Quantitative SUMO-1 modification of a vaccinia virus protein is required for its specific localization and prevents its self-association.

Abstract

Vaccinia virus (VV), the prototype member of the Poxviridae, a family of large DNA viruses, carries out DNA replication in specialized cytoplasmic sites that are enclosed by the rough endoplasmic reticulum (ER). We show that the VV gene product of A40R is quantitatively modified by SUMO-1, which is required for its localization to the ER-enclosed replication sites. Expression of A40R lacking SUMO-1 induced the formation of rod-shaped cytoplasmic aggregates. The latter likely consisted of polymers of nonsumoylated protein, because unmodified A40R interacted with itself, but not with the SUMO-1-conjugated protein. Using a bacterial sumoylation system, we furthermore show that unmodified A40R is mostly insoluble, whereas the modified form is completely soluble. By electron microscopy, the A40R rods seen in cells were associated with the cytosolic side of the ER and induced the apposition of several ER cisternae. A40R is the first example of a poxvirus protein to acquire SUMO-1. Its quantitative SUMO-1 modification is required for its proper localization to the viral "mini-nuclei" and prevents its self-association. The ability of the nonsumoylated A40R to bring ER membranes close together could suggest a role in the fusion of ER cisternae when these coalesce to enclose the VV replication sites.

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Molecular Biology of the Cell
Vol. 16, 2822–2835, June 2005
Quantitative SUMO-1 Modification of a Vaccinia Virus
Protein Is Required for Its Specific Localization and
Prevents Its Self-Association
Silvia Palacios,*†Laurent H. Perez,*†Sonja Welsch,‡Sibylle Schleich,*
Katarzyna Chmielarska,§Frauke Melchior,§and Jacomine Krijnse Locker*
*European Molecular Biology Laboratory, Cell Biology and Biophysics Programme, 69117 Heidelberg,
Germany;‡Faculty of Medicine, Institute for Hygiene, University of Heidelberg, 69120 Heidelberg, Germany;
and§Department of Biochemistry, University of Goettingen, 37073 Goettingen, Germany
Submitted November 16, 2004; Revised February 24, 2005; Accepted March 21, 2005
Monitoring Editor: Peter Walter
Vaccinia virus (VV), the prototype member of the Poxviridae, a family of large DNA viruses, carries out DNA replication
in specialized cytoplasmic sites that are enclosed by the rough endoplasmic reticulum (ER). We show that the VV gene
product of A40R is quantitatively modified by SUMO-1, which is required for its localization to the ER-enclosed
replication sites. Expression of A40R lacking SUMO-1 induced the formation of rod-shaped cytoplasmic aggregates. The
latter likely consisted of polymers of nonsumoylated protein, because unmodified A40R interacted with itself, but not
with the SUMO-1–conjugated protein. Using a bacterial sumoylation system, we furthermore show that unmodified A40R
is mostly insoluble, whereas the modified form is completely soluble. By electron microscopy, the A40R rods seen in cells
were associated with the cytosolic side of the ER and induced the apposition of several ER cisternae. A40R is the first
example of a poxvirus protein to acquire SUMO-1. Its quantitative SUMO-1 modification is required for its proper
localization to the viral “mini-nuclei” and prevents its self-association. The ability of the nonsumoylated A40R to bring
ER membranes close together could suggest a role in the fusion of ER cisternae when these coalesce to enclose the VV
replication sites.
INTRODUCTION
Posttranslational modification of proteins is an important
mechanism for the regulation of their function, activity, or
localization. The structurally related modifications ubiquiti-
nation and sumoylation are unusual because the modifier is
a small polypeptide. Although ubiquitination is predomi-
nantly involved in protein turnover (Hershko and Ciech-
anover, 1998), the exact function of SUMO conjugation is
less clear. A number of cellular proteins have been reported
to acquire SUMO-1, most of which reside in the nucleus.
Sumoylation of these proteins can alter their intracellular
localization, their activity, their stability, and their interac-
tion with other proteins (Melchior, 2000; Muller et al., 2001;
Johnson, 2004). In mammalian cells, three SUMO family
members have been described: SUMO-1, SUMO-2, and
SUMO-3 (Melchior et al., 2003). Human SUMO-1, a 101-
amino acid polypeptide, shares ?50% sequence identity
with SUMO-2/3 and with the yeast Saccharomyces cerevisiae
protein Smt3. Although the pathway of sumoylation is
mechanistically similar to ubiquitination, the enzymes are
different. SUMO-1 is activated in an ATP-dependent manner
by an E1-activating enzyme, which consists of a heterodimer
of the Aos1 and Uba2 proteins. Activated SUMO is trans-
ferred to the E2-conjugating enzyme Ubc9, which catalyzes
the formation of an isopeptide bond between the C terminus
of SUMO-1 and the ?-amino group of a lysine residue of the
target protein (Melchior, 2000; Muller et al., 2001; Johnson,
2004). Unlike ubiquitin conjugation, SUMO-1 modification
in vitro is not strictly dependent on an E3 protein ligase,
although E3 ligase activity is usually required to increase the
efficiency of modification in vitro and in vivo (Johnson and
Gupta, 2001; Sachdev et al., 2001; Pichler et al., 2002; Kagey et
al., 2003). A growing number of viral proteins have been
described that are either SUMO-1 conjugated themselves or
that interfere with the sumoylation machinery (reviewed in
Wilson and Rangasamy, 2001). These proteins are invariably
encoded by DNA viruses that replicate in the nucleus, and
interfering with the sumoylation of these proteins interferes
with their localization to the nucleus.
Vaccinia virus (VV), the prototype member of the Poxviri-
dae, was used successfully to eradicate variola virus, the
cause of smallpox. Poxviridae are large DNA viruses with a
double-stranded DNA genome encoding for ?200 proteins.
They are unique among DNA viruses because DNA repli-
cation occurs entirely in the cytoplasm rather than in the
nucleus of the infected host cell. The cytoplasmic life cycle of
VV involves virion entry, transcription, replication, and the
assembly and egress of virions (Moss, 2001). VV replication
is known to occur in discrete cytoplasmic structures called
“factories” (Cairns, 1960). These sites become gradually en-
wrapped by the rough endoplasmic reticulum (ER) resem-
bling cytoplasmic “mini-nuclei,” a process that facilitates
viral replication (Tolonen et al., 2001). Cellular inner nuclear
This article was published online ahead of print in MBC in Press
(http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E04–11–1005)
on March 30, 2005.
†These authors contributed equally to this work.
Address correspondence to: Jacomine Krijnse Locker (krijnse@
embl.de).
2822© 2005 by The American Society for Cell Biology

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Figure 1.
at 3 h postinfection, and double labeled with the anti-A40R antibody (B) and Hoechst (A). (C) Thawed cryosections prepared from HeLa cells
fixed at 3 h postinfection were labeled with anti-A40R followed by protein A coupled to 10-nm gold particles. The image shows a replication
site (RS) surrounded by ER membranes (large arrows). The central part, but not the ER membrane, is decorated with gold particles. M,
mitochondrion. Bar, 250 nm. (D) HeLa cells were infected at a multiplicity of infection of 10, and cell lysates prepared at the indicated times
postinfection. Equal amounts of total protein were analyzed by Western blotting with the anti-A40R antibody.
Characterization of a newly raised antibody to A40R. (A and B) HeLa cells were infected with a multiplicity of infection of 25, fixed
Sumoylation in the Poxvirus Life Cycle
Vol. 16, June 20052823

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membrane proteins, known to bind to DNA, are, however,
not recruited to the viral replication sites. We have therefore
proposed that the recruitment of ER cisternae to the viral
DNA sites is mediated by viral (ER-resident) membrane
proteins. To find such proteins, we have previously tagged
viral genes of unknown function having a putative trans-
membrane domain with green fluorescent protein (GFP) and
selected these by their localization to the DNA replication
sites upon expression in infected cells. In this manner, we
identified an early VV membrane protein, the gene product
of E8R, and based on its behavior in infected cells, we have
proposed that this protein is a possible candidate that me-
diates ER recruitment (Tolonen et al., 2001; Doglio et al.,
2002).
In this same GFP screening, the gene product of A40R, a
protein with a single putative transmembrane domain at its
N terminus, also localized to the viral replication sites. The
aim of the present study was therefore to characterize this
protein and to define its potential role in the ER-wrapping
process. Here, we show that A40R is modified by SUMO-1,
both in vivo and in vitro. We identify the residue in A40R
required for sumoylation and show that the modification is
essential for its proper localization to the DNA replication
sites. Specifically, overexpression of A40R lacking the
SUMO-1 modification, induces electron-dense cytoplasmic
structures that mediate the apposition of ER cisternae.
This report thus represents the first example of a poxvirus
protein that is SUMO-1 modified and the first example of a
DNA virus for which this modification does not lead to
nuclear localization but instead is a prerequisite for its loca-
tion to the cytoplasmic viral mini-nuclei.
MATERIALS AND METHODS
Cells, Viruses, and Antibodies
HeLa cells (ATCC Cl3) were grown as described previously (Sodeik et al.,
1993). VV strain western reserve (WR) or VVT7 were propagated and semi-
purified as described previously (Pedersen et al., 2000). The polyclonal anti-
body to A40R was raised in rabbit by using the peptide corresponding to
amino acids 71–86 of the A40R sequence according to the manufacturer
instructions (Neosystems, Strasbourg, France). The goat antibodies to
SUMO-1 and SUMO-2/3 have been described previously (Pichler et al., 2002).
The mouse antibodies to myc and hemagglutinin (HA) were from American
Type Culture Collection (Manassas, VA) and BAbCO (Richmond, CA), re-
spectively; anti-GMP-1, used for Western blotting, was from Zymed Labora-
tories, South San Francisco, CA). The antibodies to the VV proteins H5R and
A14L have been described previously (Salmons et al., 1997; Tolonen et al.,
2001), and anti-protein disulfide-isomerase (PDI) was a kind gift of Stephen
Fuller (Oxford University, Oxford, United Kingdom).
DNA Constructs
A40R was amplified by PCR from the VV WR genome by using the following
primers: 5?-cggaagcttctaaccgaagtagtggta-3? (forward) and 5?-cggaagcttctaac-
cgaagtagtggta-3? (reverse). After digestion with NotI and BamHI, it was
cloned into the pGEX-KG vector (Amersham Biosciences, Piscataway, NJ) cut
with the same enzymes. For the cloning of A40R in pCDNA-3.1 (Invitrogen,
Carlsbad, CA), pRSET (Invitrogen) and pFTX5 (a kind gift of Dr. Angel
Nebreda, Spanish National Cancer Centre, Madrid, Spain), A40R was ex-
tracted from pGEX-KG by BamHI/XhoI digestion and cloned on the BamHI/
XhoI sites of the digested vectors. The generation of an N-terminal six histi-
dines A40R was performed by cloning A40R in the pHAT2 vector (BamHI/
HindIII).
The site directed mutagenesis of the K95to A was done using the
QuikChange mutagenesis kit (Stratagene, La Jolla, CA) with the following
oligonucleotides: 5?-tccaaattcggatctaattgcgatagagactccaaac-3? (forward) and
5?-gtttggagtctctatcgcaattagatccgaatttgga-3? (reverse). Glutathione S-trans-
ferase (GST)-Ulp1 was kindly provided by Dr. Mark Hochstrasser (Yale
University, New Haven, CT; Li and Hochstrasser, 1999). Cloning, expression,
and purification of the recombinant E1 (His-Aos1 and Uba2) and E2 (Ubc9)
enzymes and SUMO-1 were described previously (Pichler et al., 2002).
Infections, Transfections, Immunofluorescence, and
Electron Microscopy
HeLa cells were infected with VV WR or VVT7 for 45 min at 37°C and fixed
at the indicated times. Transfections using Lipofectin (Invitrogen) were done
as described previously (Mallardo et al., 2001). Immunofluorescence was
essentially carried out as described previously (Den Boon et al., 1991), except
that the cells also were labeled with 0.5 mg/ml Hoechst (Sigma-Aldrich, St.
Louis, MO) to visualize viral and cellular DNA. For electron microscopy,
HeLa cells infected for the indicated times, were fixed and processed for
cryosectioning (van der Meer et al., 1999) or Epon embedding (Griffiths, 1993).
Thawed cryosections were labeled with affinity-purified antibodies followed
by 10-nm protein A gold, and images were taken using a Zeiss EM10 electron
microscope.
Sample Preparation, Immunoprecipitation, and Western
Blotting
For Western blotting, HeLa cells were scraped from the dishes with ice-cold
phosphate-buffered saline, pelleted, resuspended in 100 ?l of lysis buffer (1%
NP-40 in 10 mM Tris-Cl, pH 9), and incubated for 30 min on ice. The nuclei
were pelleted by centrifugation, and the cleared supernatant was mixed with
sample buffer. The preparation of postnuclear supernatants (PNSs), Na2CO3,
and TX-114 treatment were performed as described previously (Jensen et al.,
1996). For immunoprecipitation, cell pellets were resuspended in radioimmu-
noprecipitation assay (RIPA) buffer (10 mM sodium-phosphate buffer, pH 7.2,
150 mM NaCl, 1% sodium-deoxycholate, 1% Triton X-100, 0.1% SDS, 1 mM
dithiothreitol, protease inhibitors mix). Equal amounts of total protein extract,
usually 500 ?g, were precleared and immunoprecipitated 2 h at 4°C with 1 ?g
of anti-myc monoclonal antibody (mAb) immobilized on agarose beads
(Santa Cruz Biotechnology, Santa Cruz, CA). The beads were washed four
times with RIPA buffer and then resuspended sample buffer. The extracts or
immunoprecipitates were separated by SDS-PAGE and blotted on to nitro-
cellulose as described previously (Perez et al., 2002).
In Vitro Translation, SUMO-1 Modification of A40R, and
GST Pull-Down
In vitro translation of A40R was done according to the instructions of the
manufacturer (Promega, Madison, WI). In vitro modification assay using cell
extracts was performed as described previously (Mahajan et al., 1997). After
incubation with cell extracts, A40R was immunoprecipitated with anti-myc
and digested with GST-Ulp1 for 30 min at room temperature (RT). In vitro
modification assays using the recombinant enzymes (Pichler et al., 2002) were
performed in a total volume of 20 ?l with 150 ng of Aos1/Uba2, 200 ng of
Ubc9, 500 ng of SUMO-1, and 1 ?l of the in vitro-translated protein. The
reactions, containing 1 mM ATP, 0.05% of Tween 20 and 0.2 mg/ml ovalbu-
min, were incubated at 30°C for 60 min and stopped by addition of sample
buffer. For the GST pull-down, the reticulocyte lysate (20 ?l) was diluted 1:10
in immunoprecipitation (IP) buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl,
20 mM NaF, 5 mM EDTA, 5 mM EGTA, 100 ?M sodium-vanadate, 1%
NP-40), supplemented with microcystin, protease inhibitors, and 1% bovine
serum albumin and incubated for 60 min at 4°C with 1 ?g of GST or
GST-A40R, isolated from Escherichia coli, prebound to 5 ?l of glutathione
beads (New England Biolabs, Beverly, MA). The beads were washed four
times in IP buffer and resuspended in sample buffer. All samples were
analyzed by SDS-gels followed by autoradiography. For SUMO-1 modifica-
Figure 2 (facing page).
only upon expression in eukaryotic cells. (A) A40R tagged with 6His
or with myc was translated in vitro, and migration in SDS-gels of
the tagged proteins was analyzed by autoradiography. Control is
the in vitro transcription/translation mix without DNA. The num-
bers on the left refer to the approximate molecular mass of 18 and 20
kDa, inferred from the migration of molecular mass markers. (B)
Expression of A40R tagged with GST or an HA epitope was induced
in bacteria. Bacteria were lysed in sample buffer 3 h after the
addition of IPTG. The samples were analyzed by Western blotting
with the anti-A40R antibody. (C) Same myc-tagged A40R construct
used in A was transiently expressed in VVT7-infected HeLa cells.
Extracts of infected/untransfected cells (lane 1) or infected/trans-
fected cells (lane 2) were prepared at16 h posttransfection and
analyzed by Western blots with anti-myc. Note that both the pre-
dicted molecular mass form (asterisk) and the higher molecular mass
form (double asterisk) are present under this condition. (D) A40R was
transiently expressed in uninfected cells (lane 3) and detected by West-
ern blot with the anti-A40R antibody. Lane 1 is from uninfected and
nontransfected cells, and lane 2 is from infected cells and serves as
a negative and positive control, respectively.
Modification of A40R modification occurs
S. Palacios et al.
Molecular Biology of the Cell 2824

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Figure 2.
Sumoylation in the Poxvirus Life Cycle
Vol. 16, June 20052825

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tion of A40R in bacteria, BL21 (DE3) competent cells were transformed with
either pHAT2-A40R alone or cotransformed with pT-E1E2S1 (expressing
SUMO-1, Ubc9, and Aos1/Uba2, a kind gift of Drs. Hisato Saitoh and Yasu-
hiro Uchimura; Uchimura et al., 2004). A single colony was selected and
transferred to 100 ml of LB media containing chloramphenicol and the ap-
propriate antibiotics. Bacterial cultures were kept at 37°C with shaking for 8 h
until an OD600of at least 1.0. Isopropyl-?-d-thiogalactopyranoside (IPTG) was
then added at a concentration of 0.2 mM. After incubation for another 6 h at
30°C, the bacteria were collected by centrifugation and lysed by incubation
with lysozyme for 30 min on ice followed by sonication. The same protein
quantity of supernatant and pellet was run on a 15% SDS-PAGE and unmod-
ified or SUMO-1–modified A40R was detected by Western blotting by using
anti-His mAb (Sigma-Aldrich).
RESULTS
The Gene Product of A40R Acquires a Modification That
Increases Its Predicted Molecular Mass
The A40R gene product has previously been described as a
membrane protein that is N-glycosylated and transported to
Figure 3.
incubated with HeLa cell extracts supplemented with 1 mM ATP for 0, 30, and 60 min at RT. (B) A40R was translated in vitro and incubated
with (?) or without (?) HeLa cells extracts for 30 min. Half of the reaction incubated with cellular extract was immunoprecipitated with the
anti-myc antibody and digested for 30 min with Ulp-1. (A and B) Degradation product of the SUMO-1–modified form that occurs after the
incubation with cell extracts is marked with an asterisk. (C) HeLa cells were infected with VVT7, transfected with mycA40R, or mock
transfected. After 16 h of expression, cells were processed for immunoprecipitation with anti-myc. Samples were resolved by SDS-PAGE,
transferred to a nitrocellulose membrane, and probed with the rabbit anti-A40R antibody (right) or the mouse anti-SUMO-1 GMP-1(left). The
IgG light chain is marked with two asterisks.
A40R can acquire SUMO-1 modification in vitro and is SUMO-1 modified in eukaryotic cells. (A) In vitro-translated A40R was
S. Palacios et al.
Molecular Biology of the Cell2826