increasing concentra-
tions of TuMV GST–VPgPro or of GST protein. Luciferase
luminescence was used to measure translation efficiency of the
m7G-luciferase RNA. Translation of the reporter mRNA
decreased sharply when increasing amounts of GST–VPgPro
were added to the translation systems. As a control for the
presence of contaminants in our protein purifications, the same
amounts of GST protein alone, produced and purified using
the same procedure as GST–VPgPro, were added to the in
vitro translation systems; no inhibition of translation was
observed.
Fig. 1. Expression and purification of GST–VPgPro and D77N of TuMV, GST–
VPg of NVand wheat eIF(iso)4E as described under Materials and methods. In
both TuMV VPgPro and D77N protein preparations the prominent 55 kDa band
corresponds to the self-cleavage product of VPgPro (or D77N) while the 82 kDa
fainter band corresponds to the full length VPgProspecies. Sampleswere loaded
on a SDS-polyacrylamide gel and were stained with Coomassie blue.
93S. Cotton et al. / Virology 351 (2006) 92–100

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Role of the eIF(iso)4E–VPgPro interaction in translation
inhibition
We tested the D77N mutant of VPgPro on its capacity to
interfere with translation in vitro. The D77N mutant contains
an asparagine at position 77 of the protein instead of an
aspartic acid residue; the resulting protein is unable to interact
with eIF(iso)4E and virus infectivity is abolished (Léonard et
al., 2000). We reasoned that if the eIF(iso)4E–VPgPro
interaction is essential for the inhibition of translation, the
addition of D77N into WGE or RRL translation systems
would not interfere with translation of the m7G-luciferase
RNA. Increasing amounts of GST–VPgPro, GST or GST–
D77N were added to translation reactions (WGE and RRL).
GST–VPgPro or GST–D77N both decreased translation of the
reporter gene in the same fashion, in both the RRL and WGE
systems. Addition of GST had no effect on translation of the
reporter RNA (Fig. 2).
Furthermore, to test whether the VPgPro-mediated transla-
tion inhibition was linked to the sequestration of eIF(iso)4E by
VPgPro, different concentrations of VPgPro were pre-incubated
with T. aestivum eIF(iso)4E before addition to the translation
reaction. Addition of 24 or 48 pmol of VPgPro inhibited capped
luciferase RNA translation even in the presence of excess eIF
(iso)4E (Fig. 3A). Myoshi et al. (2005) have previously reported
that the GST portion of GST–VPg interferes with the binding of
TuMV VPg to eIF(iso)4E from A. thaliana in pull-down assays.
However, in our study, GST-tagged TuMV VPgPro did interact
withwheateIF(iso)4EinELISA-basedbindingassays(Fig.3B).
Binding was specific as no signal was detected in absence of
primary antibody or when eIF(iso)4E was replaced with T7-
tagged β-galactosidase.
TuMV VPgPro and NV VPg degrade reporter RNA
We monitored the fate of reporter RNA during in vitro
translation to investigate how VPgs affect protein translation.
Fig. 3. Inhibition of translation of luciferase reporter RNA by GST–VPgPro in
the presence of eIF(iso)4E. (A) Ratio of the luciferase light units (synthesized
from capped luciferase RNA) from translation reactions containing GST–
VPgProover that of reactions containing GSTin WGE incubated or not with eIF
(iso)4E. (B) GST–VPgPro interaction with wheat T7-eIF(iso)4E as demon-
stratedby ELISA-based bindingassay. Wells werecoated with1.5 μg of purified
GST–VPgPro and incubated with 1.5 μg of E. coli recombinant T7-tagged eIF
(iso)4E (bar 1) or T7-tagged β-galactosidase (bar 3). Protein retention was
detected using a monoclonal anti-T7-tag antibody. Non-specific binding of the
secondary antibody was verified by incubating VPgPro and eIF(iso)4E in
absence of anti-T7 tag antibody (bar 2). Error bars represent the standard error of
the mean. Groups a and b are statistically different (P < 0.001).
Fig. 2. Translation inhibition of reporter RNA by GST–VPgPro. (A) Relative
light units (RLU) of luciferase obtained when adding capped luciferase RNA in
WGE translation system in the presence of GST, GST–VPgPro or GST–D77N.
(B)CappedluciferaseRNAwas addedtoRRLtranslationsysteminthe presence
of GST, GST–VPgPro or GST–D77N and luciferase RLU was measured. These
experiments were repeated at least three times.
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After addition of luciferase RNA in the RRL in presence of
GST or GST–VPgPro from TuMV, or in presence of NV VPg,
RNAwas collected at different times and purified. Fig. 4 shows
that reporter RNA is degraded when TuMV GST–VPgPro or
NV GST–VPg, but not GST, is added to the translation assay.
An incubation period as short as 5 min was sufficient to allow
TuMV GST–VPgPro to degrade the reporter RNA. Ribosomal
RNAwas not affected by the addition of either protein to the in
vitro RRL.
mRNA turnover can be substantially increased when
translation is arrested (Stanssens et al., 1986). We tested
whether or not RNA degradation was triggered by the absence
of translation. Reporter RNA was incubated without added
protein or with 48 pmol of GSTor GST–VPgPro from TuMVin
WGE in presence of cycloheximide. The addition of 600 μM of
cycloheximide to the in vitro translation system completely
inhibited translation of the reporter luciferase RNA since no
light emission was detected (data not shown). Samples were
collected at different times and RNA was electrophoresed,
blotted and hybridized with a
complementary to the luciferase RNA. Fig. 5 shows that the
reporter luciferase RNA was degraded more rapidly in the
presence of TuMV GST–VPgPro compared to controls where
no protein was added or where GST was added.
32P-labelled RNA probe
TuMV VPgPro and NV VPg degrade total plant RNA
To test the involvement of cellular factors for the VPgPro
ribonucleolytic activity, 48 pmol of TuMV GST–VPgPro, NV
GST–VPgand GSTwere incubated withtotalplant RNA.Fig.6
shows that TuMV GST–VPgPro and NV GST–VPg degraded
total plant RNA within 30 min. In contrast with the RNA
degradation observed in the in vitro translation system (see Fig.
4), ribosomal RNA was degraded by GST–VPgPro and GST–
VPg.TotalplantRNAincubatedwithGSTwasnotdegradednor
was the RNA sample incubated with no added protein.
The VPg domain of TuMV is sufficient for the degradation of
total plant RNA
VPgPro auto-catalytically cleaves itself into two functional
domains: VPg and Pro (Laliberté et al., 1992). Since NV VPg
degraded RNA, we verified if the VPg domain of VPgPro was
responsible for the observed ribonucleolytic activity. We
constructed and purified the VPg domain of TuMV VPgPro
and added the protein to total plant RNA. TuMV GST–VPg was
able to degrade total plant RNA as efficiently as GST–VPgPro
(Fig. 7). No RNA degradation was observed when total RNA
was incubated without protein or with GST.
Effect of EDTA and heat treatment on TuMV VPgPro
ribonucleolytic activity
The requirement for divalent cations for VPgPro catalytic
activity was tested by incubating total plant RNA with TuMV
GST–VPgPro in increasing concentrations of EDTA. The
results showed that GST–VPgPro nuclease activity was not
completely inactivated by the addition of EDTA concentrations
ranging from 1 to 10 mM (Fig. 8A). However, degradation
products increased in size with increasing amounts of EDTA. To
investigate whether RNA degradation is due to enzymatic
Fig. 4. Reporter RNA degradation in the presence of GST, TuMV GST–VPgPro
or NV GST–VPg in a RNA stability assay. The proteins were incubated in a
RRLin the presenceof cappedluciferaseRNA andRNAsamples werecollected
at different time points. Total RNAwas run on an agarose-formaldehyde gel and
transferred to a nylon membrane which was incubated with a32P-labelled RNA
probe complementary to luciferase RNA. The 18S rRNAwas used as a loading
reference.
Fig. 5. Reporter RNA degradation in the presence of GST or TuMV GST–
VPgPro in absence of translation. 48 pmol of GST or TuMV GST–VPgPro
proteins were incubated in WGE in the presence of capped luciferase RNAwith
600 μM of cycloheximide to arrest translation of the reporter RNA. Samples
were collected at different times and RNAwas purified. Total RNAwas run on
an agarose-formaldehyde gel and transferred to nylon. The membrane was
incubated with a32P-labelled RNA probe complementary to luciferase RNA.
18S rRNA was used as a loading reference.
Fig. 6. Plant total RNA degradation by GST–VPgPro of TuMVand GST–VPg
of NV. Total RNA was incubated without protein or with 48 pmol of GST,
TuMV GST–VPgPro or NV GST–VPg for 30 min. The RNAwas purified, run
on an agarose gel and stained with ethidium bromide.
95S. Cotton et al. / Virology 351 (2006) 92–100

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cleavage, heat-denatured GST–VPgPro was incubated with
total plant RNA; no RNA degradation was observed (Fig. 8B).
Discussion
One of the roles of the cap structure found at the 5′ end of
most eukaryotic mRNAs and some viral RNAs is to interact
with the cellular host translation initiation machinery, namely
the eIF4F complex (Pestova and Hellen, 2000). eIF4E is part
of this complex and recognizes the 5′ cap structure which will
eventually lead to the recruitment of the small ribosomal
subunit (reviewed by Sachs et al., 1997). The presence of a
VPg at the 5′ end of many viral RNAs suggests that a different
mechanism is at play for translation initiation of these viral
genomes.
Potyvirus VPgs have been shown to interact with eIF4E
isoforms (Kang et al., 2005; Myoshi et al., 2005; Wittmann et
al., 1997), as is also the case for calicivirus VPg (Goodfellow
et al., 2005). The VPg–eIF4E interaction is important for virus
infection since mutations in either VPg or eIF4E of
potyviruses lead to the reduction of symptoms or the absence
of infection (Duprat et al., 2002; Lellis et al., 2002; Léonard et
al., 2000). TuMV VPgPro can also interact with PABP in
planta (Léonard et al., 2004). Interaction of VPg proteins with
translation factors has lead to the suggestion that they may
play a critical role in assembly of the viral translation initiation
complex (Daughenbaugh et al., 2003; Léonard et al., 2004).
The VPg of caliciviruses was shown to act as a ‘cap substitute’
for viral RNA translation and the effect is dependent on the
interaction with eIF4E. However, it is paradoxical that the
addition of NV VPg to an in vitro translation system was
found to inhibit translation of reporter RNAs (Daughenbaugh
et al., 2003).
We report here on the effect of TuMV VPgPro on mRNA
translation in vitro. We show that recombinant VPgPro
inhibits translation of capped reporter RNA, both in plant
and animal in vitro translation systems. One hypothesis that
would explain the effect of VPg on translation is that
sequestration of eIF(iso)4E by VPgPro in the extract could
interfere with translation. eIF4E has been reported to be the
least abundant translation factor, and perhaps the rate limiting
one, in animal cells (Duncan et al., 1987). However, the
inhibition of translation was not relieved by addition of
supplementary eIF(iso)4E in the translation system. AVPgPro
mutant that could no longer interact with eIF(iso)4E (Léonard
et al., 2000) was also tested. The mutant could still inhibit
translation. Our results do not support the hypothesis that the
interaction between VPgPro–eIF(iso)4E is responsible for the
inhibition of translation.
Given these results, we investigated whether the presence
of VPg or VPgPro in the translation system could have a
destabilizing effect on the reporter RNA. The addition of
TuMV GST–VPgPro led to the degradation of reporter RNA
over time. RNA remained intact when GST protein purified in
the same way as VPgPro was added to the translation system.
The effect was linked to the presence of VPgPro as the rest of
the fusion protein expressed without viral sequences did not
lead to RNA degradation, therefore eliminating the possibility
Fig. 7. Plant total RNA degradation by GST–VPg domain of TuMV. Total RNA
was incubated without protein or with 48 pmol of GST, TuMV GST–VPg or
TuMV GST–VPgPro for 30 min. The RNAwas purified, run on an agarose gel
and stained with ethidium bromide.
Fig. 8. Effect of EDTA and heat denaturation on the RNase activity of TuMV
VPgPro. (A) Total RNA was incubated with 48 pmol of GST or TuMV GST–
VPgPro for 30 min at 25 °C with increasing concentrations of EDTA (0, 1, 5,
and 10 mM). (B) Total RNA was incubated with 48 pmol either native or heat
denatured (95 °C for 15 min) of GST or TuMV GST–VPgPro for 30 min at
25 °C.
96 S. Cotton et al. / Virology 351 (2006) 92–100

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that a contaminating RNase from E. coli was co-purified along
with the fusion tags. RNA degradation was not the result of
translation inhibition as it was observed in translation
reactions arrested by the addition of cycloheximide; RNA
degradation was observed only in samples that contained
VPgPro protein.
Daughenbaugh et al. (2003) have also observed an inhibition
of translation when NV VPg was added to an in vitro translation
system. We speculated that both the NV VPg and TuMV
VPgPro proteins inhibit translation through RNA destabiliza-
tion and/or degradation. In our experiments, the addition of NV
to the in vitro translation system lead to the degradation of
reporter RNAs as was observed for TuMV. Given that the
TuMV VPgPro and NV VPg proteins were purified according
to different protocols and share little homology, it is unlikely
that both proteins interact with the same contaminating RNAse
from E. coli. Since the VPg protein was always predominant
relative to the full length VPgPro protein in the protein
preparations, we examined the capacity of the VPg domain
alone to degrade RNA. As seen with NV, we also observed that
the VPg domain of TuMV was sufficient for RNA degradation.
The proteinase portion of VPgPro was not required for
ribonuclease activity.
We studied the effect of purified recombinant proteins
incubated with total plant RNA, without the components of a
translation reaction. Purified RNAwas degraded in the presence
of either TuMV GST–VPgPro or NV GST–VPg. This suggests
that both proteins have a ribonucleolytic activity in vitro
without the need for cellular factors. The ribonuclease activity is
therefore associated with the VPg, and is not the result of the
activation of a latent RNAse activity from a cellular protein
present in the translation system.
Since both NV and TuMV VPg displayed RNAse activity,
we examined the two sequences for homology; alignment of the
two sequences revealed less than 13% overall identity (Fig. S1).
A stretch of 15 amino acids is well conserved between the two
proteins (60% identity and 80% similarity). This region
overlaps with the nucleotide tri-phosphate binding domain
identified and experimentally confirmed for Potato virus A,
another potyvirus (Puustinen and Mäkinen, 2004). A phos-
phate-binding site may be correlated with a ribonuclease
activity as was observed in other plant ribonucleases with no
clear homology to known ribonucleases (Bantignies et al.,
2000; Hoffmann-Sommergruber et al., 1997).
The RNAse activity displays the characteristic feature of an
enzyme, i.e. sensitivity to heat denaturation. The ribonuclease
activity of VPgPro was not completely inactivated by the
addition of EDTA. This is not uncommon as EDTA insensitive
RNases are widespread and have been previously reported in
different organisms (Mishra, 2002; Yen and Green, 1991).
It was previously shown that potyvirus replication is
associated with disappearance of cellular mRNAs. It was
suggested that host mRNA shutoff could be achieved, in part,
through the degradation of host transcripts (Aranda et al., 1996).
Interestingly, viral proteins inducing host shutoff through their
ability to degrade host mRNAs have been reported for many
animal viruses (Hulst and Moormann, 2001; Laidlaw et al.,
1998; Smiley et al., 2001). For example, proteins of RNA
viruses such as the Influenza virus, Leishmania RNA virus 1–4,
of flaviviruses and of coronaviruses were shown to have
ribonuclease activity (Bhardwaj et al., 2004; Li et al., 2005;
Hulst and Moormann, 2001; Klumpp et al., 2001; Ro and
Patterson, 2000). We hypothesize that TuMV VPg contributes
to host mRNA degradation through its ribonucleolytic activity.
TuMV could enhance its access to the translation and
replication components of the cell by degrading host mRNAs.
For example, Influenza virus proteins, using a similar strategy,
bind to capped mRNA and hnRNA molecules in the nucleus of
infected cells and cleave the capped host RNA molecules
(Klumpp et al., 2001).
The VPgPro of PVBV was shown to have DNase activity
(Anindya and Savithri, 2004) and could explain in part the
transcriptional shutdown associated with potyviral infection
(Aranda and Maule, 1998). The D77N mutation, which
impairs eIF(iso)4E binding, did not result in reduction of
translation inhibition or ribonuclease activity. This is interest-
ing in light of the results obtained by Anyanda et al., 2004)
with the D81N mutant of PVBV (equivalent to D77N mutant
of TuMV) which showed reduced DNase activity. The eIF4E-
interacting domain does not seem to be important for VPg
ribonuclease activity; this suggests that distinct regions of VPg
might be important for its nuclease activities.
It is interesting that rRNA was degraded when VPgPro was
added to purified plant RNA (i.e. where proteins had been
removed). However, rRNA remained intact but reporter RNA
was degraded when VPg was added to RRL translation
system. The unspecific RNase activity displayed by VPg
raises the question of how TuMV protects its own RNA
against degradation. It is possible that cellular factors present
in infected plant cells (and absent from the in vitro translation
systems) regulate VPg's ribonucleolytic activity. Consistent
with this hypothesis is the observation that the RNase activity
associated with the Herpes simplex virus (HSV) vhs protein
exhibits a higher specificity in vivo (Krikorian and Read,
1991; Kwong and Frenkel, 1987; Oroskar and Read, 1989;
Zelus et al., 1996) than in an in vitro RRL (Lu et al., 2001).
Vhs-mediated host shutoff is characterized by disruption of
pre-existing polyribosomes, and accelerated turnover of host
mRNA. Vhs displays little sequence specificity in vitro and
targets most, if not all, cellular and viral mRNAs, in vivo other
cytoplasmic transcripts such as rRNA, tRNA and 7SL RNA
are spared during infection. We speculate that rRNA was not
degraded in our in vitro translation system because of the
protection offered by the ribosome ribo-nucleoprotein com-
plex. The number of host proteins involved in replication/
translation of virus RNA is growing and the involvement of a
large ribonucleoprotein complex is an emerging theme across
positive-strand RNA viruses (reviewed by Thivierge et al.,
2005). It is also possible that the ribonucleoprotein complex
formed during replication and translation of the viral RNA
may protect that RNA from degradation, as we observed with
rRNA embedded in ribosomes. The association of viral RNA
with different cellular factors, perhaps as a result of different
subcellular localizations, may regulate the specificity of the
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nucleolytic activity. It is also possible that the conditions used
in vitro altered the specificity of the activity. The localization
of ribonuclease activity to the nucleus, and not the cytoplasm,
would allow an extensive reprogramming of host gene
expression while protecting viral RNA. The VPgPro protein
of potyviruses is normally found in the nucleus of infected
cells (Schaad et al., 1996).
In this report, we have shown in vitro that TuMV VPg
exhibits an RNase activity. The involvement of VPg as
stimulator of viral translation by acting as a cap substitute and
its participation in host mRNA translation shutoff by acting as a
nuclease are not mutually exclusive activities. VPg may interact
with eIF4E isoforms to facilitate the recruitment of the host
translation apparatus to its RNA, while removing host mRNAs
to reduce competition. Further work is needed however to
assess the role of VPg RNase activity during potyvirus infection
in planta and how TuMV's RNA, if its the case, avoids
degradation.
Materials and methods
Expression and purification of recombinant proteins
Sequences encoding TuMV VPgPro and TuMV D77N were
PCR-amplified from the full-length TuMV cDNA clone
(p35Tunos) and its mutant form p35Tunos-D77N, respectively
(Sanchez et al., 1998; Léonard et al., 2000), in order to construct
vectors for the expression of GST-fused VPgPro and D77N
proteins.Primersused for amplification were VPgPro–NcoI(5′-
ATCGTACCATGGCGAAAGGTAAGAGGCAAAG-3′) and
VPgPro–EcoRI (5′-ATCTTCGAATTCTTATTGTGCTA-
GACTGCCGTG-3′). The amplified fragments were digested
with NcoI/EcoRI and cloned into similarly digested pET41(b)
(Novagen). Both constructions resulted in the expression of a
fusion protein containing a S-Tag, six histidine residues and a
GST tag at the N terminus.
For construction of the vector coding for the N-terminus
GST–VPg fusion protein, TuMV VPg sequences were PCR-
amplifiedfromp35TunosusingprimersVPg–EcoRI(5′-ATCC-
GAATTCCGGAAAGGTAAGAGGCAAAG-3′) and VPg–
NotI (5′-CTTCGCGGCCGCTTACTCGTGGTCCACTGG-
GAC-3′). The amplified fragments were digested with EcoRI/
NotI and cloned into similarly digested pGEX-6P1 (Amersham
Biosciences).
BL21(DE3) (for pET41(b)-based constructs) and BL21 (for
the pGEX-6P1-derived construct) E. coli cells containing
recombinant plasmids were cultured at 37 °C to an OD600of
0.6 and protein expression was induced with 1 mM IPTG for 3 h
at 30 °C. Bacterial cells were collected by centrifugation and
resuspended in buffer A (4.3 mM Na2HPO4, 1.47 mM KH2PO4,
137 mM NaCl, 2.7 mM KCl, pH 7.3). The cells were disrupted
by sonication and the lysate was centrifuged at 39,000 × g for
20 min. The supernatant was used for affinity purification of
either GST–VPgPro, GST–VPg or GST–D77N.
The protein extract was added to GST-Bind resin (Novagen)
according to the manufacturer's protocol and incubated at room
temperature with agitation for 30 min. Beads were washed three
times with buffer A and collected by centrifugation for five min
at 500×g. The fusion proteins were eluted from the resin in a
buffer containing 10 mM reduced glutathione and 50 mM Tris–
HCl pH 8.0. Protein concentration was measured using a
Bradford assay (Bio-Rad) using bovine serum albumin as
standard. GST controls (the pET41(b) or pGEX-6P1 vectors
without inserts) were expressed and purified using the same
conditions.
The bacterial clones for expression of NV GST–VPg
(pGEX–4T1 NV GST–VPg) and wheat eIF(iso)4E (pETtag
(iso)4ETa) were kindly provided by M.E. Hardy (Montana State
University, MT, USA) and J.-F. Laliberté (INRS-Armand
Frappier, QC, Canada) respectively. NV GST–VPg and wheat
eIF(iso)4E were expressed and purified as described previously
(Daughenbaugh et al., 2003; Léonard et al., 2000).
In vitro translation
The pGEM-luc vector (Promega) containing a luciferase
cDNA was linearized with XhoI and used as template for
synthesis of capped RNA. m7G-luciferase RNA was tran-
scribed using the mMessage mMachine SP6 system (Ambion).
RNA was denatured for three min at 65 °C before use. One μg
of reporter RNA was translated in a 50-μl reaction containing
25 μl of wheat germ extract (WGE), 40 U of RNAGuard
RNase inhibitor (Amersham Biosciences) and 10 μM of amino
acid mixture (Promega). Different concentrations of GST,
TuMV GST–VPgPro, TuMV GST–D77N and NV GST–VPg
protein were added to the translation mix. The reactions were
incubated at 25 °C for 2 h and light emission was measured
after the addition of 100 μl of luciferase substrate (Promega).
The experiment was conducted at least three times. The in
vitro translation assays using RRL were performed similarly
but incubated at 30 °C for 90 min. Experiments on the effect
of the VPgPro–eIF(iso)4E interaction on translation of
reporter RNA in WGE were conducted using the same
protocol but the GST and GST–VPgPro proteins were pre-
incubated with eIF(iso)4E at 25 °C for 15 min before addition
to the translation system. In these assays, concentrations of 24,
48 or 96 pmol of eIF(iso)4E were used with 12, 24 or 48 pmol
of GST or of GST–VPgPro.
VPgPro–eIF(iso)4E ELISA binding assays
GST–VPgPro protein (100 μl of protein at 15 ng μl−1in PBS
buffer) was adsorbed to wells of a polystyrene plate (Costar) by
overnight incubation at 4 °C and wells were blocked with 5%
milk PBS solution for 2 h at room temperature. T7-labelled
wheat eIF(iso)4E or β-galactosidase proteins were diluted in
PBSwith1%milkand0.1%Tween20andincubatedfor1.5hat
4°Cinthepreviouslycoatedwells.Detectionofretainedprotein
was achieved with a mouse monoclonal anti-T7-tag antibody
(Novagen) and horseradish peroxidase-coupled goat anti-mouse
immunoglobulin (Pierce). Between each incubation, wells were
washed five times with PBS supplemented with 0.04% Tween
20.Enzymaticreactionswereperformedin100μlofOPDcitrate
buffer (50 mM citric acid, 100 mM sodium phosphate dibasic,
98S. Cotton et al. / Virology 351 (2006) 92–100

Page 8

pH 5.0, 0.5 mg/ml o-phenylenediamine dihydrochloride (OPD)
and0.1%hydrogenperoxide)andstoppedwithasolutionof3M
H2SO4. Absorbance was measured at 492 nm. Statistical
analyses were performed using the GLM procedure of SAS in
a randomized complete block design (RCBD). ANOVA was
used to detect statistical differences and LSD method used to
determine significant differences among means. SEM and
statistics were calculated for three biological replicates from a
minimum of three technical replicates (replicate of the assay on
the same microplate).
RNA stability assays
To assess RNA stability in translation reactions that were
arrested (in presence or not of VPgPro protein), in vitro
translation of luciferase RNA in WGE was performed in
presence of 600 μM of cycloheximide, an inhibitor of
ribosome translocation. Five μl of each translation reaction
were removed at 0, 5, 15 and 60 min after the addition of
luciferase RNA and RNA degradation was monitored using
Northern blot hybridizations with a
RNA probe.
32P-labelled luciferase
Total plant RNA degradation assays
RNA was extracted from Brassica perviridis using the
RNeasy Plant Mini Kit (Qiagen). Five μg of RNA (eluted in
RNase-DNase free water) were incubated with 48 pmol of
GST, TuMV GST–VPgPro, GST–VPg or NV GST–VPg
(eluted in 10 mM reduced glutathione and 50 mM Tris–HCl
pH 8.0) for 30 min at 25 °C. The volume of the reaction was
completed with RNase-DNase free water. RNA degradation
experiments following addition of EDTA and heat denaturation
(15 min at 95 °C) were carried out similarly. Samples were run
on an agarose gel and stained with ethidium bromide.
Agarose gel electrophoresis and Northern blot analysis
RNA samples were purified using the RNeasy MinElute
cleanupkit (Qiagen). Awash step with 200 μl of the RW1 buffer
(Qiagen) was added after the application of the sample on the
column to remove residual protein. RNA samples were eluted in
14 μl of RNase-free water and combined with four μl of RNA
sample buffer (20 mM HEPES, 1 mM EDTA, pH 7.8, with 50%
formamide and 6% formaldehyde). Following a 10 min
incubation at 65 °C, 2 μl of RNA loading buffer (95%
formamide, 0.025% xylene cyanol, 0.025% bromophenol blue,
18 mM EDTA pH 8, 0.025% SDS) were added and the RNA
samples were separated through a 1.5% agarose gel containing
6% formaldehyde in running buffer (20 mM HEPES, 1 mM
EDTA, pH 7.8, 6% formaldehyde). The gel was washed for 1 h
in diethyl pyrocarbonate-treated water. RNA was transferred to
a nylon membrane (Zeta-Probe, Bio-Rad) in 10× SSC pH 7
(1.5 M sodium chloride, 0.15 M sodium citrate). Following UV
cross-linking, the membrane was stained for five min in a
solution of 0.02% methylene blue and 0.3 M sodium acetate and
washed in water. The membrane was scanned and the coloration
was removed in 1 mM EDTA pH 8, 1% SDS. The membrane
was incubated for 4 h at 65 °C in 10 ml of hybridization buffer
(1 mg/ml BSA, 50% formamide, 5% SDS, 1 mM EDTA,
400 mM NaPO4pH 7.2) and incubated for 16 h with the32P-
labelled riboprobe. The membrane was washed twice with
washing buffer (0.1× SSC pH 7, 0.1% SDS, 1 mM EDTA) and
exposed to Kodak Biomax MS film.
Synthesis of32P-labelled riboprobes
The plasmid pGEM-luc (Promega) was linearized with
EcoRV. The riboprobe was synthesized for 2 h at 37 °C in T7
polymerase buffer (10 mM of DTT, 20 U of RNAGuard RNase
Inhibitor (Amersham Biosciences), 500 μM of CTP, GTP and
ATP (Invitrogen), 50 μCi of [α-32P]-UTP (Amersham Bio-
sciences), 500 ng of pGEM-luc/EcoRV) and 50 units of T7
polymerase (Invitrogen). RNase-free DNase I (10 U; Qiagen)
was added and incubated for 15 min at 37 °C. The probe was
purified with the QIAquick nucleotide removal kit (Qiagen).
Acknowledgments
We thank M.E. Hardy and J.-F Laliberté for the generous gift
of expression clones, F. Ponz for TuMV clones and J.-F.
Laliberté for critical comments. This work was supported by
grants to MGF from the National Science and Engineering
Research Council of Canada and from the Québec FQRNT fund
and the SEVE Centre in the form of fellowships.
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version at, doi:10.1016/j.virol.2006.03.019.
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