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Update on Ubiquitin and Plant Viruses
Ubiquitin and Plant Viruses, Let’s Play Together!1
Catherine Alcaide-Loridan and Isabelle Jupin*
Laboratory of Molecular Virology, Institut Jacques Monod, Centre National de la Recherche ScientifiqueUniversité Paris Diderot Sorbonne Paris Cité, 75205 Paris cedex 13, France
Over the last 30 years, posttranslational modification
of proteins by ubiquitin (Ub) and degradation by the
ubiquitin proteasome system (UPS) has emerged as a
major regulatory process in virtually all aspects of cell
biology (Glickman and Ciechanover, 2002). Ub-mediated
degradation is widely conserved across the eukaryotic kingdoms, and judging from the large number of
Arabidopsis (Arabidopsis thaliana) genes involved in
Ub-dependent protein turnover, as well as accumulating biochemical or genetic studies, protein degradation
by the UPS plays a central role in many processes in
plants (Bachmair et al., 2001; Vierstra, 2009).
The involvement of the UPS in the signaling and
regulation of interactions between plants and pathogens is also becoming increasingly clear (for review,
see Zeng et al., 2006; Dreher and Callis, 2007; Citovsky
et al., 2009; Dielen et al., 2010; Marino et al., 2012).
Given the importance of Ub attachment in regulating
the fate and function of proteins, it is not surprising
that a wide range of pathogens, particularly viruses,
have found many ways to exploit and interfere with
the UPS (Shackelford and Pagano, 2005; Isaacson and
Ploegh, 2009; Randow and Lehner, 2009).
In the case of plant viruses, a connection between
the Ub system and virus infection was suggested by
early observations indicating that perturbation of the
Ub conjugation pathway altered plant responses to
infection with Tobacco mosaic virus (TMV; Becker et al.,
1993). However, appreciation of the full involvement
of the UPS in the regulation of plant-virus interactions
has long been limited by the extreme paucity of known
targets. It is only recently that a number of viral proteins acting in these processes have been identified and
possible mechanisms proposed. It is likely that many
more examples remain to be discovered.
In some cases, viral proteins appear to usurp the
UPS by targeting cellular proteins for degradation,
presumably to the benefit of the virus. In other instances, viral proteins are themselves the target of Ub
conjugation events. At present, it is not clear whether
these ubiquitination events are obligatory steps in the
viral life cycle or whether they represent failed attempts
1
This work was supported by the Centre National de la Recherche
Scientifique, the Universite Paris Diderot, and the Agence Nationale
de la Recherche (contract nos. ANR–06–BLAN–0062 and ANR–11–
BSV8-011).
* Corresponding author; e-mail [email protected].
www.plantphysiol.org/cgi/doi/10.1104/pp.112.201905
72
of the host cell to interfere with viral multiplication.
Our recent data indicate that some of these conjugation
events can be reversed by dedicated viral proteins,
highlighting their remarkable plasticity and suggesting
that their reversal may constitute an additional level of
regulation during viral infection.
Because such findings point to the importance of
Ub-related pathways during viral infection in plants,
the aim of this review is to summarize current knowledge and discuss different aspects of the involvement
of the UPS in plant-virus interactions. The exquisite
versatility of Ub conjugation, combined with the remarkable capacity of viruses to manipulate regulatory
pathways, hold promise for exciting discoveries in the
future.
THE Ub PROTEASOME PATHWAY
Ub is a 76-residue protein that is highly conserved
throughout the eukaryotic kingdom. Attachment of
Ub to cellular proteins (referred to as ubiquitination or
ubiquitylation) is involved in the regulation of many
signaling pathways and also plays an important role
in protein homeostasis (Glickman and Ciechanover,
2002). Three distinct phases in the ubiquitination process are controlled by three classes of enzymes (Fig. 1):
(1) activation of Ub via a Ub-activating enzyme (E1),
during which Ub is transferred onto the E1; (2) transfer
of Ub from the E1 enzyme to a Ub-conjugating enzyme
(E2); and (3) transfer of Ub from the E2 enzyme onto
the protein substrate, a process achieved by an E3 ligase, which coordinates ubiquitination by providing a
binding platform for E2 enzymes and specific substrates. E3 ligases constitute a large family of proteins
that mediate the specificity of substrate ubiquitination,
and as such, they constitute very appealing targets, not
only for pharmaceutical companies but also for viral
pathogens.
Ub is linked to the target protein via an isopeptide
bond between its C-terminal Gly residue and an acceptor amino acid of the target protein substrate, in
most cases a Lys residue. This modification (monoubiquitination) can then be extended by the ligation of
additional Ub molecules. In that case, a Lys residue of
Ub serves as a conjugation site for the addition of the
next Ub molecule, generating polyubiquitinated chains.
Any of the seven Lys residues (Lys-6, Lys-11, Lys-27,
Lys-29, Lys-33, Lys-48, and Lys-63) present in Ub can
serve as an acceptor, resulting in different branching
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Ubiquitin and Plant Viruses, Let’s Play Together!
Figure 1. The UPS. Ubiquitination of a
target protein substrate occurs through
the sequential action of three classes of
enzymes: a Ub-activating enzyme (E1),
a Ub-conjugating enzyme (E2), and a
Ub ligase (E3). It can be reversed by the
action of DUBs. By reiterative rounds
of ubiquitination, polymeric Ub chains
can be generated, whose function depends on the type of Ub chains attached to the target substrate.
patterns with different topologies (Xu and Peng, 2006).
Depending on the type of ubiquitination event (monoubiquitination versus polyubiquitination), the length of
the chain (fewer than or more than four Ub molecules),
and the type of chain branching, ubiquitination of proteins may have different functions in the cell. Whereas
the most studied polyubiquitin Lys-48 linkage is associated with degradation by the proteasome, alternative
ubiquitination events are destined for other cellular
processes, such as subcellular localization, protein activation, or protein-protein interactions, thus illustrating
the exquisite versatility of Ub conjugation (Ikeda and
Dikic, 2008). Specific functions associated with noncanonical chain types remain so far undetermined in plants.
Ubiquitination can be reversed by the action of enzymes known as Ub hydrolases or deubiquitinating
enzymes (DUBs; Komander et al., 2009). Most of these
enzymes are Cys proteinases that cleave isopeptidase
bonds. They either trim poly-Ub chains or remove
them from substrate proteins, thus contributing to the
reversal of signaling events, to protein stabilization,
and to Ub homeostasis in cells. The importance of
DUBs in the regulation of cellular processes is only
now beginning to emerge.
HIJACKING OF THE UPS BY PLANT VIRUSES
Members of several different groups of plant viruses
have been shown to exploit the UPS, in all likelihood
for their own benefit, using a variety of mechanisms
(Table I). Reports have described their ability to induce, inhibit, or modify the specificity of Ub-related
host enzymes, in particular E3 ligases. In addition, an
example of a virus-encoded DUB was reported recently, indicating that plant viruses, like their animal
counterparts, may also encode for Ub-related enzymes. Such mechanisms may be beneficial to the
virus either by creating a more favorable cellular
environment or by inhibiting host defense mechanisms. Similar strategies have also been reported in
the case of bacterial and fungal pathogens, as
reviewed in this issue (Marino et al., 2012).
Induction of the Expression of Host UPS-Related Proteins
A number of proteins related to the UPS, including
Ub itself, have been reported to be induced upon viral
infection (Aranda et al., 1996; Whitham et al., 2003;
Takizawa et al., 2005; Ye at al., 2011), but in most cases,
it is unclear whether increased expression is necessary
to degrade specific cellular or viral proteins or whether
it is part of the general cellular stress response due to
viral protein expression and accumulation (Aparicio
et al., 2005; Vitale and Boston, 2008; Sugio et al., 2009).
One example, however, points to the importance of
the induction of a specific E3 ligase for the control of
viral infection. In the geminivirus Beet severe curly top
virus (BSCTV), expression of the C4 protein, a major
determinant of pathogenesis affecting cell division,
was found to promote expression of the RING-type
E3 ligase RKP (for related to KPC1; Lai et al., 2009).
Interestingly, RKP regulates the cell cycle through
degradation of the cell cycle inhibitors ICK/KRPs (for
interactors/inhibitors of Cdc2 kinases/Kip-related
proteins; Ren et al., 2008), and modification of the
accumulation level of these protein substrates was also
observed in BSCTV-infected plants. As impairment
of viral multiplication was observed in plants knocked
down for RKP as well as in those overexpressing ICK/
KRP, Lai et al. (2009) thus proposed a convincing model
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Alcaide-Loridan and Jupin
Table I. Usurping of the UPS by plant virus proteins
Proposed Mechanism
Proposed Function
References
Induction of E3
ligase RKP
Impairment
of E3 ligase
Degradation of cell
cycle regulator ICK
Inhibition of SAMDC1
degradation
Lai et al. (2009)
bC1
Inhibition of
E2 enzyme
S1UBC3
Decrease in global
polyubiquitination
TYLCSV
TYLCV
BCTV
C2/L2
Inhibition of CSN
activity
Modification of
cell cycle?
Inhibition of
methylation-dependent
gene silencing
Perturbation of
developmental and
hormonal signaling
pathways?
Perturbation of hormonemediated (jasmonate) defense
responses
TYLSCV
TGMV
ACMV
Rep
FBNYV
CLINK
Virus
Geminivirus
BSCTV
C4
BDCTV
C2
CLCuMV
Nanovirus
Motifs
of
Interest/
Activity
Cellular
or Viral
Target
Viral Genus
Viral
Protein
Impairment of SCF E3
ligase
via neddylation status
Induction of
Modulation of SCF E3
specific
ligase
F-box proteins
via overexpression of
specific
F-box proteins
Interaction with E1 Limited modification of
SUMO-conjugating
SUMOylation
enzyme
F-box
protein
Usurping SCF
E3 ligase
Polerovirus
BWYV
CABYV
P0
F-box
protein
Usurping SCF
E3 ligase
Enamovirus
PEMV-1
P0
F-box
protein
Potexvirus
PVX
P25
Benyvirus
BNYVV
P25
Tymovirus
TYMV
98K
Usurping SCF
E3 ligase?
Usurping SCF
E3 ligase?
Interaction with
F-box protein
Usurping SCF
E3 ligase?
Interaction with
TYMV 66K
polymerase
PRO/
DUB
activity
Interaction with SKP1
Interaction with pRB
degradation of pRB?
Interaction with ASK1
and ASK2
Proteasomeindependent
degradation of AGO1
Degradation of AGO1
Proteasome-dependent
degradation of AGO1
Degradation of
unknown protein
Inhibition of TYMV
66K polymerase
ubiquitination
and degradation
in which C4 affects BSCTV infection by regulating the
host cell cycle, to which viral replication is coupled,
through controlling the accumulation of ICK/KRPs.
Inhibition of Host E2 enzymes
In the geminivirus Cotton leaf curl Multan virus
(CLCuMV), the bC1 protein, a pathogenicity factor
encoded by the satellite b DNA, was reported to interact with the host Ub-conjugating (E2) enzyme
S1UBC3 (Eini et al., 2009). Overexpression of bC1 in
Zhang et al.
(2011)
Eini et al. (2009)
Lozano-Durán
et al. (2011a,
2011b)
Lozano-Durán
and Bejarano
(2011)
Castillo et al.
(2004)
Sánchez-Durán
et al. (2011)
Aronson et al.
(2000)
Lageix et al.
(2007)
Pazhouhandeh
et al. (2006)
Baumberger et al.
(2007)
Bortolamiol et al.
(2007)
Csorba et al.
(2010)
Fusaro et al.
(2012)
Chiu et al. (2010)
Modification of
cell cycle?
Inhibition of RNA
silencing
Inhibition of RNA
silencing
Inhibition of RNA
silencing
Regulation of host
defenses?
Thiel et al. (2012)
Regulation of viral RNA
replication
Chenon et al.
(2012)
transgenic plants led to a decrease in the global accumulation level of polyubiquitinated proteins, supporting the idea that bC1 inhibits the Ub conjugation
step in a nonspecific manner. The interaction of bC1
with S1UBC3 was also found to correlate with the severity of symptoms in plants, which are reminiscent of
those observed upon perturbation of the Ub system in
plants (Bachmair et al., 1990), and thus may reflect the
numerous perturbations in developmental and hormonal signaling pathways that are regulated by the
UPS (Kelley and Estelle, 2012).
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Ubiquitin and Plant Viruses, Let’s Play Together!
Usurping Host E3 Ligases through Recruitment
of F-Box-Containing Proteins
There is increasing evidence that usurping of host E3
ligases is a common strategy used by plant viruses. In
all cases reported so far, viruses were observed to
hijack a particularly versatile class of E3 Ub ligases,
designated SCF complexes (for SKP1/Cullin1/F-box/
RBX1; Lechner et al., 2006; Fig. 2). Such complexes
are composed of four components: RBX1 (for RING
BOX1), Cullin, and SKP1 (for S-phase kinase-related
protein1), which form a conserved scaffold that assembles with one particular F-box protein. This class of
E3 is the most prevalent in plants, with more than 700
F-box proteins having been identified through genome
analysis and domain homology (Gagne et al., 2002).
F-box proteins serve as substrate-specific adapters,
binding to SKP1 through their F-box motif and recruiting protein targets to the core ubiquitination
complex by means of a specific protein-protein interaction domain.
Encoding a protein bearing an F-box motif may allow viruses to hijack the host E3 ligase core complex to
promote the degradation of specific key cellular proteins.
The identification of such proteins remains challenging,
as their substrates are expected to be ubiquitinated
and degraded as a result of their association with the
enzyme.
The first example of this putative viral hijacking was
reported for the multicomponent single-stranded DNA
nanovirus Faba bean necrotic yellow virus (FBNYV). The
virally encoded CLINK (for cell cycle link) protein
contains an F-box motif that interacts with SKP1 in
vitro and in vivo, suggesting its involvement in a bona
fide SCF complex (Aronson et al., 2000). Interestingly,
CLINK also harbors a motif known to interact with
the retinoblastoma tumor-suppressor protein pRB
(Aronson et al., 2000). Through alteration of pRB
Figure 2. Schematic representation of an SCF-type E3 ligase. The generic architecture of an SCF complex is shown. The F-box protein
serves as a substrate recognition subunit. The activity of the SCF
complexes is regulated by conjugation of the Cullin subunit to the UbL
RUB1. The COP9 signalosome contributes to this regulation due to its
derubylating activity.
activity, the virus acquires the capacity to modify cell
cycling and to force cells into DNA synthesis (Lageix
et al., 2007), thus creating a cellular environment favorable for efficient replication of the viral genome.
However, whether CLINK inactivates pRB by targeting its degradation, as described for animal papillomavirus, remains to be established.
Another example of viral proteins bearing an F-box
motif are the P0 proteins encoded by the poleroviruses
Beet western yellows virus (BWYV) and Cucurbit aphidborne yellow virus (CABYV; Pazhouhandeh et al., 2006),
which are potent suppressors of RNA silencing, one of
the major antiviral defense systems in plants (for review, see Ding, 2010). Both proteins were found to
interact with the Arabidopsis homologs of SKP1, ASK1
and ASK2 (for Arabidopsis SKP1-like). Interestingly,
the suppressor activity of P0 was found to correlate
with the functionality of its F-box, supporting the idea
that P0 targets an essential component of the host RNAsilencing pathway for degradation (Pazhouhandeh et al.,
2006). This component was identified as being ARGONAUTE1 (AGO1; Baumberger et al., 2007; Bortolamiol
et al., 2007), the core component of the RISC complex (for
RNA-induced silencing) involved in the RNA-silencing pathway (for review, see Vaucheret, 2008). Interaction of P0 with AGO1 is proposed to interfere with
its assembly within RISC complexes (Csorba et al.,
2010), leading to its degradation by an as yet unclear
proteasome-independent mechanism (Baumberger et al.,
2007).
Such findings are particularly exciting because they
not only exemplify a novel viral strategy to counteract
antiviral plant defenses but also raise the provocative
possibility of an interplay between protein degradation and the RNA-silencing pathway.
The capacity of virus-silencing suppressors to affect
AGO1 protein stability has been reported for two additional examples: the enamovirus Pea enation mosaic
virus-1 (PEMV-1) protein P0 (Fusaro et al., 2012) and
the potexvirus Potato virus X (PVX) protein P25 (Chiu
et al., 2010). In the case of PVX, targeting of AGO1
appeared dependent on proteasome activity (Chiu et al.,
2010).
Knockdown of the expression of the SKP1 subunit in
the SCF complex was found to severely impair BWYV
infection (Pazhouhandeh et al., 2006) and to cause a
delay in PVX systemic infection (Ye at al., 2011), thus
confirming the importance of this type of E3 ligase in
viral infectivity. However, further studies are required
to determine whether the effect on PVX infectivity relates to AGO1-mediated suppression of RNA silencing
or to another, possibly coordinated, effect of the UPS
on viral cell-to-cell movement (see below; Ye and Verchot,
2011).
More recently, an alternative pathway was proposed, where the virus itself does not encode an F-box
protein but instead interacts with a plant F-box protein. The protein P25 encoded by the benyvirus Beet
necrotic yellow vein virus (BNYVV) has been described
as a pathogenicity factor leading to the appearance of
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Alcaide-Loridan and Jupin
necrosis in susceptible sugar beet (Beta vulgaris). Yeast
two-hybrid experiments evidenced the interaction of
P25 with a number of candidate proteins involved in
ubiquitination (Thiel and Varrelmann, 2009), in particular a protein containing an F-box domain and two
Kelch repeats. The biological relevance of BNYVV P25
in terms of degradation remains unclear, but rather
than P25 being a substrate of this host F-box protein, it
has been proposed that P25 might inhibit the interaction between the SKP1 homolog ASK1 and the F-box
protein, thereby altering proper target recognition and
leading to cell necrosis in an as yet undefined manner
(Thiel et al., 2012).
Preventing Degradation by Impairment of Host E3 Ligases
In contrast to the examples given above, which exemplify a viral strategy aimed at destroying cellular
proteins, an alternative strategy consists of protecting
from degradation host proteins that are usually unstable.
In this context, S-adenosyl-methionine decarboxylase1
(SAMDC1) was identified as an interaction partner of
the geminivirus BSCTV-silencing suppressor protein C2
(Zhang et al., 2011). SAMDC1 proteasomal degradation
appears inhibited by BSCTV C2, a process that in turn
impacts host and viral DNA methylation, providing a
way to negatively regulate the gene silencing-mediated
antiviral defense mechanism in planta and to facilitate
viral multiplication (Zhang et al., 2011).
How BSCTV C2 achieves the stabilization of
SAMDC1 is unknown at present. However, data obtained in the case of other geminiviruses, such as Tomato yellow leaf curl Sardinia virus (TYLCSV), Tomato
yellow leaf curl virus (TYLCV), and Beet curly top virus
(BCTV), support the idea that the C2/L2 protein can
usurp the UPS by targeting a broad range of E3 ligases
all at once by acting on their neddylation/rubylation
status (Lozano-Durán et al., 2011a). Nedd8 (for neuronal
precursor cell-expressed developmentally downregulated8, also called RUB1 [for Related to Ubiquitin]
in plants) is a ubiquitin-like protein (UbL) whose reversible conjugation to the Cullin subunit of E3 ligase
is required for its activation (Hotton and Callis, 2008;
Fig. 2). RUB1 is conjugated to cullins via specific enzymes in a manner similar to that of Ub conjugation,
while deconjugation is achieved by the derubylation
activity of the COP9 signalosome complex (CSN;
Schwechheimer and Isono, 2010). Expression of C2/L2
proteins in transgenic Arabidopsis plants was found to
compromise the activity of the CSN over CUL1, which
thus accumulates in its rubylated form. As a consequence, plant pathways that are regulated by these
SCF complexes are altered (Lozano-Durán et al.,
2011a). Given the pleiotropy of physiological and developmental processes regulated by SCFs (Hua and
Vierstra, 2011), this capability of geminivirus proteins
to interfere with the activity of SCF complexes is an
extremely powerful strategy to interfere with plant
physiology, and in particular with hormone-mediated
plant defense responses. Processes related to jasmonate
biosynthesis and perception were found to be the major
targets of the TYLCSV C2 protein (Lozano-Durán et al.,
2011a).
Even more attractive is the observation that particular SCF complexes can escape this inhibition, provided that the corresponding specific F-box protein
is overexpressed, a process that indeed appears to occur during geminivirus infection (Lozano-Durán and
Bejarano, 2011). These results thus raise the tantalizing
idea that geminiviruses may have the ability to regulate distinct SCF complexes both positively and negatively simultaneously by playing with their constituent
subunits. It should be noted that the use of global
approaches has been instrumental in producing these
exciting findings.
Preventing Degradation by Virally Encoded
Deubiquitinating Enzymes
Ubiquitination processes can be reversed by the action of DUBs. Although several DUBs have now been
described in animal viruses, it is only recently that
such an activity was demonstrated to be encoded by
the plant virus Turnip yellow mosaic virus (TYMV;
Chenon et al., 2012). TYMV DUB activity is carried by
the Cys proteinase domain of the replication protein,
which is also involved in endoproteolytic processing of
viral proteins. It displays structural homologies with
the ovarian tumor protein, but, in contrast to the homologous DUBs encoded by animal viruses, TYMV
DUB does not exhibit a global effect on the accumulation level of polyubiquitinated proteins. Instead, it
was reported to specifically target the viral polymerase, shown previously to be a proteasomal substrate
(Camborde et al., 2010; see below), leading to its deubiquitination and subsequent stabilization. It is worth
noting that many plant viruses encode Cys proteinase
domains, and a subset of Flexiviridae members also
contain proteinase domains with homologies to cellular
ovarian tumor-like proteins (Makarova et al., 2000;
Martelli et al., 2007). If these viral enzymes could act as
DUBs, this would suggest an important function for
such an activity also in these viruses.
SUMOylation of Viral Replication Proteins
SUMO (for small ubiquitin-related modifier) is a
UbL protein that can be covalently attached to Lys
residues of target proteins via an enzymatic cascade
mechanistically similar to that of Ub (Ulrich, 2009).
The functions of SUMO conjugation (referred to as
SUMOylation) vary depending on the target protein,
as a major consequence of SUMOylation is to inhibit,
modify, or enable protein-protein interactions. Because
SUMO is conjugated to Lys residues, it can also compete with Ub conjugation, thereby modulating protein
stability and subcellular localization (Ulrich, 2009).
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Ubiquitin and Plant Viruses, Let’s Play Together!
The importance of SUMOylation is illustrated by
the cases of the geminiviruses Tomato golden mosaic
virus (TGMV), African cassava mosaic virus (ACMV),
and TYLSCV, whose replication proteins (Rep) interact with the host SUMO-conjugating enzyme E1
(Castillo et al., 2004). Altering SUMO expression (either positively or negatively) strongly reduced viral
replication (Castillo et al., 2004). Further analysis
revealed that the interaction between Rep and SUMO
E1 is required for viral DNA replication and viral
infectivity. Overexpression of Rep did not alter the
general SUMOylation pattern of plant proteins, but as
few additional conjugated proteins were detected, it
was proposed that modulation of SUMOylation may
be limited to specific host proteins, which remain to be
defined (Sánchez-Durán et al., 2011).
VIRAL PROTEIN TARGETS: IS Ub FRIEND OR FOE?
Information from genome-wide genetic or proteomic screens (Kushner et al., 2003; Panavas et al., 2005;
Serviene et al., 2006; Li et al., 2008; Gancarz et al., 2011;
Lozano-Durán et al., 2011b) supports the idea that
ubiquitination of viral proteins is important for viral
multiplication. Direct demonstration of Ub conjugation of viral proteins has also been reported in a few
cases (described further below; Table II). Yet, our
current understanding of the precise role of this posttranslational modification is limited: in addition to
the modification of protein turnover by proteasomal
degradation, Ub conjugation may affect protein localization, may serve as a molecular switch between
different functions, or may affect the ability of the viral
proteins to interact with specific host factors. This diversity of functions stems from the myriad ways in
which target proteins can be modified (e.g. monoubiquitination, multimonoubiquitination, or polyubiquitination; Fig. 1) and, in the latter case, also in the
type of Ub chain linkages or their length (Ikeda and
Dikic, 2008). However, such information is scarce in
the case of plant or plant viral proteins, and thus the
role(s) of such posttranslational modification(s) is often
undetermined. The interpretation of such data is
complicated further by the fact that UPS degradation
can correspond either to the regulation of functional
proteins carrying specific destruction signals or to direct the removal of damaged, misfolded, or overexpressed proteins. Such processes are reversible, thanks
to viral or cellular DUBs, and can also be competed out
by UbL proteins (such as SUMO), which may form
conjugates with specific target Lys residues, thus leading
to protection from Ub-mediated proteolysis. Finally,
whether viruses take any advantage of these degradation processes, or whether they correspond solely to
cellular defense responses, or both, is still unclear.
Monoubiquitination of Viral Replication Proteins
Viral replication is the central step of the infection
cycle, with the rapid production of huge numbers of
viral progeny in the infected cell. Viral genome replication requires the assembly of replication complexes
featuring the close association of both viral and host
components, in particular subcellular compartments.
In the case of the tombusvirus Tomato bushy stunt
virus (TBSV), Cdc34p E2 Ub-conjugating enzyme was
described as a novel component of the viral replication
complex and as critical for efficient replicase activity
(Li et al., 2008). Cdc34p was found to interact directly
with the TBSV p33 replication protein, leading to its
monoubiquitination and biubiquitination (Li et al.,
2008). Ubiquitination of p33 seems to have no effect on
its metabolic stability (Barajas and Nagy, 2010). Instead, it was found to contribute to the interaction with
ESCRT (for endosomal sorting complexes required for
transport) proteins (Barajas and Nagy, 2010), whose
temporary recruitment to sites of viral replication is
required for optimal replicase activity and protection
of the viral RNA template (Barajas et al., 2009a).
Ubiquitination of p33 thus appears to be required for
the proper targeting of TBSV replication complexes.
Polyubiquitination and Degradation of Viral
Replication Proteins
The assembly of viral replication complexes depends
on many critical interactions between various partners.
Modifying the proper stoichiometry of replication
complex subunits via selective degradation is thus
likely to affect the outcome of viral replication. In some
instances, proteasome subunits, E3 ligases, or cellular
DUBs have been shown to affect the efficiency of viral
replication, but such effects could not be linked directly to the degradation of viral proteins by the UPS
and possibly could be due to indirect effects caused by
cellular stress and/or perturbation of Ub homeostasis
(Barajas et al., 2009b; Yamaji et al., 2010; Gancarz et al.,
2011; Wang et al., 2011).
More direct evidence for the involvement of the UPS
in the metabolic stability and accumulation level of
viral replication proteins, and hence in the efficiency of
viral replication, has been demonstrated recently in
the case of TYMV.
TYMV 66K polymerase accumulates in infected cells
(Prod’homme et al., 2001), but this accumulation is
transient due to the degradation of 66K by the UPS at
late time points in viral infection (Camborde et al.,
2010). Degradation of the polymerase appeared to be a
limiting factor during viral infection, but as the polymerase bears a degradation signal, identified as a
PEST-like sequence (i.e. a region enriched in Pro, Asp,
Glu, Ser, and Thr residues; Rechsteiner and Rogers,
1996), that is conserved among tymoviruses (Héricourt
et al., 2000; Camborde et al., 2010), it is likely that
ubiquitination and/or proteasomal degradation of the
polymerase is important for the regulation of viral replication. Several hypotheses have been put forward
(Camborde et al., 2010): (1) low levels of polymerase
may help maintain the integrity of the viral genome, as
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Alcaide-Loridan and Jupin
Table II. Plant virus proteins targeted by the UPS
Viral Genus
Virus
Viral
Protein
Tombusvirus
TBSV
Motifs of
Interest
Type of Ubiquitination
Proposed Mechanism
Proposed Function
Reference
p33
Monoubiquitination
Interaction with Cdc34p
E2 enzyme and
ESCRT proteins
Proper targeting of
replication complexes
TBSV
p98
Polyubiquitination
Interaction with Rsp5
E3 ligase
Proteasome-independent
degradation
Regulation of viral
RNA regulation?
Host defense
mechanism?
Li et al. (2008)
Barajas et al.
(2009a)
Barajas and
Nagy (2010)
Barajas et al.
(2009b)
SPMV
CP
Monoubiquitination
TYMV
69K
Polyubiquitination
TYMV
66K
TMV
30K
Polyubiquitination
TMV
CP
Monoubiquitination
TMV
CP
Polyubiquitination
Hordeivirus
BSMV
CP
Monoubiquitination
Bromovirus
BMV
CP
Monoubiquitination
CP
Monoubiquitination
Tymovirus
Tobamovirus
Comovirus
CPMV
CPSMV
Caulimovirus CaMV
Polerovirus
PLRV
Potexvirus
PVX
PEST
sequence
Polyubiquitination
CP
PEST
precursor sequence
MP17
TGBp3
polymerase crowding favors viral RNA recombination; (2) the amount of polymerase available may
constitute a switch either in the replication process
itself or (3) during other steps of the viral multiplication cycle; or (4) ubiquitination as a means of escape
from the host surveillance mechanisms.
However, the most surprising finding was the recent
report that such ubiquitination and degradation processes can be counteracted by another TYMV-encoded
Proteasomal degradation
Dunigan et al.
(1988)
Drugeon and
Jupin (2002)
Preventing cell toxicity?
Regulation of viral
movement?
Host defense
mechanism?
Inhibition of RNA
silencing?
Proteasomal degradation
Regulation of viral
Camborde et al.
RNA replication?
(2010)
Host defense
mechanism?
Proteasomal degradation Preventing cell toxicity?
Reichel and
Beachy (2000)
Regulation of viral
Gillespie et al.
movement?
(2002)
Host defense
mechanism?
Dunigan et al.
(1988)
Proteasomal degradation
Removal of misfolded
Jockusch and
and insoluble proteins
Wiegand
(2003)
Dunigan et al.
(1988)
Dunigan et al.
(1988)
Dunigan et al.
(1988)
Proteasome-dependent and
Karsies et al.
-independent degradation
(2001)
Proteasomal degradation
Regulation of viral
Vogel et al.
movement?
(2007)
Induction of ERAD
Preventing cell toxicity Ju et al. (2008)
Translocation from the
Regulation of viral
Ye et al. (2011)
ER to the cytoplasm
movement?
Induction of SKP1
Host defense
Ye and Verchot
mechanism?
(2011)
Proteasomal degradation
Inhibition of RNA
silencing?
replication protein bearing a DUB activity (Chenon
et al., 2012). Because proteasomal degradation of 66K
could have been avoided easily by mutation of the
PEST sequence during evolution, this result points to
the importance of the dynamics and reversibility of
ubiquitination events and underscores the idea that the
virus has evolved to take advantage of both ubiquitination and deubiquitination events for implementing
precise temporal and/or spatial control of its life cycle.
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Ubiquitin and Plant Viruses, Let’s Play Together!
Polyubiquitination and Degradation of Viral
Movement Proteins
Following infection of an individual cell, plant viruses
spread from cell to cell through intercellular connections,
the plasmodesmata, by exploiting virus-encoded movement proteins (MPs; Lucas, 2006; Ueki and Citovsky,
2011). MPs are thought to form complexes with the viral
genome, docking it to plasmodesmata, where they then
increase plasmodesmatal permeability to allow its transport into neighboring cells. Strikingly, a number of viral
MPs accumulate only transiently during the early to mid
stages of viral infection, and the timing of expression
appears critical for viral infection (Maule, 1991). The
selective degradation of proteins is a recurrent theme
in regulatory mechanisms involving timing control
(Glickman and Ciechanover, 2002), and the UPS degradation of virus MPs indeed appears to be a rather
common phenomenon.
TMV MP was the first viral MP reported to be degraded in vivo, as proteasome inhibitors lead to increased stability of this protein, which then accumulates
in a polyubiquitinated form, presumably in the endoplasmic reticulum (ER; Reichel and Beachy, 2000). TYMV
MP was also shown to be very unstable in vitro, to be
polyubiquitinated, and to be a substrate of the proteasome (Drugeon and Jupin, 2002). More recently, Potato
leafroll virus (PLRV) MP and the PVX protein TGBp3
were also shown to be degraded by the proteasome
(Vogel et al., 2007; Ju et al., 2008). In the latter case,
translocating TGBp3 from the ER to the cytoplasm for
degradation was demonstrated to involve an endoplasmic reticulum-associated protein degradation (ERAD)
pathway, a component of the protein quality control
system that normally eliminates misfolded or unassembled proteins from the ER (for review, see Smith et al.,
2011). This was so far unprecedented in plant viruses (Ju
et al., 2008; Ye et al., 2011).
A number of hypotheses have been put forward to
explain the relevance of such observations during viral
infection. UPS degradation may be considered as a
specific host cell defense pathway against viral infection, as MP is central to the spread of viruses and, in
some instances, also appears to be a suppressor of
RNA silencing (Voinnet et al., 2000; Chen et al., 2004),
thus constituting a good target for plant defense responses. Such a hypothesis would be consistent with
the observation that improved TMV trafficking functions correlate with evasion of the host degradation
pathway (Gillespie et al., 2002).
In the case of PVX, however, degradation does not
seem to be a limiting factor in viral infection (Ju et al.,
2008), and it has been proposed that ERAD induction
is primarily a stress response, preventing the cytotoxicity and cell death that are linked to ER stress (Ye
et al., 2011). In turn, this process may also be considered as a viral strategy to maintain host viability,
which is both in the interest of the virus and its host
cell. That induction of the UPR (for unfolded protein
response) as a consequence of continued ER stress may
also be used by PVX as a means to stimulate AGO1
degradation, as reported above, constitutes an interesting possibility, although not yet demonstrated (Ye
and Verchot, 2011).
Monoubiquitination of Viral Structural Proteins
Monoubiquitination of the TMV coat protein was
described almost 25 years ago (Dunigan et al., 1988)
after observing that a minor fraction (estimated at an
average frequency of one per virion) was conjugated
to a host protein. This observation was soon extended
to the structural proteins of other viruses (i.e. Barley
stripe mosaic virus [BSMV], Brome mosaic virus [BMV],
Cowpea mosaic virus [CPMV], Cowpea severe mosaic virus
[CPSMV], and Satellite panicum mosaic virus [SPMV];
Hazelwood and Zaitlin, 1990), but the significance of
these observations has not been elucidated to date.
Polyubiquitination and Degradation of Viral
Structural Proteins
During viral infection, structural proteins are produced in enormous amounts and can constitute up to
20% of total cell protein content. As the error rate of
viral polymerases generates mutations at high frequency, it is not unexpected that misfolded and insoluble viral structural proteins are produced during
viral multiplication. It was shown for TMV that such
proteins are massively polyubiquitinated (Jockusch
and Wiegand, 2003). As functional proteins are not
targeted, these ubiquitination events are likely to correspond to a cellular protein response to stress (Sugio
et al., 2009) rather than a specific regulatory process.
In contrast, the precursor of the caulimovirus Cauliflower mosaic virus (CaMV) coat protein (CP) was
reported to contain three specific instability determinants (Karsies et al., 2001), which make this protein
likely to be the target of a regulatory process. One
uncharacterized degradation signal targets the CP
precursor for degradation by the proteasome, whereas
the other two, which display characteristics of PESTrelated sequences (Rechsteiner and Rogers, 1996), seem
to induce a proteasome-independent degradation
pathway. Although mutation of the degradation signals affected viral infectivity, how these data relate to
our current knowledge of the CaMV multiplication
cycle remains unclear.
CONCLUSION
Ub can intervene at each and every step of the viral
multiplication cycle, and in turn, viruses have developed many tools to usurp the UPS. There is converging
evidence on the importance of the UPS in the regulation
of viral infection, and it is likely that the development of
molecular or protein tools (i.e. tagged versions of Ub,
high-affinity Ub-binding traps, or sensitive antibodies)
will precipitate many more reports. Efforts to identify
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Alcaide-Loridan and Jupin
specific degradation signals may help discriminate between specific degradation and clearing of misfolded
proteins. As mentioned above, ubiquitinated substrate
proteins are not necessarily targeted for degradation,
as monoubiquitination or “atypical” chain types have
various other functions. Characterization of the linkage
type of Ub chains and determining their functions in
plants, as well as the existence of possible cross-talk
between Ub and UbL(s) or between Ub and other
posttranslational modifications, are missing at present,
and there is an urgent need to explore these questions in
the near future.
Regarding usurping of the UPS, this appears to be
done mostly by hijacking E3 ligases; the identification
and characterization of host substrates will confirm
the importance of this tactic, either for promoting a
favorable cellular environment or for blocking the activation of defense mechanisms. In this respect, it will
not be surprising if key plant defense regulators are
identified as substrates of the UPS upon viral hijacking. This is likely to provide exciting new insights into
the molecular mechanisms associated with plant-virus
interactions.
In recent years, our appreciation of the dynamic
aspects of Ub modifications has increased dramatically. It now appears that viruses probably benefit
from the spatial and temporal fine-tuning occasioned
by such reversible and versatile modification. Understanding where and when the ubiquitination of host
and viral targets operates could thus turn out to be as
important as identifying the substrates themselves.
Finally, the main question that remains open regards
the function of such modifications in the viral life cycle:
do they represent a viral strategy to enhance infectivity, a
host defense reaction, and a combination of the two?
To decipher this entanglement, a system-wide analysis
of the ubiquitination networks that are linked to viral
infection will be necessary. This will require a combination of modern proteomics approaches, reverse genetic
screens, and biochemical characterization of enzyme activities. We expect such studies to provide mechanistic
insights into the complexity of virus infection but also to
reveal how these mechanisms could be exploited to target viruses. The development of small molecules that
interfere with the activity of the Ub-related enzymes that
are essential for viral infectivity or are misregulated in
disease may thus possibly be exploited for the development of new antiviral strategies.
ACKNOWLEDGMENTS
We thank Prof. V. Citovsky for the invitation to write this review and Dr.
H. Rothnie for careful editing of the manuscript.
Received June 14, 2012; accepted July 14, 2012; published July 16, 2012.
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