Viral manipulation of the cellular sumoylation machinery
Lowrey et al. Cell Communication and Signaling
Viral manipulation of the cellular sumoylation machinery
Angela J. Lowrey 0
Wyatt Cramblet 0
Gretchen L. Bentz 0
0 Division of Biomedical Sciences, Mercer University School of Medicine , Macon , Georgia
Viruses exploit various cellular processes for their own benefit, including counteracting anti-viral responses and regulating viral replication and propagation. In the past 20 years, protein sumoylation has emerged as an important post-translational modification that is manipulated by viruses to modulate anti-viral responses, viral replication, and viral pathogenesis. The process of sumoylation is a multi-step cascade where a small ubiquitin-like modifier (SUMO) is covalently attached to a conserved ΨKxD/E motif within a target protein, altering the function of the modified protein. Here we review how viruses manipulate the cellular machinery at each step of the sumoylation process to favor viral survival and pathogenesis.
Viruses; Small ubiquitin-like modifier; Sumo; Ubc9; Senp; Pias; RanBP2; SAE1; SAE2
Post-translational modification of proteins is important
to numerous cellular events, allowing cells to respond
to both external and internal stimuli. The most
understood modifications include ubiquitination,
phosphorylation, acetylation, methylation, and glycosylation. In
1997, a new type of modifying protein (small
ubiquitinlike modifier or SUMO) was identified [
]. Since then,
four SUMO isoforms (SUMO-1, −2, −3, and −4) have
been characterized in humans. Sequence alignment
revealed that SUMO-2 and SUMO-3 are approximately
97% similar, so they are often referred to as SUMO-2/3.
SUMO-4 shares around 86% identity with SUMO-2/3,
while SUMO-1 has approximately 46% identity with
SUMO-2/3. SUMO-1 and SUMO-2/3 are ubiquitously
expressed in the body; however, SUMO-4 has been
detected only in the kidney, dendritic cells, and
]. Each SUMO can be covalently conjugated
to the lysine residue found within the conserved
ΨKxD/E motif, where Ψ represents a hydrophobic
residue of the target protein, resulting in its
The sumoylation process begins with the
transcription and translation of the sumo genes to yield the
SUMO pro-peptide (Fig. 1). A SUMO protease (see
below) removes a small number of amino acids from
the C-terminus of the pro-peptide to reveal the SUMO
C-terminal di-glycine motif. The end result is the
mature form of SUMO, which can be used to modify a
target protein (Fig. 1). Mature SUMO is activated by
the SUMO-activating enzyme, which is a heterodimer
consisting of two subunits (SAE1 and SAE2, Fig. 1).
Using ATP as a donor/substrate, SUMO E1 catalyzes
the adenylation of the di-glycine motif of the mature
SUMO, forming a SUMO-AMP intermediate. During
this step, the SUMO E1 undergoes a conformational
change, which then allows for the formation of a
transient intermediate thioester bond between SUMO and a
cysteine residue on SAE2 (C173).
Following activation, SUMO is passed to the
SUMOconjugating enzyme Ubc9 (Fig. 1), a 158-aa protein that
forms a single domain structure similar to other
ubiquitin conjugating proteins [
]. Ubc9 consists of four core
β-sheets that are surrounded at the ends by four
]. Within the pocket formed by these structures
is the conserved catalytic cysteine residue of Ubc9
(C93). SAE1/2 transfers the SUMO to Ubc9 C93,
forming a second transient intermediate thioester bond [
The Ubc9 pocket also identifies the canonical ΨKxD/E
motif within the target protein (Fig. 1) [
]. The catalytic
site of Ubc9 catalyzes formation of an isopeptide bond
with the C-terminal SUMO di-glycine motif and the
εamino group of the lysine residue within the SUMO
motif of the target protein (Fig. 1) [
]. In addition to the
interaction of SUMO with the pocket of Ubc9, the target
protein interacts through non-covalent interactions with
the surface of Ubc9. This surface is composed of
numerous patches with positive and hydrophobic residues [
While Ubc9 has several SUMO-independent functions,
it is proposed that the non-covalent SUMO-Ubc9
interactions enable the formation of poly-SUMO chains [
In some cases, the attachment of SUMO to the target
protein also requires a SUMO ligase (E3) (Fig. 1), such
as Ran binding protein (RanBP2) [
], a member of the
protein inhibitor of activated STAT (PIAS) protein
], or the polycomb protein Pc2 [
]. These SUMO
E3 ligases confer specificity towards the target protein
and may help mediate the sumoylation of target
proteins, including residues outside of the canonical
ΨKxD/E motif. The SUMO E3 ligases are thought to
interact with SUMO and Ubc9 and serve as adaptors
between the Ubc9-SUMO intermediate and the target
The entire process can be reversed by SUMO
proteases or Sentrin-specific proteases (SENPs) (Fig. 1). In
mammals, six SENP isoforms (SENP 1–3 and 5–7) with
de-sumoylating activity have been identified [
isoforms are divided into sub-families based on their
cellular distribution, role in maturation of the SUMO
pro-peptides, and/or their specificity in cleavage of
SUMO-1- or SUMO-2/3-modified proteins. SENP1 and
SENP2 make up the first sub-family due to their ability
to cleave SUMO-1, −2, and −3 [
]. The second and
third sub-families are SENP3 and SENP5 or SENP6 and
SENP7, respectively, which preferentially cleave
SUMO2/3-modified proteins over SUMO-1-modified proteins
]. In addition to de-conjugating sumoylated proteins,
SENP1, SENP2, and SENP5, are also responsible for the
maturation of the SUMO pro-peptides (Fig. 1) [
The SENPs share a conserved C-terminal cysteine
protease catalytic domain [
], which has the typical catalytic
triad (cysteine-histidine-aspartic acid). The C-terminal
domain is formed by anti-parallel five-stranded β-sheets
surrounded by two α-helices [
]. This structure interfaces
with SUMO, allowing for the interaction between the
SENP and SUMO precursors or sumoylated proteins [
Within the catalytic site, tryptophan residues form a
tunnel that allow for the accurate position of the SUMO
diglycine motif and scissile bond [
]. Within the tunnel,
the scissile bond creates a kink in the isopeptide linkage
or within the SUMO pro-peptide and promotes the
cleavage of the bond, resulting in protein de-sumoylation or
SUMO maturation, respectively [
In a manner similar to ubiquitination, proteins can be
mono- or poly-sumoylated. SUMO-2/3 contains the
canonical SUMO motif, granting mature SUMO-2/3 the
ability to sumoylated, forming poly-SUMO chains on
target proteins [
]. However, SUMO-1 lacks this motif,
so it can only be used to mono-sumoylate a target
protein or act as a terminator of a SUMO-2/3 chain [
Little is known of how these forms differ in regulating
protein function [
While only 5–10% of a target protein is found in a
sumoylated form at any given time, the effect
sumoylation has on protein function can be long-lived, even
affecting protein function after it is de-sumoylated [
Sumoylation can regulate protein activity through
altering a protein’s intracellular location, affecting a protein’s
ability to interact with other proteins, and modifying a
protein’s ability to interact with DNA [
3, 19, 20
sumoylation also modulates cellular processes, including
nuclear trafficking, cell division, DNA replication, DNA
damage responses, transcription, and chromosome
]. Because of the multitude of cellular
processes affected by protein sumoylation, dysregulation
of sumoylation processes can significantly alter normal
cellular events, such as cell motility and survival, and
result in extremes, including cancer progression and
Viruses and sumoylation processes
While the intracellular pool of free SUMO-1 is
considered to be limited due to its conjugation to
highaffinity targets such as RanGap1 [
SUMO-2/3-mediated sumoylation is primarily inducible by stress [
Global changes in protein sumoylation (by SUMO-1
and/or SUMO-2/3) occur following heat shock [
DNA damage [
], and inhibition of the proteasome
], and other cellular stimuli, such as viral
infection. During infection and replication, viruses can
manipulate the sumoylation process to ensure viral
persistence within the host. In addition, protein
sumoylation has a role in mediating the antiviral effect of the
interferons . Through inhibition or induction of
protein sumoylation, viruses have a multitude of
mechanisms by which they manipulate this cellular process to
ensure their survival and propagation.
Numerous viruses benefit from impaired sumoylation
processes. Decreased sumoylation of specific antiviral
proteins (promyelocytic leukemia protein/PML and
Sp100) has been suggested to be important in regulating
anti-viral immune responses. For example, herpes
simplex virus-1 (HSV-1) infection results in a three-fold
decrease in the modification of over 100 cellular
proteins, including PML and Sp100, by SUMO-2/3
(Table 1) [
]. The observed changes were dependent on
the viral ubiquitin ligase ICP0, which targeted the
SUMO-2/3-modified proteins to the proteasome for
]. Many of the ICP0-targeted proteins
are involved in the regulation of transcription, chromatin
assembly, and chromatin modification, which suggests
the importance of decreased protein sumoylation for
lytic HSV replication [
]. Similarly, the Epstein-Barr
virus (EBV) protein kinase BGLF4 suppresses global
cellular sumoylation processes in order to facilitate EBV
lytic replication [
], which suggests inhibition of
sumoylation processes aids herpesvirus propagation.
In contrast, some viruses benefit from increased
protein sumoylation. Influenza virus (Type A and Type B;
IAV and IBV) infection leads to a viral
replicationdependent global increase in cellular sumoylation
(Table 1) [
]. While select influenza viral proteins
are targeted for sumoylation , IAV infection
substantially increased the modification of 76 cellular
substrates by SUMO-1 and 117 cellular substrates by
]. This increase was paralleled by
decreased sumoylation of over 500 cellular proteins
], suggesting the exchange of SUMO-1/2/3 from
pre-existing targets to a restricted set of new targets
]. Furthermore, because increased gross SUMO
conjugation was not observed following infection with
multiple cytoplasmic-replicating RNA viruses,
Domingues et al. propose that the induction of SUMO
remodeling is a specific response to nuclear-replicating
]. Similarly, we documented a global increase
in cellular sumoylation during EBV latency, which is
mediated by the principal viral oncoprotein latent
membrane protein-1 (LMP1; Table 1) [
LMP1induced sumoylation of cellular proteins contributes to
the oncogenic potential of LMP1 [
], modulation of
innate immune responses [
], and the maintenance of
], all of which suggest the importance of
increased sumoylation during latent infections.
While understanding global changes in sumoylation
processes is critical to understanding global cellular
changes that occur during viral infection, it is also
important to identify the changes in sumoylation of
specific cellular targets to elucidate mechanisms by which
viruses modulate cellular responses that are pathogenic
and ensure their propagation. Deciphering how viruses
manipulate components of the sumoylation machinery
may lead to new therapeutic targets that inhibit
sumoylation processes, thus inhibiting viral replication and
Viruses and sumo/SUMO levels
The first potential target of the SUMO machinery is the
expression of the sumo genes. The four SUMO isoforms
are found on different chromosomes, specifically
chromosomes 2, 17, 21, and 6. To date, only the promoter
for sumo-1 has been identified [
], which has potential
Viral Abbreviations: CELO Chicken embryo lethal orphan, ADV adenovirus, HSV Herpes Simples virus, HCMV human cytomegalovirus, HHV-6 human herpesvirus-6,
EBV Epstein-Barr virus, KSHV Kaposi’s Sarcoma-associated herpesvirus, HPV human papilloma virus, VACV Vaccinia virus, HCV hepatitis C virus, JEV Japanese
encephalitis virus, EBOV Ebola virus, IAV Influenza A virus, IBV Influenza B virus, HPIV human parainfluenza virus, FMDV foot-and-mouth disease virus,
HIV human immunodeficiency virus;? – Probable manipulation
NF-κB, FOXP3, p53, and TCF-4E binding sites. While
the transcriptional activity of these factors can be
activated or repressed by a multitude of viruses and we have
preliminary data that suggests the sumo promoters are
activated during EBV latency through activation of
NFκB by LMP1 (unpublished data) (Fig. 2), the ability of
any particular virus to activate the sumo promoters
remains to be reported.
SUMO levels can also be regulated post-transcriptionally.
For example, interferon-α treatment increases unconjugated
SUMO-1 levels, but not sumo-1 mRNA levels [
]. In this
case, agonist binding to the toll-like receptors results in the
activation of IFN and NF-κB signaling, which inhibits let-7
family microRNA levels and increases SUMO levels in
treated cells [
]. This increase in SUMO levels suppresses
HSV and HIV replication (Fig. 2) [
], highlighting one
mechanism by which the sumoylation process mediates the
antiviral effect of the interferons [
Influenza virus infection has also been shown to
increase SUMO levels without increasing SUMO mRNA
ADV E4-ORF3 [
KSHV bKZIP [
KSHV LANA [
HSV ICP0: PIAS1/ HSV [
60, 65, 66
High risk E6:
NP: PIAS1 [
HCV: PIAS3? [
] JEV [
transcripts (Fig. 2) . As explained above, IAV
infection results in the sumoylation of nearly 200 cellular
proteins, which is correlated with the de-sumoylation of
over 500 cellular proteins [
]. While the distinct
mechanism of the regulation of SUMO levels by influenza
virus was not determined, it was shown to require the
viral RNA, which led to the hypothesis that
nuclearreplicating viruses trigger a stress response that induces
SUMO remodeling and results in increased SUMO
These findings identify three different mechanisms by
which viruses can regulate sumo/SUMO levels following
infection. First, the sumo promoters can be regulated by
viral infection. Second, viruses can inhibit let-7 family
microRNA levels to increase translation of sumo
mRNAs. Third, viruses can regulate intracellular SUMO
pools post-translationally by inducing SUMO
remodeling. However, a better understanding of how
intracellular SUMO levels are affected by viral infections is
required to truly comprehend the function of
sumoylation processes in viral replication and develop tools to
manipulate protein sumoylation for therapeutic gains.
Viruses and the SUMO-activating enzyme
The SUMO-activating enzyme is a common target for
several sumoylation inhibitors, including ginkgolic acid
(an alkylphenol from Ginko biloba) and Davidiin (an
ellagitannin from Davidia involucrata) (Fig. 2) [
These inhibitors bind to the SUMO-activating enzyme
(SAE1/2) and impair the formation of the E1-SUMO
]. While there are additional cellular
targets for these drugs, their effects on sumoylation
processes have been documented [
37, 39, 40
]. Viruses have
also evolved mechanisms by which they can impair the
formation of the E1-SUMO intermediate. For example,
infection of HeLa cells with avian adenovirus CELO
(chicken embryo lethal orphan) induces a reduction of
SAE1 and SAE2 (Fig. 2) [
]. Mechanistically, this
happens through the recruitment of the cullin RING
ubiquitin ligases by the essential viral early protein
Gam1 and the formation of a complex with SAE1/2 .
The cullin RING ubiquitin ligases ubiquitinate SAE1,
resulting in the degradation of SAE1 by the proteasome
]. The consequence of SAE1 degradation is an
increase in unpaired SAE2, which leads to the subsequent
proteasome-mediated degradation of SAE2 [
end result is the accumulation of SUMO-unmodified
substrates, increased localization of SUMO-1 in the
cytoplasm, and destruction of PML nuclear bodies all of
which contribute to enhanced viral propagation [
While the avian adenovirus is the only virus to date
reported to target the SUMO-activating enzyme, it is
highly likely that multiple viruses have the ability to
inhibit, or possibly induce, the SUMO-activating enzyme,
and thus the ability to modulate sumoylation processes.
Viruses and Ubc9
The E2 SUMO-conjugating enzyme, Ubc9, has been
proposed as an ideal target for therapies targeting the
sumoylation pathway [
]. Two proposed methods of
targeting Ubc9 include knockdown of Ubc9 levels by
siRNA and over-expression of an enzymatically inactive
Ubc9 (Ubc9 C93S) [
]. Recently the antibiotic
Spectomycin B1 was documented to bind directly to Ubc9 and
inhibit the formation of the Ubc9-SUMO intermediate
]. Viruses have been shown to be able to manipulate
Ubc9 through direct interactions, Ubc9 degradation, or
altered Ubc9 localization in order to promote viral
infectivity and viral pathogenesis.
During the sumoylation process, potential target
proteins interact with Ubc9. Some viruses are able to hijack
Ubc9 for their own gain. The first viral gene expressed
following human adenovirus infection (E1A) interacts
with the N-terminus of Ubc9 (Fig. 3) [
], which results
in competition between E1A and mono-sumoylated
target proteins and inhibits the poly-sumoylation of the
target proteins. While the direct Ubc9-interacting residues
have not been mapped, EBV LMP1 also hijacks Ubc9
during latent viral infections (Fig. 3) [
documented that the understudied C-terminal activating
region 3 (CTAR3) was necessary and sufficient for this
]. While this interaction does not induce
the sumoylation of LMP1 itself, the LMP1/Ubc9
interaction did result in increased sumoylation of other
cellular proteins, which we subsequently showed to be
important in modulating innate immune responses,
maintaining viral latency, and the oncogenic potential of
The human papillomavirus (HPV) protein (E2), which
aids viral replication and genome segregation and
downregulates expression of the oncogenic E6 and E7, also
interacts with Ubc9 (Fig. 3) [
]. However, instead of
using this interaction to affect the sumoylation of other
proteins, the viral E2 is itself sumoylated [
of this sumoylation decreases the transcriptional activity
of papillomavirus E2 and abrogated its repressive effects
on E6/7 expression [
]. These findings suggests that
the hijacking of Ubc9 by HPV E2 and the sumoylation
of E2 has an inhibitory effect on viral promoters but an
activating effect on select cellular promoters [
ability of a viral protein to interact with Ubc9 and be
sumoylated is not unique to HPV. Numerous viral
proteins, including the immediate early herpesvirus proteins
], have also been shown to interact with the
SUMO conjugating enzyme and be sumoylated.
Independent of the enzymatic function of Ubc9 and
the covalent attachment of SUMO to a target protein,
human herpesvirus-6 immediate early protein 2 (IE2)
interacts with Ubc9, which induces the repression of
IE2-mediated promoter activation [
]. The viral IE2
lacks a consensus SUMO motif, and sumoylation of the
viral protein has not been reported [
]. Instead, it
appears that the viral IE2 hijacks Ubc9 in the nucleus
to facilitate recruitment of a repressive transcription
]; however, the role of this interaction in
viral replication remains to be determined.
Instead of hijacking Ubc9 to increase protein
sumoylation, some viruses induce the degradation of Ubc9. The
HPV oncogenic protein E6 binds and leads to the
degradation of Ubc9 by the proteasome (Fig. 3) [
E6induced degradation of Ubc9 requires the cellular
ubiquitin ligase E6AP [
]. Interestingly, Ubc9 levels
increase during cervical lesion progression, suggesting
the possibility of using Ubc9 levels to diagnose cervical
]. While these findings seem somewhat
contradictory, it is possible that E6-induced degradation
of Ubc9 is altered during cervical lesion progression, or
as Heaton et al. propose, the reduction of Ubc9 may lead
to subcellular region-specific substrate effects [
Similarly, the avian adenovirus early protein Gam1
interacts with Ubc9 (Fig. 3) [
]. While ubc9 RNA levels
were unaltered, Gam1 expression greatly reduced the
stability of Ubc9 [
proteasomemediated degradation of Ubc9 did require an
enzymatically active Ubc9 [
], suggesting there are unidentified
sumoylation-specific aspects to this interaction. The end
result of inhibition of sumoylation processes by Gam1 is
the activation of transcription and the induction of an
environment favorable for viral replication [
These data suggest that viruses have multiple
mechanisms by which they can target the E2
SUMOconjugating enzyme: they can hijack Ubc9 to regulate
protein sumoylation; they can interact with Ubc9 to
induce their own sumoylation and regulate their activity;
they can interact with Ubc9 independent of cellular
sumoylation processes; and they can induce the
degradation of Ubc9 by the proteasome. Because Ubc9 has
been suggested to be an ideal target for therapies
targeting the sumoylation pathway [
], deciphering how
specific viral proteins target the expression and function
of Ubc9 may reveal potential interventions to treat
Viruses and SUMO E3 ligases
While SUMO E3-ligases can guide substrate specificity,
they are not required for sumoylation in vitro.
Interestingly, the ability of viruses to manipulate SUMO E3
ligases, specifically the protein inhibitor of activated
STATs (PIAS) family and RANBP2, has been
investigated and reported more often than any other member
of the SUMO machinery. Levels of members of the
PIAS family are increased following some viral
infections, resulting in dysregulation of antiviral immune
responses. For example, PIAS4 levels are upregulated
during HSV-1 infection (Fig. 4) [
]. During lytic
replication PIAS4 is recruited to nuclear foci
containing viral genomes and positively regulates intrinsic
anti-viral immune responses to HSV infection [
Parvovirus B19 infection increases PIAS3 levels (Fig. 4)
]. Specifically, the nonstructural viral protein NS1
transactivates numerous cellular promoters, including
]. PIAS3 can act as a negative regulator for
STAT3, which is also phosphorylated and activated by
]. The NS1-mediated modulation of
STATtargeted gene expression exacerbates inflammatory
responses and results in endothelial cell dysfunction
and viral pathogenesis, including viral cardiomyopathy
]. In a similar correlation of PIAS3 levels and viral
pathogenesis, increased PIAS3 levels are also associated
with relapse of chronic hepatitis C virus infection and
resistance to interferon-α treatment (Fig. 4) [
however, the mechanism for the events remain to be
clarified. Overall, these reports suggest that some
viruses induce PIAS expression in order to regulate
viral replication and viral pathogenesis.
While some viruses induce the expression of SUMO
E3 ligases, other viruses can inhibit cellular SUMO E3
ligase activity. For example, HPV E6 targets PIASy to
inhibit the sumoylation of p53 and prevent cellular
senescence (Fig. 4) [
]. Only E6 from high-risk
papillomaviruses inhibited PIASy, which suggests that
this selective targeting of the SUMO E3 ligases has a
role in oncogenesis [
]. Similarly, human
cytomegalovirus immediate-early protein (IE)-2 hijacks PIAS1 to
inhibit the SUMO E3 ligase from sumoylating IE1 (Fig. 4)
]. Decreased sumoylation of IE1 corresponded with
enhanced repression of interferon-stimulated genes [
which suggests the IE2-PIAS1 interaction is an
important step in inhibiting inflammation and promoting viral
Both adenoviruses and HSV-1 are thought to regulate
the activity of SUMO E3 ligases by altering their
localization (Fig. 4). First, the adenoviral E4-ORF3,
which disrupts the anti-viral PML nuclear bodies (a
process regulated by sumoylation), specifically targets
PIAS3 to the nuclear scaffolds associated with viral
genome replication domains [
], suggesting sumoylation
processes are being redirected to aid viral replication.
Second, the disruption of anti-viral PML nuclear bodies
is mediated by HSV-1 ICP0 which, in addition to having
E3 ubiquitin ligase activity, possess SUMO-targeted
ubiquitin ligase properties that target sumoylated
proteins for degradation [
]. While PIAS1 restricts viral
replication, ICP0 disrupts the recruitment of PIAS1 to
viral replication domains, allowing lytic replication to
]. PIAS1, along with PIASxβ, interacts with
the nucleocapsid protein of hantaviruses (Fig. 4),
specifically Seoul virus and Hantann virus [
]. Analysis of the
hantavirus nucleocapsid protein sequence revealed a
conserved sumoylation motif; however, sumoylation of
the nucleocapsid protein remains to be documented
]. While the direct function of this protein-protein
interaction during hantavirus replication remains
unknown, it may be similar to the ICP0/PIAS1
interaction in modulating antiviral responses and aiding
In contrast to infection by HPV, adenoviruses, HSV,
and hantaviruses, Ebola virus infection increases the
activity of the SUMO E3 ligase PIAS1 (Fig. 4) [
viral VP35 suppresses the production of type I
interferons due to its interaction with interferon regulatory
factors (IRF)-3 and −7 [
]. Ubc9 and PIAS1 are
recruited to the VP35/IRF interaction, resulting in the
sumoylation of these transcription factors and their
transcriptional repression [
]. The modulatory effect
that Ebola virus VP35 has on IRF7 is similar to our
findings of the regulation of this transcription factor by
EBV LMP1 [
]. This leads us to propose that LMP1
may also interact with PIAS1 or another SUMO E3
ligase to increase the sumoylation of select cellular
proteins. However, this remains to be tested.
While there have been multiple investigations into the
viral manipulation of PIAS family members, fewer studies
have examined how viruses affect the nuclear pore protein
RanBP2, which has SUMO E3 ligase activity [
levels are upregulated following Japanese encephalitis
virus infection (Fig. 5) [
]. Knockdown of RanBP2
increases viral replication and decreases production of Type
I interferons, which suggests that Japanese encephalitis
virus-induced RanBP2 levels have an anti-viral function
], limiting viral replication and viral-mediated immune
Virus-induced manipulation of RanBP2 is thought to
regulate the movement of proteins and viral genomes
into and out of the nucleus. HSV infection induces the
glycosylation of RanBP2 and reduces the interaction of
RanBP2 with other members of the nuclear pore
complex (Fig. 5) [
], leading to the proposal that
glycosylated RanBP2 does not associate with the nuclear pores
during HSV infection and alters the trafficking of
proteins or complexes into and out of the nucleus [
During HIV-1 infection, RanBP2 is essential for the
nuclear import of the viral genome (Fig. 4) [
binding to the viral capsid (Fig. 5) . Supporting the
importance of RanBP2 to HIV replication is the finding
that certain RanBP2 point mutations that map to the
capsid-RanBP2 interacting domains have evolved in
primates under positive selection [
]. While primates with
these select RanBP2 mutations display enhanced HIV
replication, the role of these mutations in the interaction
of RanBP2 and the viral capsid and the nuclear import
of viral genome is unknown.
Interestingly, a naturally occurring RanBP2 mutation
has been associated with increased susceptibility to
recurrent acute necrotizing encephalopathy following
infection by influenza, HHV-6, Coxsackievirus,
enteroviruses, or parainfluenza virus [
]. Similar to HIV
infection, the naturally selected mutation enhances viral
replication, which suggests that RanBP2 also aids
delivery of the influenza genome to the nucleus. While the
role of RanBP2 in sumoylation processes during HIV
and influenza virus infection remains to be investigated,
it is likely that protein sumoylation and the nuclear
import of viral genomes is positively affected by these
naturally occurring mutations.
Viral mimics of the SUMO E3 ligases
Perhaps the most novel mechanism by which a virus
can regulate the function of a SUMO E3 ligase is by
encoding its own ligase, which is the case for Kaposi’s
sarcoma-associated herpesvirus (KSHV), adenovirus,
and foot-and-mouth disease virus (Fig. 5) [
First, KSHV encodes the early gene K-bZIP (open
reading frame K8 spliced to adjoin the ZIP domain) that
belongs to the basic region-leucine zipper family of
transcription factors [
]. Multiple functions have been
identified for K-bZIP [
], including a role as a
SUMO adaptor where it recruits Ubc9 to induce the
sumoylation and transcriptional repression of specific
viral promoters [
]. While this is similar to our work
investigating the hijacking of Ubc9 by Epstein-Barr
virus LMP1 [
], K-bZIP is unique in that it has been
identified to have SUMO E3 ligase activity that is
specific for SUMO-2/3, which suggests K-bZIP may have a
role in mediating the poly-sumoylation of target
]. K-bZIP auto-sumoylates itself and catalyzes
the sumoylation of its interacting proteins (ex. p53 and
], which ultimately inhibits the activation of the
Type I interferons and regulates KSHV reactivation
and lytic replication [
98, 107, 108
]. During KSHV
latency, the latency-associated nuclear antigen (LANA)
also acts as a SUMO E3 ligase recruiting the
SUMOUbc9 intermediate and inducing the sumoylation of
cellular histones (Fig. 5) [
]. While the exact function
of this viral SUMO E3 ligase activity is unclear, it is
likely to be critical in the maintenance of viral latency
similar to our results on EBV LMP1 [
findings highlight the importance of sumoylation processes
in both lytic and latent herpesvirus infections.
Like KSHV, adenoviruses also encode two SUMO E3
ligase mimics. The adenoviral protein E1B-55 K, which
regulates late viral gene expression, can also function as
a SUMO E3 ligase that specifically induces the
sumoylation of p53 and its localization of PML nuclear bodies
(Fig. 5) [
]. The end result is the inactivation of p53,
which is then exported from the nucleus and targeted
for degradation by the proteasome [
]. Recently the
adenoviral early protein E4-ORF3 was shown to possess
SUMO E3 ligase activity due to its ability to mediate the
sumoylation of transcription intermediate factor
(TIF)1γ, which has a role in transcriptional regulation and
DNA damage repair (Fig. 5) [
specifically induces the modification of TIF-1γ by SUMO-3
and aids poly-SUMO chain elongation, eventually
leading to its degradation by the proteasome . The
eventual degradation of proteins targeted for
sumoylation by both E1B-55 K and E4-ORF3 may help promote
early and late adenoviral gene expression, facilitating
The SUMO E3 ligase mimic for foot-and-mouth
disease virus is different from the DNA viral mimics in that
it has been assigned a specific PIAS-like function (Fig. 5)
]. The viral proteinase (leader protein; Lpro) is a
papain-like proteinase that auto-catalytically self-cleaves
from the viral polyprotein [
]. Lpro inhibits anti-viral
responses and helps the virus evade host immune
], in part due to its ability to act as a
de-ubiquitinating enzyme . Recently, Lpro was
shown to have a domain with PIAS-associated function
]. Mutation of the Lpro PIAS-like domain
significantly inhibits viral replication and viral pathogenesis
]. In addition mutation of the Lpro PIAS-like domain
increased the production of virus-specific neutralizing
]. While proteins involved with
sumoylation processes were not specifically examined, analyses
showed that when compared with the mutant virus,
wild-type virus was able to upregulate expression of
numerous proteins associated with post-translational
]. It is probable that Lpro can act as a
SUMO E3 ligase, targeting proteins involved with
antiviral responses, in order to allow viral replication to
Together, these findings show that although the
SUMO E3 ligases may not be required for sumoylation
in vitro, they have an important role in regulating viral
replication, anti-viral responses, and viral pathogenesis.
Therefore, in the future the identification of additional
mechanisms by which viruses can regulate the
expression and activity of SUMO E3 ligases or even mimic the
ligases will contribute to our knowledge of viral
pathogenesis and suggest novel interventions in the treatment
of viral disease.
Viruses and SUMO proteases
The SENPs regulate the intracellular pools of free
SUMO as well as the de-sumoylation of modified target
proteins, making them critical in the regulation of
sumoylation processes. Interestingly, SENP inhibitors
inhibit HIV replication [
], which suggests the SUMO
proteases may be ideal targets for regulating viral
infection or replication in vivo. Further, this finding also
indicates that viruses have evolved mechanisms by which
they use the SENPs in order to ensure viral
dissemination. However, the effect viral infection has on SENPs
remains to be documented. We have preliminary data
suggesting that EBV LMP1 inhibits SENP activity during
viral latency (unpublished data) (Fig. 6). When we
specifically focused on SENP2, we found that LMP1
induced the sumoylation of SENP2, resulting in the
inability of SENP2 to interact with sumoylated proteins,
thus inhibiting its activity (unpublished data).
Just as some viruses encode their own SUMO E3
ligase, certain viruses are thought to encode SENP
mimics. Vaccinia virus and fowlvirus encode a protease
(I7) that is expressed late in infection (Fig. 6) [
viral I7 protease has a C-terminal region with many
structural similarities to the SENPs [
]; however the
ability of this protease to actually cleave sumoylated
proteins remains to be documented. African swine fever
virus, a similar large, double-stranded DNA virus,
encodes a cysteine protease (pS273R), which is a 31-kDa
protein that has the conserved catalytic residues
characteristic of SENPs [
]. pS273R specifically cleaves the
viral polyproteins following a di-glycine motif (Fig. 6)
], which coincides with the specificity of the SENPs
]. Interestingly, S273R associates with the core of
mature viral particles [
], suggesting a possibility that
it, through its di-glycine motif-targeted cysteine protease
activity, may have a function in the early steps following
In some cases, the SENP mimics lack the ability to
deconjugate sumoylated proteins. For example, the
adenoviral protease processes viral proteins and has
structural similarity to the Saccharomyces cerevisiae
SUMO protease (Ulp1) [
]. While the adenoviral
protease is essential for viral infectivity, it has been
shown that it does not actually have the ability to
desumoylate modified target proteins [
]; however, the
possibility exists that the viral protease competes with
the SENPs in interacting with sumoylated proteins. This
could result in decreased SENP activity. Regardless, this
study highlights the importance of elucidating functional
targets of viral SENP mimics. Due to their role in
regulating the maturation of the SUMO precursor and
protein de-sumoylation [
], functional SENPs can affect
target protein sumoylation and de-sumoylation. The
critical role that dysregulation of cellular sumoylation
processes has in viral infection, replication, and egress,
suggests that SENPs may be an ideal viral target for
manipulation of this cellular process.
Other pathogens and manipulation of the SUMO machinery
The ability of a pathogen to manipulate sumoylation
processes and members of the sumoylation machinery is
not unique to just viruses of vertebrates. Sumoylation
processes are important for White spot syndrome virus
(WSSV) infection in crustaceans [
infection increases levels of SUMO and Ubc9 at the mRNA
and protein levels [
]. Silencing of SUMO and
Ubc9 expression using RNA interference inhibits viral
gene expression, viral replication, and shrimp mortality
], which highlights the importance of sumoylation
processes in the life cycle of WSSV. Another example is
the geminivirus Rep protein [
], which binds to
double-stranded DNA and catalyzes the cleavage and
ligation of single-stranded DNA. Rep also hijacks the
Ubc9 homolog in plants, increasing sumoylation
processes, and aiding plant virus replication [
Outside of viruses, Shigella spp. also targets Ubc9
]. As a gram-negative bacterium, Shigella has a Type
3 secretion system that can deliver proteins into the
cytosol of infected cells. As a result of the delivery of
bacterial proteins to cells, Shigella decreases cellular
Ubc9 levels along with the level of sumoylated proteins
within the infected cell [
]. This suggests that one
mechanism by which Shigella infection causes the
pathology associated with shigellosis is through inhibition of
cellular sumoylation processes.
As we have outlined here, viruses possess multiple
different mechanisms by which they manipulate the
sumoylation machinery for their benefit and to the detriment of
the host. Many investigations into sumoylation processes
during viral infection have focused on the covalent
modification of specific viral and cellular proteins. We
have focused on how viruses affect the different steps of
the sumoylation processes to regulate viral replication
and viral pathogenesis. Viruses are able to target each
step of the sumoylation process (Table 1), from the
activation of the sumo promoters and altering the
intracellular pools of free SUMO available for conjugation to
the regulation of the expression/function of the SENPs.
Most previous research has focused on the
manipulation of Ubc9 and the SUMO E3 ligases. However, as we
have shown, there are additional possible targets by
which viruses can influence sumoylation processes, and
we have not addressed the role of SUMO-interacting
motifs in the SUMO machinery and in viral proteins and
SUMO-targeted ubiquitin ligases. Together, these
findings highlight the importance of sumoylation processes
in the viral life cycle and reveal the necessity of
deciphering unknown mechanisms by which viruses target
the cellular sumoylation machinery and sumoylation
processes during their infection cycle.
The same pathway can be manipulated by different
pathogens in different ways to achieve the same end
goal, which is viral replication and propagation.
Therefore, elucidating how viruses manipulate each step of the
sumoylation process may reveal new targets for specific
antiviral therapies. The viral-mediated targeting of the
SUMO machinery to enhance/inhibit sumoylation
processes could also have potential therapeutic effects in
designing new treatments for cancer and other diseases
where sumoylation processes are dysregulated.
ADV: Adenovirus; CELO: Chicken embryo lethal orphan; EBOV: Ebola virus;
EBV: Epstein-barr virus; FMDV: Foot-and-mouth disease virus; HCMV: Human
cytomegalovirus; HCV: Hepatitis C virus; HHV-6: Human herpesvirus-6;
HIV: Human immunodeficiency virus; HPIV: Human parainfluenza virus;
HPV: Human papilloma virus; HSV: herpes simples virus; IAV: Influenza A virus;
IBV: Influenza B virus; ICP0: Infected cell protein 0; IE: Immediate early;
JEV: Japanese encephalitis virus; KSHV: Kaposi’s sarcoma-associated
herpesvirus; LANA: Latency-associated nuclear antigen; LMP1: Latent membrane
protein-1; NP: Nucleocapsid protein; ORF: Open reading frame; PIAS: Protein
inhibitor of activated STAT; RanBP2: Ran binding protein-2; VACV: Vaccinia
We apologize to those researchers whose work could not be cited for lack of
space. We would like to thank E.I. Snyder, J. Heckman, and R.M. Bentz for
their support and R. McCann and R. McKallip for their assistance editing the
This work was supported by the National Cancer Institute (CA160786).
Availability of data and materials
AL and GB wrote the manuscript and made the figures. AL, WC, and GB revised
the manuscript and figures. All authors read and approved the final manuscript.
Ethics approval and consent to participate
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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