HERC6 Is the Main E3 Ligase for Global ISG15
Conjugation in Mouse Cells
Diede Oudshoorn1, Sander van Boheemen1¤, Maria Teresa Sa ´nchez-Aparicio1, Ricardo Rajsbaum1,
Adolfo Garcı ´a-Sastre1,2,3., Gijs A. Versteeg1*.
1Department of Microbiology, Mount Sinai School of Medicine, New York, New York, United States of America, 2Department of Medicine, Mount Sinai School of
Medicine, New York, New York, United States of America, 3Global Health and Emerging Pathogens Institute, Mount Sinai School of Medicine, New York, New York, United
States of America
Type I interferon (IFN) stimulates expression and conjugation of the ubiquitin-like modifier IFN-stimulated gene 15 (ISG15),
thereby restricting replication of a wide variety of viruses. Conjugation of ISG15 is critical for its antiviral activity in mice.
HECT domain and RCC1-like domain containing protein 5 (HerC5) mediates global ISGylation in human cells, whereas its
closest relative, HerC6, does not. So far, the requirement of HerC5 for ISG15-mediated antiviral activity has remained unclear.
One of the main obstacles to address this issue has been that no HerC5 homologue exists in mice, hampering the
generation of a good knock-out model. However, mice do express a homologue of HerC6 that, in contrast to human HerC6,
can mediate ISGylation. Here we report that the mouse HerC6 N-terminal RCC1-like domain (RLD) allows ISG15
conjugation when replacing the corresponding domain in the human HerC6 homologue. In addition, sequences in the C-
terminal HECT domain of mouse HerC6 also appear to facilitate efficient ISGylation. Mouse HerC6 paralleled human HerC5 in
localization and IFN-inducibility. Moreover, HerC6 knock-down in mouse cells abolished global ISGylation, whereas its over
expression enhanced the IFNb promoter and conferred antiviral activity against vesicular stomatitis virus and Newcastle
disease virus. Together these data indicate that HerC6 is likely the functional counterpart of human HerC5 in mouse cells,
suggesting that HerC62/2mice may provide a feasible model to study the role of human HerC5 in antiviral responses.
Citation: Oudshoorn D, van Boheemen S, Sa ´nchez-Aparicio MT, Rajsbaum R, Garcı ´a-Sastre A, et al. (2012) HERC6 Is the Main E3 Ligase for Global ISG15
Conjugation in Mouse Cells. PLoS ONE 7(1): e29870. doi:10.1371/journal.pone.0029870
Editor: Volker Thiel, Kantonal Hospital St. Gallen, Switzerland
Received July 26, 2011; Accepted December 5, 2011; Published January 17, 2012
Copyright: ? 2012 Oudshoorn et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work has been partly funded by National Institute of Allergy and Infectious Diseases grant U19AI083025. The funders had no role in study design,
data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: Gijs.Versteeg@mssm.edu
. These authors contributed equally to this work.
¤ Current address: Department of Virology, Erasmus Medical Center, Rotterdam, The Netherlands
Type I interferons (IFN) are induced in response to infection
and mediate the expression of antiviral IFN-stimulated genes
(ISGs) . ISG15 is an 15 kDa ubiquitin-like modifier consisting
of two ubiquitin-like domains that is early and abundantly induced
by type I IFN . Similar to ubiquitin, ISG15 is conjugated to
lysines on numerous target proteins through the action of specific
E1-activating, E2-conjugating, and E3-ligase enzymes, all of which
are also IFN-inducible [3,4,5,6]. Previous knock-down studies
demonstrated that HerC5 is the main ISG15 E3 ligase to mediate
global conjugation in human cells, whereas HerC6 -its closest
relative- was devoid of any ISG15 conjugating activity [3,7].
The HerC protein family consists of six members, two of which
(HerC1 and HerC2) are substantially larger than the others
(HerC3–6) [8,9]. They share a C-terminal HECT ubiquitin E3-
ligase domain and an N-terminal RCC1-like domain (RLD). The
exact function of the RLDs in these proteins has remained elusive
. Phylogenetic analysis of the HerC protein family indicated
that HerC5 is only present in higher vertebrates .
So far, ISG15-mediated antiviral activity has been shown most
convincingly in vivo by means of infections in ISG152/2mice
[11,12]. In addition, overexpression of ISG15 by a recombinant
Sindbis virus results in in vivo attenuation. Interestingly, recombi-
nant Sindbis viruses overexpressing unconjugatable ISG15
mutants were not attenuated, suggesting that attachment of mouse
ISG15 to lysines of target proteins is critical for antiviral function
. A recent report indicated that HerC5 may globally target de
novo synthesized proteins for ISG15 conjugation, thereby making
viral proteins major targets for ISGylation . However, the
question remains if HerC5 and global ISGylation are important
for antiviral activity in vivo as ISGylation-mediated antiviral effects
might be due to other minor ISG15 E3 ligases with more narrow
specificity, such as EFP .
Development of a mouse model to address this question has
been hampered by the fact that mice do not possess a HerC5
homologue. The closest mouse gene to human HerC5 is the
mouse homologue of human HerC6 . Since human HerC6
does not exert any ISG15 conjugating activity, the question
remains what the functional counterpart of human HerC5 is in
Recently, we reported that mouse HerC6, but not human
HerC6 could mediate ISG15 conjugation onto cellular proteins in
a transfection system . Here we set out to establish if HerC6 in
PLoS ONE | www.plosone.org1 January 2012 | Volume 7 | Issue 1 | e29870
mice functionally parallels human HerC5 and report that HerC6
acts as the main ISG15 E3 ligase to mediate global ISG15
conjugation in mouse cells.
The mouse HerC6 RLD confers efficient ISG15
conjugation to human HerC6
In order to assess what moiety of the mouse HerC6 protein
confers ISG15 E3 ligase activity in comparison to human HerC6,
which doesn’t possess ISG15 E3 activity, we constructed plasmids
expressing chimeric human/mouse HerC6 proteins. Either the N-
terminal RLD or the C-terminal HECT domain was exchanged
with the equivalent of the alternate species (Fig. 1a). Analysis of the
chimeric proteins by Western blot demonstrated that the chimeras
express to similar levels as their wild-type equivalents (Fig. 1b).
Next, these constructs were tested for their ability to conjugate
ISG15 to cellular targets in the presence of over-expressed E1 and
E2 enzymes. As previously described, both hHerC5 and mHerC6
effectively conjugated both human and mouse ISG15, whereas no
ISG15 conjugates were detected in cells overexpressing hHerC6
(Fig. 1c, lanes hH6, hH5 and mH6). Replacement of the human
HerC6 N-terminal RLD by its mouse equivalent almost
completely restored ISGylation (Fig. 1c, compare lanes mH6
and MNHC), suggesting that mainly differences in the RLD
between hHerC6 and mHerC6 allow mHerC6 to conjugate
ISG15. However, exchange of the mouse HerC6 C-terminus
partially restored ISGylation by hHerC6 (Fig. 1c, compare lanes
hH6 and HNMC), indicating that the ability of mHerC6 to
function as an E3 ligase compared to hHerC6 also partially relies
on differences in the protein’s C-terminal half that contains the
HECT domain. The same constructs were analyzed in the
presence of the mouse E1, E2 and ISG15. Despite the fact that
mouse ISG15 was much more efficiently conjugated in the
presence of its native conjugation machinery, similar results were
obtained with the HerC6 chimeras (data not shown).
The results described in figure 1c suggested that mainly the
mHerC6 N-terminal half is required for efficient ISG15
conjugation, irrespective of the species origin of ISG15. Conju-
gation of ISG15 chimeric proteins harboring either the N- or C-
terminal Ub-like domain of mouse or human ISG15 confirmed
and strengthened this observation. The mHerC6 N-terminal half
almost completely restored E3 activity in hHerC6 for both
chimeric ISG15 proteins (Fig. 1d, compare lanes mH6 and
MNHC), although the mHerC6 C-terminal half also partially
restored ISG15 conjugation (Fig. 1d, compare lanes hH6 and
HNMC). Moreover, the mISG15 and HNMCISG15 molecules
were consistently conjugated with higher efficiency than hISG15
or the MNHCISG15 chimera. Together with the observation that
conjugation of endogenous ISG15 upon IFN treatment is
consistently more robust in mouse cells than in human cells (data
not shown), these latter data seem to indicate that mISG15 may be
more efficiently conjugated than hISG15 and that this property
seems to reside in its C-terminal Ub-like domain (Fig. 1., compare
panel c and d).
In conclusion, these data demonstrate that mouse HerC6 has
ISG15 E3 ligase activity and that this activity mainly depends on
features in the N-terminal half of the protein compared to human
Figure 1. The mouse HerC6 RLD confers efficient ISG15 conjugation. A. Schematic overview of the HerC6 chimeras with the RLD and HECT
domains. B. HerC expression levels were determined in HEK-293T cells transfected with plasmids expressing wild-type HA-tagged HerC proteins and
human-mouse chimeric HerC6 proteins. C/D. HEK-293T cells transfected with plasmids expressing human E1 and E2 enzymes, indicated HerC proteins
and either V5-tagged human or mouse wild-type ISG15 (C) or human-mouse ISG15 chimeras (D) were analyzed for global ISGylation by V5-specific
Mouse HERC6 Mediates Global ISGylation
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HerC6. Next we investigated if expression of endogenous mHerC6
is also stimulated by type I IFN and localizes similar to human
HerC5 and HerC6.
mHerC6 is induced by type I interferon and localizes
exclusively in the cytoplasm
Although human HerC5 and HerC6 differ in their ability to
conjugate ISG15, they share their inducibility by type I IFN and
their localization in the cytoplasm . We investigated if mouse
HerC6 is also IFN-inducible and localizes to a sub-cellular site
similar to its human relatives. To this end, HerC5 and hHerC6
mRNA upregulation was measured in human Hep2 and HeLa
cells stimulated with IFNb and compared to mHerC6 mRNA
regulation in several IFN-stimulated mouse cell lines (Fig. 2a).
IFN stimulation upregulated human HerC5 and HerC6 mRNA
in both human cell lines, albeit to a lesser extent in Hep2 than in
HeLa cells (Fig. 2a). HerC5 and HerC6 were both upregulated 9-
fold in Hep2 cells, whereas HerC5 mRNA increased slightly more
(20-fold) compared to HerC6 mRNA (14-fold) in HeLa cells
(Fig. 2a, left panel). The same amount of IFN induced an average
12-fold upregulation of mHerC6 mRNA levels in the six mouse
cell lines tested (Fig. 2a, right panel), varying from 7-fold in NIH-
3T3 cells to 25-fold in the macrophage cell line RAW 264.7.
Subsequently, localization of HerC proteins was examined by
immuno-fluorescence assays of HeLa cells transfected with
HerC5/6 expression plasmids. Since all tested HerC5/6 proteins
are IFN-inducible, we investigated sub-cellular localization both
under non-induced conditions, as well as during infection with
SeV, a potent inducer of the type I IFN system .
Infection with SeV translocated IFN-regulatory factor 3 (IRF-3),
a transcription factor critical for efficient IFNb induction , to
the nucleus (Fig. 2b). These results indicated that the assay
conditions were correct for detecting sub-cellular changes of
molecules important for the IFN-response. In agreement with
previously published observations, human HerC5 and HerC6
localized exclusively to the cytoplasm in mock-induced cells
(Fig. 2c). Despite the nuclear translocation of IRF-3, which is
considered a hallmark of productive IFN induction and hence SeV
infection, human HerC5 and HerC6 localization was unchanged
during SeV infection (Fig. 2c). Similarly, mouse HerC6 localized
in the cytoplasm and its localization remained unchanged during
SeV infection (Fig. 2c). Together these data show that mouse
HerC6 IFN-inducibility and localization in mouse cells are very
similar to that of hHerC5/6 in human cells.
mHerC6 is essential for global ISG15 conjugation in
Since mouse HerC6 was able to facilitate ISG15 conjugation in
an overexpression system, we next set out to determine if HerC6 is
the main E3 ligase mediating global ISGylation in mouse cells. To
that end mouse L-929 cells were transfected with siRNAs
specifically targeting mHerC6. As a control siRNAs targeting
hHerC6 (but not mHerC6) were used.
Firstly, RNA was extracted from siRNA transfected cells and
specific knock-down was determined by real-time RT-PCR with
species-specific HerC6 primers. We compared IFN treated
samples to mock samples to assure that IFN induction would
not abolish knock-down. Efficiency of knock-down was deter-
mined by normalizing the observed knock-down to the human
specific siRNA controls. Four different commercial siRNAs
targeting mHerC6 were tested. Efficient and specific knock-down
was observed for all of them, whereas controls had no effect on
mHerC6 mRNA levels (data not shown). The assay was repeated
with the two most efficient siRNAs (mHerC6a and –b) which
specifically reduced mHerC6 mRNA in mouse cells by .85%
compared to the control (Fig. 3a).
Finally, global ISGylation was analyzed in the context of
mHerC6 knockdown. We hypothesized that if mHerC6 were the
functional equivalent of human HerC5, knock-down of mHerC6
in L-929 would abolish global ISGylation. We transfected siRNAs
targeting either mHerC6, hHerC6, mouse ISG15 or a scrambled
control in L-929 cells and analyzed ISGylation 48 h after IFN
stimulation. ISG15 expression was only upregulated in IFN-
treated cells. Moreover, ISGylation was detected when a non-
targeting control siRNA was transfected, whereas ISGylation was
specifically abolished by knockdown of mHerC6, indicating that
mHerC6 is essential for global ISGylation in mouse cells. Two
different mHerC6-specific siRNAs demonstrated the same effect
(Fig. 3b), thus making off-target effects by the siRNAs unlikely.
The increase of unconjugated ISG15 upon knockdown of
mHerC6 is likely due to accumulation of ISG15 that would
otherwise be conjugated.
To validate these results in a transfection system in which
ISG15 expression is controlled, we subsequently co-transfected a
V5-tagged ISG15 expression plasmid together with a mHerC6-
specific or control siRNA and stimulated the cells with IFN. ISG15
was expressed from a plasmid and thus at a constant level in all
samples (Fig. 3c). However, global ISGylation was only detected
upon IFN treatment (Fig. 3c), indicating that upregulation of the
endogenous E1, E2 and E3 enzymes was IFN-dependent as
expected. Moreover, transfection of four different siRNAs
targeting mouse HerC6 abolished global ISGylation in compar-
ison with the control siRNA (Fig. 3c, IFN treated lanes and Fig.
S1). Taken together these data confirm that HerC6 is the main
ISG15 E3-ligase in mouse cells and in that respect functionally
parallels human HerC5, even though genetically it is more closely
related to hHerC6. The use of four different mHerC6-specific
siRNAs strongly demonstrated that the loss of global ISGylation
upon mHerC6 knock-down is specific and not resulting from off-
mHerC6 enhances IFNb promoter induction and confers
antiviral activity in mouse cells
Previous studies have demonstrated that hHerC5 positively
regulates the IFNb promoter and confers antiviral activity . To
investigate if the HerC5/6 proteins could enhance an innate
immune signal initiated by the RNA sensor RIG-I, HEK-293T
cells were transfected with an IFNb reporter construct controlling
firefly luciferase, a constitutively active renilla luciferase internal
control plasmid, a limiting amount of a plasmid expressing a
constitutively active form of RIG-I (RIG-I(2CARD)) and the
indicated HerC5/6 proteins. TRIM25, a well-established activator
of RIG-I, was used as a positive control . As previously
reported, hHerC5 enhanced IFNb promoter activity (by 8.7 fold;
Fig. 4a) over the unspecific control protein GST, whereas its
hHerC6 relative without ISG15 E3 ligase activity did not further
enhance promoter activity. In contrast, mHerC6 also further
enhanced IFNb activity by 8.5 fold, similar to its hHerC5
counterpart (Fig. 4a).
To address the antiviral properties of mHerC6 in mouse cells,
we determined if HerC5/6 expression would confer antiviral
resistance to VSV-GFP and NDV-GFP infection. We transfected
L-929 cells with the indicated constructs and subsequently infected
the cells with either vesicular stomatitis virus (VSV) or Newcastle
disease virus (NDV) expressing GFP, and analyzed the produced
virus secreted into the supernatant by TCID50 assay. In
correlation with what has been previously reported, hHerC5
Mouse HERC6 Mediates Global ISGylation
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Mouse HERC6 Mediates Global ISGylation
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Figure 2. mHerC6 is induced by type I interferon and localizes exclusively in the cytoplasm. A. Indicated murine and human cell lines
were stimulated with recombinant type I IFN and subsequently analyzed for HerC mRNA regulation by RT-qPCR. Values are relative fold-change over
mock-induced samples. Experiments were reproduced at least twice; a representative experiment is shown. Error bars represent standard deviation of
qPCR replicates. B. HeLa cells transfected with an empty control plasmid and subsequently infected with SeV were immuno-stained with IRF-3
specific antibodies. C. Localization of overexpressed HA-tagged HerC proteins in the presence of subsequent SeV infection was determined in HeLa
cells by immuno-fluorescence assay using HA- and SeV-specific antibodies.
Figure 3. mHerC6 is essential for global cellular ISG15 conjugation in mouse cells. (A) mRNA knock-down of HerC6 was analyzed by RT-
qPCR in mouse L929 cells transfected with siRNAs specifically targeting human or mouse HerC6 and subsequently treated with recombinant type I
IFN. mRNA levels in mHerC6 knock-down cells are plotted relative to mRNA levels in cells with non-targeting hHerC6 siRNA. Experiments were
reproduced at least twice; a representative experiment is shown. Error bars represent standard deviation of qPCR replicates. B/C. L-929 cells were
transfected with (B) indicated siRNAs, stimulated with IFN for 48 h and probed for endogenous ISG15 on a Western blot or (C) simultaneously
transfected with a V5-tagged mouse ISG15 plasmid and indicated siRNAs, stimulated with IFN for 48 h and subsequently analyzed for global ISG15
conjugation by V5-specific immunoblot.
Mouse HERC6 Mediates Global ISGylation
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Figure 4. mHerC6 stimulates the IFNb promoter and confers antiviral activity against VSV and NDV. (A) HEK-293T cells were transfected
with an IFNb reporter construct controlling firefly luciferase, a constitutively active renilla luciferase internal control plasmid, a limiting amount of a
plasmid expressing a constitutively active form of RIG-I (RIG-I(2CARD)) and the indicated HerC5/6 proteins. After 24 h, cells were lysed and luciferase
measured. Values were normalized to the internal control and plotted relative to the GST control. (B) L-929 cells were transfected with the indicated
plasmids. After 48 h, the cells were infected with VSV-GFP or NDV-GFP at an m.o.i. of 5. At 7 h p.i. supernatant was harvested from the VSV-GFP
infected cells and at 16 h p.i. from the NDV-GFP infected cells. The supernatants were tittered by TCID50 on HEK-293T cells.
Mouse HERC6 Mediates Global ISGylation
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significantly reduced both VSV and NDV virus production by
100-fold, whereas its hHerC6 relative without E3 activity did not
(Fig. 4b). Moreover, mHerC6 also reduced the titers of both
viruses, highly similar to its human counterpart, hHerC5.
Taken together, these results indicate that mHerC6 parallels
hHerC5 in its ability to enhance the IFNb promoter and confer
antiviral activity. In contrast, the hHerC6 protein did not have
these activities, which correlates with its lack of E3 ligase activity.
Moreover, the fact that the hHerC5 and mHerC6 proteins had
activity in both human and mouse cells, is in line with our previous
observation that hHerC5 and mHerC6 can mediate global
ISGylation using ISG15 and the conjugation machinery of the
Here we demonstrate that mouse HerC6 parallels human
HerC5 in its localization, induction by type I IFN, antiviral activity
and its role as main ISG15 E3 ligase in mouse cells. Together these
findings indicate that humans and mice developed different HerC
proteins to facilitate global ISG15 conjugation during evolution.
The results from the silencing studies strongly suggest that both
humans and mice only express a single protein to globally attach
ISG15 to a wide range of target proteins during IFN induction
(Fig. 3b and c). The fact that only higher vertebrates express both
HerC5 and an IFN-induced human HerC6-like protein without
global ISGylation activity, suggests that HerC5 may have arisen as
an evolutionary response against pathogens specific to higher
vertebrates, taking over the role of global ISGylation that HerC6
has in lower vertebrates.
No distinct function has been attributed to hHerC6 so far.
Although it is clearly not essential for global ISGylation, we cannot
rule out that it may possess E3 ligase activity for a more select
range of target proteins or that it conjugates Ub-like modifiers
different from ISG15. The results obtained with the human-mouse
HerC6 chimeras show that the enzymatic HECT domain of
human HerC6 is capable of ISG15 conjugation in the presence of
the mouse HerC6 N-terminus (Fig. 1c). Therefore, a function of
hHerC6 in the regulation of antiviral effects of ISG15 as an E3-
ligase cannot be completely excluded, although in that case its N-
terminal RLD would probably target a more selective set of
proteins. It should be noted that the predicted binding site of the
E2 enzyme also resides in the C-terminal half of HerC proteins,
just upstream of the HECT domain. Thus, some of the increased
ISGylation in chimeras containing the human HerC6 N-terminus
and the mouse HerC6 C-terminus may indicate that mice and
humans could use E2 enzymes that may be differentially
compatible with global ISGylation.
We demonstrate that the previously reported IFNb-inducing
and antiviral effect exerted by hHerC5 , is shared by HerC6 in
mouse cells (Fig. 4). Yet, hHerC6, which does not possess E3 ligase
activity, failed to reduce VSV and NDV production or stimulate
an IFNb reporter. Shi et. al. also described antiviral effects of
hHerC5 expression on SeV , which could potentially have
lowered the virus levels in our localization studies (Fig. 2).
Nevertheless, we demonstrate strong SeV antigen expression by
immuno-fluorescence, which is highly similar in all infected
samples, suggesting potentially only a minor effect by SeV
reduction in our assay (Fig. 2).
The fact that the main global E3-ligases differ between mice and
humans raises the question whether there are more differences in
the ISG15 antiviral system. Conjugation of endogenous ISG15
and experiments with the ISG15 chimeras indicate that global
conjugation of mISG15 under the conditions tested is more
efficient than that of hISG15 and depends on its C-terminal Ub-
like domain (Fig. 1c and 1d). Together with the observation that
humans and mice adopted different HerC proteins during
evolution for the same function of global ISGylation, this could
indicate that other –hitherto unidentified- functional differences
between human and mouse ISG15 exist.
A recent report indicated that HerC5 mainly conjugates ISG15
to newly synthesized proteins in tissue culture and may by that
mechanism largely target de novo synthesized viral proteins during
infection . Moreover, additional studies have shown that
conjugation of ISG15 is critical for antiviral function in mice .
However, it remains unclear if global ISGylation by HerC proteins
is important to confer antiviral protection. Alternatively, conjuga-
tion by more specific E3 ligases may play an important role in
The findings in tissue culture put forward in this report indicate
that targeted gene disruption of HerC6 will likely prove a
reasonable model to study the role of global ISG15 E3 ligase
activity in ISGylation-mediated antiviral activity in vivo.
Materials and Methods
Cells, viruses and IFN-b stimulation
HEK-293T, HeLa, Hep2, EOMA, MEF, NIH-3T3, Hepa and
L-929 cells were acquired from the ATCC and maintained in
Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented
with 10% Fetal Bovine Serum (FBS) and 100 IU/mL penicillin-
streptomycin. Sendai virus Cantell strain was purchased from
ATCC, grown for two days on 10-day old embryonated chicken
eggs and tittered using HA assays with turkey red blood cells
(Lampire Biological Laboratories, Cat# 7209403). Newcastle
disease virus (NDV) expressing green fluorescent protein (GFP)
(NDV-GFP) was grown in 10-day-old embryonated eggs (Charles
River) and used for infections in the presence of TPCK trypsin
. Vesicular stomatitis virus (VSV) expressing GFP (VSV-GFP)
was grown in BHK-21 cells .
Cloning, plasmids and siRNAs
Expression plasmids for human Ube1L, UbcH8, HerC5,
HerC6, ISG15 and mouse Ube1L, UbcM8, HerC6, ISG15, as
well as plasmids expressing chimeric human/mouse and mouse/
human ISG15 have been described previously . The reporter
plasmid expressing firefly luciferase under the IFNb promoter was
described previously . Expression plasmid for flag-tagged
RIG-I(2CARD) was previously described . Plasmid pRL-TK
constitutively expressing renilla luciferase was obtained from
Promega. siRNAs specifically targeting hHerC6 (CUGUGAUG-
CAUGAUUCUAAtt), mISG15 (CCAUGACGGUGUCAGAA-
CUUU) and mHerC6 (CACCAUACCUUAUACUGAAtt and
GCAACUAUCGGUUGGAUUUtt), as well as a scrambled
control were purchased from Ambion.
For the creation of human/mouse chimeric HerC6 expression
constructs, unique KpnI and NotI sites were inserted upstream of
the start codon and downstream of the stop codon, respectively, in
both parental constructs using site directed mutagenesis (Quick-
Change XL, Stratagene). Subsequently, silent mutations were
introduced to create unique XhoI sites in the middle of the ORFs
between the RLD and HECT domain encoding sequences.
Ultimately, chimeric constructs were generated by exchanging the
KpnI-XhoI fragment or the XhoI-NotI fragment in the mHerC6
clone with the equivalent region of the hHerC6 clone. The
resulting plasmids encode HA-tagged chimeric HerC6 proteins
that are fused between the conserved amino acids L461 and E462.
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Plasmid DNA transfections were performed in 6-well format
with 1 mg of plasmid DNA at 30% confluence using either
Fugene6 (Roche) for HEK-293T or Lipofectamine 2000 (Invitro-
gen) for HeLa and L-929. siRNA was transfected using
Lipofectamine RNAiMAX (Invitrogen) in 12-well format. Trans-
fections for immunofluorescence were carried out in suspension
with 500 ng of plasmid DNA and 105cells in 12-well format.
HeLa and L-929 cells were stimulated 16–24 h post transfection
(hpt) with 1000 IU/mL (for RT-qPCR) or 10,000 IU/mL (for
protein analysis) of universal IFN-b (PBL).
For reporter assays HEK-293T cells were seeded in 24-well
clusters, to reach ,30% confluence 16 hrs later at the time of
transfection. Each well of the cluster was transfected with a total of
575 ng DNA. Each transfection contained 50 ng firefly luciferase
reporter plasmid, 25 ng renilla luciferase reporter plasmid (pRL-
TK) and 2 ng flag-RIGI(2CARD) plasmid. Each transfection mix
was supplemented with 500 ng Herc5/6 plasmid. A plasmid
expressing GST was included in each experiment as a base-line
control. All samples were transfected in triplicate. Transfection
complexes were formed with 2 ul Fugene6 (Roche) in 20 ul
OptiMEM at room temperature for 30 minutes and directly
added to complete growth medium on the cells.
Twenty-four hrs after transfection, cells were lysed in passive
lysis buffer (Promega) and dual-luciferase activity was measured
using the Dual-luciferase reporter assay system (Promega) in a
Biotek Synergy 4 plate reader. Firefly luciferase values were
normalized to renilla luciferase values. Fold increase of the firefly
reporter was calculated relative to the GST baseline control in
Samples were lysed in disruption buffer (3 M Urea, 1 M b-
mercaptoethanol and 2% SDS), boiled for 10 min. and analyzed
for ISG15 conjugate formation by Western blot. HEK-293T cells
were harvested after 24 h, whereas HeLa and L-929 cells were
collected 48 h after IFN-b stimulation. Proteins were separated by
SDS-PAGE on 4–15% gradient gels (Biorad) and subsequently
transferred to an Immobilon-P membrane (Millipore) using a
semi-dry transfer system (Biorad). Western blot analysis was
performed using SNAP i.d. (Millipore) with either an HRP-
conjugated V5 mAb (Serotec, MCA1360P), an anti-HA mouse
mAb (Sigma, H9658) or a rabbit polyclonal anti-b-actin Ab
(Sigma, A2103). Secondary HRP-conjugated sheep anti-mouse Ig
and donkey anti-rabbit Ig Abs (GE Healthcare) were used for
Total RNA was isolated from HeLa and L-929 cells8 h after IFN-
b stimulation using RNeasy columns (Qiagen) and treated for 30 min
with Turbo DNase (Ambion). One mg of DNase-treated RNA was
reverse transcribed using the iScript cDNA synthesis kit (Biorad).
Real-time PCR was performed in 384 well plates in triplicate using
gene specific primers for hHerC5 (fw_TCATTCTCCACCCCAA-
(fw_TGACACAAGCAAGCCAACTC and rev_CATCCACGAA-
GTCTTCAGA) and mHerC6 (fw_TCCGGTGTTCTGAAACC-
TTC and rev_ CATCCTTCGCATTGAGGAAT) and Light-
cycler480 SYBR green I mastermix (Roche) in a Roche LightCycler
480. Relative mRNA abundances were calculated using the DDCt
method  using 18S rRNA (fw_GTAACCCGTTGAACCC-
CATT and rev_CCATCCAATCGGTAGTAGCG) as a reference
and plotted as fold change compared to mock-control samples.
Immunofluorescence and confocal microscopy
HeLa cells were transfected in suspension as described above,
seeded on glass coverslips, 24 h post transfection infected with
Sendai virus for 8 hours and ultimately fixed in ice-cold absolute
methanol. After blocking with 1% bovine serum albumin (BSA) in
PBS, coverslips were incubated with primary antibodies diluted in
1% BSA in PBS for 1 h at room temperature (rt) and washed with
PBS. Subsequently, coverslips were incubated with secondary
antibodies as well as DAPI for nuclear staining (Invitrogen) for
30 min at rt and embedded in Prolong Gold (Invitrogen). Primary
antibodies used are an anti-IRF3 mAb (Santa Cruz, FL-425,
1:100), anti-HA mAb (Sigma, H9658, 1:500) and polyclonal anti-
Sendai serum (Charles River, 1:400). Secondary Alexa Fluor 488-
conjugated anti-mouse Ig and Alexa Fluor 633 anti-rabbit Ig Abs
(Invitrogen) were used for detection. Confocal laser scanning was
performed using a Zeiss LSM 510 Meta (Carl Zeiss) fitted with a
Plan Apochormat 636/1.4 oil objective. Images were collected at
a resolution of 1024 by 1024 pixels and processed using Zeiss LSM
Image Browser (Carl Zeiss).
attenuates global ISGylation. L-929 cells were transfected
with a V5-tagged mouse ISG15 plasmid and indicated siRNAs,
stimulated with IFN for 48 h and subsequently analyzed for global
ISG15 conjugation by V5-specific immunoblot.
Knock-down of mHerC6 in mouse cells
We are grateful to Richard Cadagan and Osman Lizardo for excellent
technical assistance. Mouse-ISG15 polyclonal antibody and RAW 264.7
cells were kindly provided by Deborah J. Lenschow.
Conceived and designed the experiments: DO GV AG-S. Performed the
experiments: DO SvB MTS-A RR GV. Analyzed the data: DO GV AG-S.
Wrote the paper: DO GV AG-S.
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