Identification of Secret Agent as the O-GlcNAc Transferase ...

JOURNAL OF VIROLOGY, Aug. 2005, p. 9381–9387 Vol. 79, No. 15
0022-538X/05/$08.00ϩ0 doi:10.1128/JVI.79.15.9381–9387.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Identification of Secret Agent as the O-GlcNAc Transferase That Downloaded from http://jvi.asm.org/ on February 11, 2016 by guest
Participates in Plum Pox Virus Infection

D. Chen,1 S. Ju´arez,1 L. Hartweck,2 J. M. Alamillo,1 C. Simo´n-Mateo,1 J. J. P´erez,1
M. R. Fern´andez-Fern´andez,1† N. E. Olszewski,2 and J. A. Garc´ıa1*

Department of Plant Molecular Genetics, Centro Nacional de Biotecnolog´ıa (CSIC), Campus Universidad
Auto´noma de Madrid, 28049 Madrid, Spain,1 and Department of Plant Biology and Plant Molecular
Genetics Institute, University of Minnesota, Saint Paul, Minnesota 551082

Received 1 March 2005/Accepted 23 April 2005

Serine and threonine of many nuclear and cytoplasmic proteins are posttranslationally modified with
O-linked N-acetylglucosamine (O-GlcNAc). This modification is made by O-linked N-acetylglucosamine trans-
ferases (OGTs). Genetic and biochemical data have demonstrated the existence of two OGTs of Arabidopsis
thaliana, SECRET AGENT (SEC) and SPINDLY (SPY), with at least partly overlapping functions, but there
is little information on their target proteins. The N terminus of the capsid protein (CP) of Plum pox virus (PPV)
isolated from Nicotiana clevelandii is O-GlcNAc modified. We show here that O-GlcNAc modification of PPV CP
also takes place in other plant hosts, N. benthamiana and Arabidopsis. PPV was able to infect the Arabidopsis
OGT mutants sec-1, sec-2, and spy-3, but at early times of the infection, both rate of virus spread and
accumulation were reduced in sec-1 and sec-2 relative to spy-3 and wild-type plants. By matrix-assisted laser
desorption ionization–time of flight mass spectrometry, we determined that a 39-residue tryptic peptide from
the N terminus of CP of PPV purified from the spy-3 mutant, but not sec-1 or sec-2, was O-GlcNAc modified,
suggesting that SEC but not SPY modifies the capsid. While our results indicate that O-GlcNAc modification
of PPV CP by SEC is not essential for infection, they show that the modification has a role(s) in the process.

Dynamic modification of serine and threonine with O-linked O-GlcNAc modifications have been found in some structural
␤-N-acetylglucosamine (O-GlcNAcylation) is widespread proteins from different animal viruses such as the cytomegalo-
among nuclear and cytoplasmic eukaryotic proteins (33). The virus basic phosphoprotein (8), the adenovirus fiber protein
modification is essential for viability in both plants and animals (20), and the baculovirus gp41 protein (34), as well as in the
(9, 23). O-GlcNAcylation is a regulatory modification that nonstructural rotavirus NS26 protein (6). The biological rele-
shares many common traits with protein phosphorylation (26). vance of these modifications is yet unknown.
In addition, it has been documented for several proteins that
phosphorylation and O-GlcNAcylation can occur at the same Plum pox virus (PPV) is a potyvirus that in nature infects
site. While the details of how O-GlcNAcylation functions in fruit trees of the Prunus genus but that is also able to infect
specific pathways remain to be elucidated, some general experimentally different herbaceous hosts (18). The messen-
themes have emerged. The level of O-GlcNAcylation is dy- ger-polarity single-stranded genomic RNA of potyviruses is
namic and is influenced by hormonal signals, stress, and met- translated into a large polyprotein that is further processed by
abolic status, and the modification has roles in the regulation three virus-encoded proteases (21, 22). The potyviral genome
of transcription and protein synthesis and degradation. Distur- is encapsidated in flexuous rod particles made up of ϳ2,000
bance of cellular processes regulated by O-GlcNAcylation ap- units of a single type of capsid protein (CP) located at the C
pears to be involved in very important pathological disorders end of the viral polyprotein (25). The N- and C-terminal re-
such as Alzheimer’s disease (17) and diabetes (31). gions of the potyviral CP are surface exposed and can be
released from the virus particles by mild proteolysis with tryp-
The enzymes responsible for O-GlcNAc addition (O-GlcNAc sin (24). The CP of PPV has been shown to be modified by
transferases [OGTs]) are highly conserved in plants and ani- O-GlcNAcylation and phosphorylation. O-GlcNAc-modified
mals (33). OGT enzymes have an N-terminal tetratricopeptide residues were mapped to the N-terminal portion of the protein
repeat domain and a C-terminal catalytic domain. While ani- (3). In this paper, we have identified SEC as the plant OGT
mals have one OGT, Arabidopsis thaliana has two: Secret involved in these modifications and investigated the relevance
Agent (SEC) and SPINDLY (SPY) (9, 29). Genetic experi- of SEC function for virus infection.
ments have demonstrated that SEC and SPY have at least
partly overlapping functions (9), but so far, there is very little MATERIALS AND METHODS
information on their target proteins.
Virus infection and purification. PPV isolate Rankovic (16) and the chimera
* Corresponding author. Mailing address: Department of Plant Mo- PPV-NK-GFP (5) were used in these experiments. Plants were grown in a
lecular Genetics, Centro Nacional de Biotecnolog´ıa (CSIC), Campus greenhouse maintained at 16 h of light with supplementary illumination and a 19
Universidad Auto´noma de Madrid, 28049 Madrid, Spain. Phone: 34 to 22°C temperature. Young Nicotiana benthamiana plants were mechanically inoc-
915854535. Fax: 34 915854506. E-mail: [email protected]. ulated by rubbing three leaves per plant with crude sap from PPV-infected plants.
Wild-type A. thaliana ecotypes Columbia (Col-0) and Wassilewskija (WS) and the
† Present address: Centre for Protein Engineering, MRC Centre, sec-1 (WS background), sec-2, and spy-3 (Col-0 background) mutants (9) were
Hills Road, CB2 2QH Cambridge, United Kingdom. inoculated by infiltration with Agrobacterium tumefaciens C58C1 harboring pBIN-
PPV-NK-GFP, which contains a full-length cDNA copy of the genome of PPV-NK-

9381

9382 CHEN ET AL. J. VIROL.

GFP cloned into pBin19 (C. Lucini, J. J. Lo´pez-Moya, J. M. Alamillo, and J. A. FIG. 1. Localization of the capsid protein coding sequence in the Downloaded from http://jvi.asm.org/ on February 11, 2016 by guest
Garc´ıa, unpublished results). Approximately 150 ␮l of the Agrobacterium cultures genome of PPV-NK-GFP. The different PPV protein products are
(optical density at 600 nm ϭ 0.5) induced with acetosyringone were applied with a shown in the box representing the PPV polyprotein. Dark gray and
syringe to the undersides of three leaves of young Arabidopsis seedlings. black boxes represent the N- and C-terminal regions and the core
region, respectively, of the PPV CP. The location of the introduced
Although PPV accumulation appears to be quite similar in N. clevelandii, N. GFP open reading frame is indicated above the map. The sequence of
benthamiana, and Arabidopsis, the protocol previously described for PPV puri- the N-terminal tryptic peptide from amino acids 1 through 39 is un-
fication from leaves of N. clevelandii (16) was not suitable for purification from derlined.
N. benthamiana or Arabidopsis, probably because of virus aggregation (data not
shown). Thus, the original protocol was slightly modified for these hosts. In brief, trometry analyses have revealed the existence of O-GlcNAcy-
10 g of N. benthamiana- or Arabidopsis-infected leaves collected 3 weeks post- lation at the N-terminal region of the CP of PPV virions pu-
inoculation was homogenized with 50 ml (Arabidopsis) or 20 ml (N. benthamiana) rified from N. clevelandii (3). In order to assess whether this
of 0.18 M McIlvain’s citric acid-phosphate buffer, pH 7, containing 0.2% thio- modification could also take place in other plant species, PPV
glycolic acid, 0.01 M sodium diethyldithiocarbamate, 0.5 M urea, and 3 mM was purified from another well-characterized PPV host, N.
EDTA first for 5 min with a mortar and pestle and then for 10 min with a Waring benthamiana, and from two different ecotypes of Arabidopsis,
blender at low speed. Next, 50 ml (Arabidopsis) or 10 ml (N. benthamiana) of cold Col-0 and WS, which has been recently shown to be susceptible
chloroform was added to the mixture and shaken in the Waring blender for to PPV infection (unpublished results from several laborato-
another 5 min. The homogenate was centrifuged for 11 min at 5,000 ϫ g, and the ries). PPV virions purified from infected plants of these species
supernatant was centrifuged for 2 h at 82,500 ϫ g. The pellet was resuspended in were partially digested with trypsin, and the resulting peptides
3 ml (Arabidopsis) or 4 ml (N. benthamiana) of 10 mM McIlvain’s citric acid- were analyzed by MALDI-TOF. Similar to the previously pub-
phosphate buffer, pH 7, containing 1 M urea and 0.2% ␤-mercaptoethanol for lished spectrum of PPV purified from N. clevelandii (3), the
2 h and centrifuged for 10 min (Arabidopsis) or 5 min (N. benthamiana) at 1,500 spectra of the three samples showed three ions corresponding
ϫ g. The supernatant was centrifuged for 1.5 h at 57,000 ϫ g (Arabidopsis) or to the partially digested tryptic peptide spanning residues 1 to
82,000 ϫ g (N. benthamiana). The resulting pellet was resuspended for 2 h in 0.5 39 of the PPV CP (Fig. 1 and 2). One ion was the size expected
ml (Arabidopsis) or 0.6 ml (N. benthamiana) of 0.1 M sodium borate buffer, pH from the unmodified peptide (m/z ϭ 4,077 Da), and the other
8.2, containing 10 mM EDTA, clarified by centrifugation for 5 min at 1,200 ϫ g two ions were the sizes expected if the peptide was modified
(Arabidopsis) or 1,500 ϫ g (N. benthamiana), layered over a 3-ml cushion of 20% with one or two O-GlcNAc residues (m/z ϭ 4,280 Da and 4,483
sucrose in the same buffer, and centrifuged at 72,000 ϫ g for 2 h. The pellet was Da, respectively) (Fig. 2). These results indicate that similar
resuspended in 50 ␮l (Arabidopsis) or 100 ␮l (N. benthamiana) of 5 mM sodium O-GlcNAcylation of PPV CP appears to take place in different
borate buffer, pH 8.2, and clarified by centrifugation for 10 min at 4,800 ϫ g host plants.
(Arabidopsis) or 7,400 ϫ g (N. benthamiana). All the purification steps were
carried out at 4°C, and the purified virus was stored at Ϫ20°C. PPV infection of sec plants is impaired relative to that of
wild-type or spy plants. Arabidopsis has two OGTs: SPY and
Matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) SEC. SEC protein is more similar to animal OGTs than to
analysis of PPV CP digested with trypsin. Approximately 10 ␮g of purified PPV SPY, while SPY shows the same level of similarity with SEC
virions was digested with 2 ng of modified porcine trypsin (Promega) in a buffer and animal OGTs (9, 14). Both SEC (9) and SPY (28) have
containing 25 mM ammonium carbonate, pH 8, in a reaction volume of 10 ␮l. shown to have OGT activity. Two T-DNA insertion mutants of
Digestion proceeded at 37°C for 20 min, and then it was rapidly stopped by SEC have been described: sec-1 is in a WS background and the
adding 1 ␮l of 0.5% trifluoroacetic acid. Reaction products were desalted with a T-DNA is inserted into the tetratricopeptide repeat domain,
Zip-Tip reverse-phase C18 column (Millipore) and eluted in 5 ␮l of 70% aqueous and sec-2 is in a Col-0 background and the T-DNA is inserted
acetonitrile and 0.1% trifluoroacetic acid. into an intron adjacent to exons coding for the putative cata-
lytic region of the protein (9). While sec-1 and sec-2 have no
About 0.4 ␮l of matrix solution (5 g/liter 2,5-dihidroxibenzoic acid in 33% obvious phenotypes (9), spy plants have defects in a number of
aqueous acetonitrile and 0.1% trifluoroacetic acid) was deposited onto a 400-␮m processes, including gibberellin and cytokinin responses, flow-
AnchorChip MALDI target (Bruker Daltonics, Bremen, Germany) and allowed ering, circadian regulation, and light inhibition of hypocotyls
to dry at room temperature. Then, 0.4 ␮l of the tryptic peptide mixture was elongation (7, 15, 27, 30). spy-3 is in a Col-0 background and
added and again allowed to dry at room temperature. Peptide mass fingerprint- has a Gly-to-Ser substitution in the C-terminal region of the
ing spectra were acquired with a MALDI-TOF Bruker Reflex IV mass spec- protein (14). Interestingly, homozygous sec-1/spy-3 and sec-2/
trometer (Bruker Daltonics) equipped with a nitrogen laser (337 nm). Analyses spy-3 double mutants die during embryogenesis, indicating that
were performed in reflector positive ion mode, accumulating 120 shots, with 400 SEC and SPY have an overlapping and essential function dur-
ns of pulsed ion delayed extraction and an acceleration voltage of 20 kV and ing embryogenesis (9).
1,600 V in the reflector detector. The mass spectra were externally calibrated
using a mixture of peptide standards (angiotensin II, substance P, bombesin, In order to investigate the possible role of plant OGTs in the
somatostatine 28, and cytochrome C). O-GlcNAcylation of PPV CP and in virus infection, sec-1,
sec-2, and spy-3 mutant plants were inoculated with PPV-NK-
Processing of the spectra and data analysis were performed with Bruker GFP, a recombinant PPV expressing the GFP, which allows an
Daltonics XTOF 5.1.1 and Biotools 2.1 software.

Assessment of spread and accumulation of PPV. Spreading of PPV-NK-GFP
was assessed by visualizing green fluorescent protein (GFP) fluorescence under
a Leica MZ FLIII fluorescence microscope with excitation and barrier filters of
480/40 nm and 510 nm, respectively. PPV accumulation was quantified by dou-
ble-antibody sandwich indirect enzyme-linked immunosorbent assay (ELISA)
using the REALISA kit (C. C. Durviz, S.L.). The samples were prepared by
grinding the infected leaves with a mortar and pestle in 5 mM sodium phosphate
buffer, pH 7.2 (2 ml/g), and storing them frozen at Ϫ20°C. A standard curve was
obtained with different amounts of purified PPV virions diluted in the extract of
healthy plants. The enzymatic reaction was developed with the Sigma FAST
p-nitrophenyl phosphate tablet set system (Sigma), and the optical density at 405
nm of the reaction product was measured in a Titertek Multiskan MCC/340
(Labsystems) spectrophotometer. Statistical analysis of the data was carried out
with PRISM 4 software (GraphPad).

RESULTS

The N-terminal region of PPV CP is O-GlcNAcylated in
Nicotiana benthamiana and Arabidopsis thaliana. Mass spec-

VOL. 79, 2005 ROLE OF SECRET AGENT IN PPV INFECTION 9383

FIG. 2. (A to F) MALDI-TOF analysis of trypsin-digested PPV virions purified from the plants indicated in each panel. The mass/charge ratio Downloaded from http://jvi.asm.org/ on February 11, 2016 by guest
(m/z, in Daltons) assigned to peaks that can derive from the peptide from amino acids 1 through 39, as well as their suggested modifications, are
indicated. O-GlcNAc, O-GlcNAc modification; PHOS, phosphorylation; AC, acetylation; a.i., arbitrary intensity; wt, wild type.

easier monitoring of the infection (5). PPV caused in wild-type plants (Fig. 5). In contrast, virus titer was not affected in spy-3.
Col-0 and WS mild symptoms consisting of leaf chlorosis, ro- In agreement with the experiments using GFP to monitor the
sette gathering, curling of cauline leaves, and shortening of the infection, the reduction in virus accumulation in sec plants was
inflorescence stems (data not shown). Similar symptoms were less pronounced at 19 dpi (Fig. 5). The infectivity of the PPV
observed in the three mutant plants inoculated with PPV-NK- virions produced in the wild-type and the mutant plants was
GFP, especially late in the infection. Although nearly 100% of assessed by a local lesion assay in Chenopodium foetidum (19).
the inoculated plants became infected, examination of GFP Virions produced in sec-1 and sec-2 plants appeared to be
localization in the leaves detected differences in the pattern of slightly less infectious than those produced in the correspond-
infection between the different genotypes (Fig. 3). Radiation of ing wild-type Arabidopsis plants. However, due to the rather
the infection from the major veins and the extent of infection low linearity and accuracy of the local lesion assay, the differ-
of the lamina were reduced in both sec alleles relative to the ences were not statistically significant (data not shown).
corresponding wild type. At 12 days postinoculation (dpi), a
similar number of leaves from spy-3 and wild-type plants were The O-GlcNAcylation of the N-terminal region of PPV-CP is
infected (Fig. 4) but the percentage of sec-1 and sec-2 plants prevented by sec but not by spy mutations. PPV was purified
with detectable GFP expression was slightly, but reproducibly, from infected sec-1, sec-2, and spy-3 plants. The purified virions
reduced. The sec-1 and sec-2 alleles had a larger effect on the were subjected to partial trypsin digestion, and the resulting
spread of an infection within a leaf. Large GFP fluorescence peptides were analyzed by MALDI-TOF. The spectrum of the
areas were observed in a smaller proportion of leaves from spy-3 sample showed the presence of the peptide from amino
sec-1 and sec-2 plants than in wild-type or spy-3 plants (Fig. 3 acids 1 through 39 in the same three forms, nonglycosylated,
and 4). The differences in the extent of virus spread among the mono-, and di-O-GlcNAcylated, found in the spectrum of viri-
different genotypes had decreased by 19 dpi (Fig. 3 and 4). ons purified from wild-type Arabidopsis (Fig. 2 and Table 1). In
contrast, only nonglycosylated peptide from amino acids 1
In order to have a more quantitative assessment of the through 39 was detected in samples prepared from virions
progress of PPV infection in the different genotypes, the purified from sec-1 and sec-2 plants (Fig. 2 and Table 1),
amount of virus in the infected leaves was determined by indicating that SPY OGT or other plant proteins were not able
ELISA. At 12 dpi, PPV accumulation was significantly lower in to glycosylate the N-terminal region of PPV CP in the absence
sec-1 and sec-2 plants than in their corresponding wild-type of the SEC protein.

9384 CHEN ET AL. J. VIROL.

FIG. 3. PPV spreading in mutant and wild-type (wt) Arabidopsis FIG. 4. Level of PPV infection in mutant and wild-type (wt) Ara- Downloaded from http://jvi.asm.org/ on February 11, 2016 by guest
plants. Plants were inoculated with PPV-NK-GFP and observed at bidopsis plants. Four or five PPV-NK-GFP-infected plants of each
different times postinoculation under a fluorescence microscope. A Arabidopsis type were collected at 12 dpi and 19 dpi. Infection was
ruler with minor divisions in mm is shown beside each picture. assessed in all leaves of each plant by monitoring GFP expression with
a fluorescence microscope. Leaves were classified as heavily infected
The presence of acetylated forms (mass increase of 42 Da) when virus infection occupied more than one-fourth of the leaf lamina
of the nonglycosylated and O-GlcNAcylated variants of the or more than half of the leaf vasculature.
peptide from amino acids 1 through 39 have been previously
reported (3). These acetylated peptides were also detected in However, sec spy double mutants died during embryogenesis,
the virions purified from N. benthamiana and wild-type and showing that OGT activity is also essential for plant viability
mutant Arabidopsis (Fig. 2 and Table 1). A careful examination and that SEC and SPY have at least one overlapping function
of the spectra also detected some signals that could correspond (9). The extent of overlap in the function between SPY and
to phosphorylated (mass increase of 80 Da) and/or phosphor- SEC is unknown. While spy plants exhibit a number of obvious
ylated plus acetylated (mass increase of 80 plus 42 Da) forms defects, including altered gibberellin and cytokinin responses,
of the nonglycosylated peptide from amino acids 1 through 39 circadian rhythms, light responses, and flowering time (7, 15,
in all the spectra (Fig. 2 and Table 1). Moreover, we also 27, 30), these defects are not detectable or are much weaker in
detected peptides with the size expected for the peptide from sec plants (9; L. Hartweck and N. E. Olszewski, unpublished
amino acids 1 through 39 mono-O-GlcNAcylated and phos- results). These results can be explained if SPY and SEC have
phorylated among the tryptic peptides of virions purified from completely overlapping substrates but SPY is responsible for
N. benthamiana, wild-type Arabidopsis Col-0 and WS, and spy-3 most of the O-GlcNAcylation or if SPY has some substrates
plants (Fig. 2 and Table 1). Interestingly, faint peaks that could that are not modified by SEC.
correspond to a phosphorylated form of the di-O-GlcNAcy-
lated peptide from amino acids 1 through 39 were found in the Very little is known of the OGT targets in plant cells. O-
spectra of virions purified from N. benthamiana and wild-type glycosylation with the terminal GlcNAc modification of several
Arabidopsis Col-0 (Fig. 2 and Table 1), suggesting that at least nuclear pore complex proteins of tobacco, including a protein
three Ser/Thr residues of the peptide from amino acids 1 that shows sequence similarity to bacterial aldose-1-epime-
through 39 can be modified at the same time. rases, has been described previously (11), but the enzyme re-
sponsible for these modifications has not been identified. We
DISCUSSION have previously reported that the CP of a plant virus, PPV, was
O-GlcNAc modified in N. clevelandii (3); now, we show that
O-GlcNAc modification has been shown to play very impor- this modification is not specific for this host, since it takes place
tant regulatory roles in animals (32), and in agreement with in a similar way in two other plant species, N. benthamiana and
this, deletion of the OGT gene of mouse was lethal (23). Loss Arabidopsis (Fig. 2). More important, the MALDI-TOF anal-
of either SPY or SEC function alone does not cause lethality. ysis of PPV virions purified from sec and spy mutants indicates
that SEC protein is the OGT responsible for the O-GlcNAc

VOL. 79, 2005 ROLE OF SECRET AGENT IN PPV INFECTION 9385

FIG. 5. PPV accumulation in infected leaves of mutant and wild- modification of the N-terminal region of PPV CP and that Downloaded from http://jvi.asm.org/ on February 11, 2016 by guest
type (wt) Arabidopsis plants. Virus amount was determined by ELISA neither SPY nor other plant proteins is able to glycosylate this
in two pools of caulinar and two pools of rosette systemically infected sequence (Fig. 2). However, we cannot rule out the possibility
leaves from five (Col-0 wt, sec-2, and spy-3) or four (WS wt and sec-1) that SPY could be involved in the O-GlcNAc modification of
PPV-NK-GFP-inoculated plants collected at 12 dpi (between 8 and 36 other regions of PPV CP or other PPV proteins. These results
caulinar and between 15 and 28 rosette-infected leaves) and 19 dpi support the hypothesis that SEC and SPY have partial func-
(between 15 and 35 caulinar and between 6 and 21 rosette-infected tional independence. Whereas O-GlcNAc modification of the
leaves). The graphs show the average values with their standard devi- N-terminal region of PPV CP, like that of animal proteins,
ations. Differences between sec-2 and Col-0 wt at 12 dpi and between adds single sugar monomers (3; J. J. P´erez, S. Ju´arez, and J. A.
sec-1 and WS wt at 12 and 19 dpi were statistically significant (P values Garc´ıa, unpublished results), the size of sugar chains of the
of 0.04, 0.01, and 0.01). Differences between sec-2 and Col-0 wt at 19 glycosylated nuclear pore proteins of tobacco corresponds to
dpi and between spy-3 and Col-0 wt at 12 and 19 dpi were not statis- more than five monosaccharides (10), suggesting that different
tically significant (P values of 0.5, 0.82, and 0.53). enzymes could be involved in each of these modifications.
Having in mind that SPY is less similar to mammalian OGTs
than SEC, it is tempting to speculate that SPY could be in-
volved in the second type of modification, which would be
specific to plants.

The ability of PPV to infect sec-1 and sec-2 mutants (Fig. 3)
demonstrates that O-GlcNAc modification by SEC OGT is not
essential for PPV viability in Arabidopsis. However, the effi-
ciency of PPV infection is markedly lower in these mutants
than in the spy-3 mutant or in wild-type plants (Fig. 4 and 5),
indicating that SEC activity plays an important role in infec-
tion. O-GlcNAc modifications at the segment from amino ac-
ids 1 through 39 of PPV CP are not essential, since deletions or
substitutions that remove all Thr and Ser residues of this re-
gion have no perceptible effects on PPV infection in N. cleve-
landii (3, 4). Therefore, O-GlcNAc modification of other por-
tions of PPV CP or other PPV proteins by SEC may play a role
in the infection process. The present data do not allow us to
discriminate whether some O-GlcNAc modifications are more
important than others or the overall level of O-GlcNAcylation
is the relevant factor. Moreover, we cannot rule out the pos-
sibility that the effect of sec mutation on PPV infection might
be an indirect result of the action of SEC on a host protein. It
is also important to point out that, although O-GlcNAc mod-
ification by SEC is not essential for the PPV infection of
Arabidopsis, the possibility exists that O-GlcNAcylation plays a
more important role in some plants (for instance, in the PPV
natural woody hosts) than in others.

The role of O-GlcNAc modification in PPV infection is still

TABLE 1. m/z of MALDI-TOF signals that could correspond to modified forms of the peptide spanning amino acids 1
through 39 from PPV CPa

Amt of ion (Da)

Nonglycosylated Mono-O-GlcNAc Di-O-GlcNAc

Plant host Genotype Non-AC, AC, Non-AC, AC PHOS AC, Non-AC, AC PHOS
non- PHOS non- (ϩ203, (ϩ203, PHOS non- (ϩ406, (ϩ406,
PHOS AC PHOS (ϩ42, PHOS ϩ42) ϩ80) (ϩ203, PHOS ϩ42) ϩ80)
(ϩ42) (ϩ80) ϩ80) ϩ42,
(ϩ203) ϩ80) (ϩ406)

A. thaliana Col-0 Wild type 4,076.21 4,118.02 4,156.14 4,198.65 4,279.04 4,321.03 4,358.84 4,401.64 4,481.98 4,524.17 4,561.44

sec-2 4,078.71 4,120.88 4,158.76 4,200.80 — — — — — — —

spy-3 4,076.36 4,118.59 — 4,198.70 4,279.48 4,321.32 4,358.62 4,400.96 4,482.41 4,525.02

A. thaliana WS Wild type 4,077.07 4,118.74 4,156.96 — 4,279.94 4,321.83 4,359.96 — 4,482.85 4,524.93 —

sec-1 4,079.27 4,121.35 4,159.15 4,201.81 — — — — — — —

N. benthamiana Wild type 4,077.13 4,118.84 4,157.39 — 4,280.2 4,321.83 4,360.88 — 4,483.32 4,524.87 4,564.34
a AC, acetylated; PHOS, phosphorylated; —, nonacetylated and nonphosphorylated.

9386 CHEN ET AL. J. VIROL.

an open question. It is well known that in mammals, O- REFERENCES Downloaded from http://jvi.asm.org/ on February 11, 2016 by guest
GlcNAcylation and phosphorylation collaborate in the regula-
tion of macromolecular interactions that control the activity of 1. Baratova, L. A., N. V. Fedorova, E. N. Dobrov, E. V. Lukashina, A. N.
many cellular processes (26). PPV CP has been shown to Kharlanov, V. V. Nasonov, M. V. Serebryakova, S. V. Kozlovsky, O. V.
contain phosphoserine and phosphothreonine residues (3). Al- Zayakina, and N. P. Rodionova. 2004. N-terminal segment of potato virus X
though MALDI-TOF does not allow a precise analysis of pro- coat protein subunits is glycosylated and mediates formation of a bound
tein phosphorylation, the MALDI-TOF spectra of PPV CP water shell on the virion surface. Eur. J. Biochem. 271:3136–3145.
subjected to trypsin digestion showed signals that could corre-
spond with phosphorylated peptides (Fig. 2 and Table 1). As 2. Comer, F. I., and G. W. Hart. 2000. O-glycosylation of nuclear and cytosolic
expected, the presence of these putative phosphorylated pep- proteins. Dynamic interplay between O-GlcNAc and O-phosphate. J. Biol.
tides was not affected by the sec or spy mutations. Both non- Chem. 275:29179–29182.
glycosylated and O-GlcNAc-modified peptides appeared to be
phosphorylated, in agreement with the fact previously ob- 3. Ferna´ndez-Ferna´ndez, M. R., E. Camafeita, P. Bonay, E. M´endez, J. P.
served in mammalian organisms that, although O-GlcNAcyla- Albar, and J. A. Garc´ıa. 2002. The capsid protein of a plant single-stranded
tion and phosphorylation are reciprocal modifications, a single RNA virus is modified by O-linked N-acetylglucosamine. J. Biol. Chem.
polypeptide can contain simultaneously phosphate and O- 277:135–140.
GlcNAc residues (2). These data suggest that the O-GlcNA-
cylation of PPV CP, like that of those mammalian systems, 4. Ferna´ndez-Ferna´ndez, M. R., J. L. Mart´ınez-Torrecuadrada, J. I. Casal, and
could function in coordination with phosphorylation. J. A. Garc´ıa. 1998. Development of an antigen presentation system based on
plum pox potyvirus. FEBS Lett. 427:229–235.
The genomic RNA is a central player in the most critical
processes of viral infection: translation, replication, encapsida- 5. Ferna´ndez-Ferna´ndez, M. R., M. Mourin˜o, J. Rivera, F. Rodr´ıguez, J. Plana-
tion, and cell-to-cell and long-distance movement. Thus, it is Dura´n, and J. A. Garc´ıa. 2001. Protection of rabbits against rabbit hemor-
likely that allocation of RNA molecules to each of these pro- rhagic disease virus by immunization with the VP60 protein expressed in
cesses is highly regulated. Ivanov and colleagues have demon- plants with a potyvirus-based vector. Virology 280:283–291.
strated that phosphorylation of the CP of the potyvirus potato
virus A reduces its ability to bind RNA and have suggested that 6. Gonzalez, S. A., and O. R. Burrone. 1991. Rotavirus NS26 is modified by
this modification might regulate the formation and/or stability addition of single O-linked residues of N- acetylglucosamine. Virology 182:
of virions and other viral ribonucleoproteins (12, 13). It is 8–16.
tempting to speculate that CP O-GlcNAc modification could
also contribute to this regulatory mechanism. The fact that the 7. Greenboim-Wainberg, Y., I. Maymon, R. Borochov, J. Alvarez, N. Olszewski,
effect of the sec alleles on PPV infection appears to be more N. Ori, Y. Eshed, and D. Weiss. 2005. Cross talk between gibberellin and
apparent at early times (Fig. 3 through 5) suggests that the cytokinin: the arabidopsis GA response inhibitor SPINDLY plays a positive
regulation of RNA distribution could be required at the be- role in cytokinin signaling. Plant Cell 17:92–102.
ginning of the infection, when there could be a limiting amount
of RNA molecules. An alternative possibility is that O-GlcNAc 8. Greis, K. D., W. Gibson, and G. W. Hart. 1994. Site-specific glycosylation of
residues modifying PPV CP could be playing a role in virion the human cytomegalovirus tegument basic phosphoprotein (UL32) at
stability similar to that proposed for the galactose and fucose serine 921 and serine 952. J. Virol. 68:8339–8849.
residues that are O-linked to the N-terminal NAcSer of the
Potato virus X CP, which has been shown to affect the water- 9. Hartweck, L. M., C. L. Scott, and N. E. Olszewski. 2002. Two O-linked
absorbing capacity of the viral particles (1). It is striking that N-acetylglucosamine transferase genes of Arabidopsis thaliana L. Heynh.
plant RNA viruses have adopted different O-glycosylation sys- have overlapping functions necessary for gamete and seed development.
tems to modify their CPs; it will be interesting in the future to Genetics 161:1279–1291.
determine whether the role of SEC is PPV specific or it also
modifies the CPs of other potyviruses or of viruses of other 10. Heese-Peck, A., R. N. Cole, O. N. Borkhsenious, G. W. Hart, and N. V.
families. Raikhel. 1995. Plant nuclear pore complex proteins are modified by novel
oligosaccharides with terminal N-acetylglucosamine. Plant Cell 7:1459–1471.
The results described in this report identify a novel target for
antiviral action. We have demonstrated that the O-GlcNAc 11. Heese-Peck, A., and N. V. Raikhel. 1998. A glycoprotein modified with
modification of PPV takes place in several plant hosts and that terminal N-acetylglucosamine and localized at the nuclear rim shows se-
viral propagation is compromised by sec mutations that do not quence similarity to aldose-1-epimerases. Plant Cell 10:599–612.
noticeably affect plant growth. This could contribute to control
the propagation of the virus in field conditions. 12. Ivanov, K. I., P. Puustinen, R. Gabrenaite, H. Vihinen, L. Ro¨nnstrand, L.
Valmu, N. Kalkkinen, and K. Ma¨kinen. 2003. Phosphorylation of the poty-
ACKNOWLEDGMENTS virus capsid protein by protein kinase CK2 and its relevance for virus infec-
tion. Plant Cell 15:2124–2139.
We thank Elvira Dom´ınguez for technical assistance and Juan Pablo
Albar for helpful advice in MALDI-TOF analysis. 13. Ivanov, K. I., P. Puustinen, A. Merits, M. Saarma, and K. Ma¨kinen. 2001.
Phosphorylation down-regulates the RNA binding function of the coat pro-
This work was supported by grants BIO2001-1434 and BIO2004- tein of potato virus A. J. Biol. Chem. 276:13530–13540.
02687 from the Spanish MEC, QLK2-CT-2002-01050 and SP22-CT-
2004 from the European Union to J.A.G., MCB-0112826 from the 14. Jacobsen, S. E., K. A. Binkowski, and N. E. Olszewski. 1996. SPINDLY, a
National Science Foundation, and DE-FG01-04ER04-02 from the tetratricopeptide repeat protein involved in gibberellin signal transduction in
U.S. Department of Energy to N.E.O. Arabidopsis. Proc. Natl. Acad. Sci. USA 93:9292–9296.

15. Jacobsen, S. E., and N. E. Olszewski. 1993. Mutations at the SPINDLY locus
of Arabidopsis alter gibberellin signal transduction. Plant Cell 5:887–896.

16. La´ın, S., J. L. Riechmann, E. M´endez, and J. A. Garc´ıa. 1988. Nucleotide
sequence of the 3Ј terminal region of plum pox potyvirus RNA. Virus Res.
10:325–342.

17. Liu, F., K. Iqbal, I. Grundke-Iqbal, G. W. Hart, and C. X. Gong. 2004.
O-GlcNAcylation regulates phosphorylation of tau: a mechanism involved in
Alzheimer’s disease. Proc. Natl. Acad. Sci. USA 101:10804–10809.

18. Lo´pez-Moya, J. J., M. R. Ferna´ndez-Ferna´ndez, M. Cambra, and J. A.
Garc´ıa. 2000. Biotechnological aspects of plum pox virus. J. Biotechnol.
76:121–136.

19. Lo´pez-Moya, J. J., and J. A. Garc´ıa. 2000. Construction of a stable and highly
infectious intron-containing cDNA clone of plum pox potyvirus and its use to
infect plants by particle bombardment. Virus Res. 68:99–107.

20. Mullis, K. G., R. S. Haltiwanger, G. W. Hart, R. B. Marchase, and J. A.
Engler. 1990. Relative accessibility of N-acetylglucosamine in trimers of the
adenovirus types 2 and 5 fiber proteins. J. Virol. 64:5317–5323.

21. Revers, F., O. Le Gall, T. Candresse, and A. J. Maule. 1999. New advances
in understanding the molecular biology of plant/potyvirus interactions. Mol.
Plant-Microbe Interact. 12:367–376.

22. Riechmann, J. L., S. La´ın, and J. A. Garc´ıa. 1992. Highlights and prospects
of potyvirus molecular biology. J. Gen. Virol. 73:1–16.

23. Shafi, R., S. P. Iyer, L. G. Ellies, N. O’Donnell, K. W. Marek, D. Chui, G. W.
Hart, and J. D. Marth. 2000. The O-GlcNAc transferase gene resides on the
X chromosome and is essential for embryonic stem cell viability and mouse
ontogeny. Proc. Natl. Acad. Sci. USA 97:5735–5739.

24. Shukla, D. D., P. M. Strike, S. L. Tracy, K. H. Gough, and C. W. Ward. 1988.
The N and C termini of the coat proteins of potyviruses are surface-located
and the N-terminus contains the major virus-specific epitopes. J. Gen. Virol.
69:1497–1508.

25. Shukla, D. D., C. W. Ward, and A. A. Brunt. 1994. Genome structure,

VOL. 79, 2005 ROLE OF SECRET AGENT IN PPV INFECTION 9387

variation and function, p. 74–112. In D. D. Shukla, C. W. Ward, and A. A. 30. Tseng, T. S., P. A. Salom´e, C. R. McClung, and N. E. Olszewski. 2004.
Brunt (ed.), The potyviridae. CAB International, Cambridge, United King- SPINDLY and GIGANTEA interact and act in Arabidopsis thaliana path-
dom. ways involved in light responses, flowering, and rhythms in cotyledon move-
26. Slawson, C., and G. W. Hart. 2003. Dynamic interplay between O-GlcNAc ments. Plant Cell 16:1550–1563.
and O-phosphate: the sweet side of protein regulation. Curr. Opin. Struct.
Biol. 13:631–636. 31. Vosseller, K., L. Wells, M. D. Lane, and G. W. Hart. 2002. Elevated nucle-
27. Sothern, R. B., T. S. Tseng, S. L. Orcutt, N. E. Olszewski, and W. L. ocytoplasmic glycosylation by O-GlcNAc results in insulin resistance associ-
Koukkari. 2002. GIGANTEA and SPINDLY genes linked to the clock ated with defects in Akt activation in 3T3-L1 adipocytes. Proc. Natl. Acad.
pathway that controls circadian characteristics of transpiration in Arabidop- Sci. USA 99:5313–5318.
sis. Chronobiol. Biol. 19:1005–1022.
28. Thornton, T. M., L. Kreppel, G. W. Hart, and N. E. Olszewski. 1999. Genetic 32. Wells, L., and G. W. Hart. 2003. O-GlcNAc turns twenty: functional impli-
and biochemical analysis of Arabidopsis SPY, p. 445–448. In A. Altman, M. cations for post-translational modification of nuclear and cytosolic proteins
Ziv, and S. Izhar (ed.), Plant biotechnology and in vitro biology in the 21st with a sugar. FEBS Lett. 546:154–158.
century. Kluwer Academic Publishers, Dordrecht, The Netherlands.
29. Thornton, T. M., S. M. Swain, and N. E. Olszewski. 1999. Gibberellin signal 33. Wells, L., K. Vosseller, and G. W. Hart. 2001. Glycosylation of nucleocyto-
transduction presents. . .the SPY who O-GlcNAc’d me. Trends Plant Sci. plasmic proteins: signal transduction and O-GlcNAc. Science 291:
4:424–428. 2376–2378.

34. Whitford, M., and P. Faulkner. 1992. A structural polypeptide of the bacu-
lovirus Autographa californica nuclear polyhedrosis virus contains O-linked
N-acetylglucosamine. J. Virol. 66:3324–3329.

Downloaded from http://jvi.asm.org/ on February 11, 2016 by guest