Cellular requirements for iron-sulfur cluster insertion into the antiviral radical SAM protein viperin

21 Pages·2017·7.24 MB·English

JBC Papers in Press. Published on June 14, 2017 as Manuscript M117.780122
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M117.780122
Iron-sulfur cluster insertion into viperin 

Cellular requirements for iron-sulfur cluster insertion into the antiviral radical SAM protein viperin

Arunkumar S Upadhyay1,2,§, Oliver Stehling3,§, Christakis Panayiotou1,2, Ralf Rösser3, Roland
Lill3,4,#, and Anna K Överby1,2,#
1Department of Clinical Microbiology, Virology, Umeå University, 90185 Umeå, Sweden, 2The
Laboratory for Infection Medicine Sweden (MIMS), Umeå University, 90187 Umeå, Sweden,
3Institut für Zytobiologie und Zytopathologie, Philipps-Universität Marburg, Robert-Kochstr. 6,
35032 Marburg, Germany, 4LOEWE Zentrum für Synthetische Mikrobiologie SynMikro, Hans-
Meerwein-Str., 35043 Marburg, Germany
Running title: Iron-sulfur cluster insertion into viperin

§ These authors contributed equally
# To whom correspondence should be addressed:
Anna K Överby, Department of Clinical Microbiology, Virology, Umea University, Building 6F, Umeå
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90185, Sweden. Phone: [phone], Fax: [phone], Email: [email protected]. w
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Roland Lill, Institut für Zytobiologie und Zytopathologie, Philipps-Universität Marburg, Robert-Kochstr. loa
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6, 35032 Marburg, Germany Phone: [phone], Fax: [phone], E-mail: [email protected] d fro
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Key words: Iron‐sulfur protein, CIA targeting complex, biogenesis, metal biology, interferon ://w
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Abstract with the C terminus of viperin and used CIA1 as the b/
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Viperin (RSAD2) is an interferon-stimulated primary viperin-interacting protein. In contrast, g
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antiviral protein that belongs to the radical S- CIA2A bound to viperin’s N terminus in a CIA1-, st o
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adenosyl methionine (SAM) enzyme family. CIA2B- and MMS19-independent fashion. Of note, A
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Viperin’s iron-sulfur (Fe/S) cluster is critical for its the observed interaction of both CIA2 isoforms ril 1
antiviral activity against many different viruses. with a single Fe/S target protein is unprecedented in , 2
0
1
CIA1 (CIAO1), an essential component of the the CIA pathway. 55Fe radiolabeling experiments 9
cytosolic iron-sulfur protein assembly (CIA) with human cells depleted of CIA1, CIA2A, CIA2B
machinery, is crucial for Fe/S cluster insertion into or MMS19 revealed that CIA1, but none of the
viperin and hence for viperin’s antiviral activity. In other CIA factors, is predominantly required for
the CIA pathway, CIA1 cooperates with CIA2A, 55Fe/S cluster incorporation into viperin.
CIA2B, and MMS19 targeting factors to form Collectively, viperin maturation represents a novel
various complexes that mediate the dedicated CIA pathway with a minimal requirement of the
maturation of specific Fe/S recipient proteins. To CIA targeting factors and represents a new
date, however, the mechanisms of how viperin paradigm for the insertion of the Fe/S cofactor into
acquires its radical-SAM Fe/S cluster to gain a radical-SAM protein.
antiviral activity are poorly understood. Using co-
immunoprecipitation and 55Fe radiolabeling
RSAD2 (radical S-adenosyl methionine
experiments, we therefore studied the roles of
domain containing 2) is one of the interferon (IFN)-
CIA2A, CIA2B, and MMS19 for Fe/S cluster
stimulated genes (ISGs) that is strongly upregulated
insertion. CIA2B and MMS19 physically interacted
1 

Copyright 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
Iron-sulfur cluster insertion into viperin 

by type I IFNs, lipopolysaccharide, responsible for binding a [4Fe-4S] cluster that uses
polyinosinic:polycytidic acid, or by different virus SAM as the fourth ligand (15,16). Mutations of the
infections (1-8). Its gene product viperin (virus conserved cysteine residues abrogate viperin’s
inhibitory protein, endoplasmic reticulum- antiviral action against HCV (2), West Nile virus
associated, interferon-inducible) has received (WNV), DENV (3) and TBEV (6). Despite the
increasing attention owing to its ability to interfere central importance of the radical SAM domain for
with the proliferation of numerous RNA and DNA viperin function, little is known about the
viruses from different families. Overexpression of maturation of its Fe/S cluster and about the
viperin has been shown to inhibit budding and assembly of SAM-coordinated [4Fe-4S] clusters in
release of influenza A virus by disrupting lipid raft general (6).
micro-domains in the plasma membrane (7). In Studies in yeast and human cells have
addition, viperin localizes to lipid droplets, a site of revealed the importance of the CIA machinery for
replication for hepatitis C virus (HCV) and Dengue maturation of cytosolic and nuclear Fe/S proteins
virus (DENV) (9), and targets positive-stranded (21-23). This essential biosynthetic system is
RNA synthesis of tick-borne encephalitis virus composed of eleven known CIA proteins that act in
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(TBEV) (6) and DENV (10). Since the broad range three main steps of the assembly reaction. First, a o
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of viperin-affected viruses use different routes of [4Fe-4S] cluster is assembled on the scaffold lo
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infection and mechanisms of replication, the complex CFD1-NBP35 (24-26). This reaction e
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antiviral mechanism of viperin is unlikely to be requires a sulfur source from mitochondria and the fro
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virus-specific, yet the molecular mechanism of electron transfer chain comprised of NDOR1 (yeast http
viperin function is unknown. Tah18) and CIAPIN1 (Dre2) (21,27,28). Second, ://w
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Viperin is highly conserved from fungi to the cluster is released from CFD1-NBP35 and w
lower vertebrates and mammals, and is a member transferred to target apoproteins for insertion into .jbc
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of the radical S-adenosyl methionine (SAM) iron- the polypeptide chain. This reaction requires the rg
b/
sulfur (Fe/S) protein family (6,11-14). Radical Fe/S protein IOP1 (yeast Nar1) and the CIA y g
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SAM proteins usually perform chemically difficult targeting complex CIA1-CIA2B-MMS19 which, as es
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reactions such as C-C and C-H bond cleavages, C- a whole or in part, is required for maturation of most n A
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S bond formations or alkylations (15,16). Human cytosolic-nuclear Fe/S clients including DNA ril 1
viperin (361 residues, molecular mass 42 kDa) is polymerases, DNA helicases, and nucleic acid , 2
0
1
composed of three domains (17): The N terminus metabolism proteins (29-33). In human cells, the 9
(residues 1-42) harbors an amphipathic α-helix that CIA2B-related protein CIA2A specifically assists
is important for viperin’s localization to the the maturation of iron regulatory protein 1 (IRP1),
endoplasmic reticulum (ER) (18) and to lipid a protein involved in cellular iron homeostasis (33).
droplets (9). The domain is also required for Third, the recently identified CIA factors Yae1-
antiviral activity against Chikungunya virus (19). Lto1 function as adapters that specifically recruit
The C-terminal domain (residues 218-361) has the Fe/S protein Rli1 (human ABCE1) to the CIA
been shown to mediate protein-protein interactions, targeting complex (34). Previous studies have
e.g., with the HCV proviral factor hVAP-33 (20), indicated that targeting and insertion of [4Fe-4S]
and with CIA1 (also known as CIAO1), a clusters into different client apoproteins require
component of the cytosolic iron-sulfur protein specific combinations of the CIA targeting factors
assembly (CIA) machinery (6). The central domain of the second step of the CIA pathway (31,33).
(residues 71-182) is homologous to the MoaA Depletion of the dedicated CIA assembly factors
subfamily of radical SAM enzymes (11). It contains impairs Fe/S cofactor insertion and results in a
a conserved cysteine-rich motif (CxxxCxxC)
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Iron-sulfur cluster insertion into viperin 

destabilization and eventually degradation of the Viperin interacts with the CIA1-CIA2B-
respective apoproteins. MMS19 targeting complex via its C terminus and
We have previously shown that viperin with CIA2A via its N terminus ̶ The finding that
requires the physical interaction with CIA1 to viperin bound to both CIA2A and CIA2B was
become matured and antivirally active (6), surprising because these proteins assist distinct
implying a critical function of the CIA machinery branches of the CIA pathway (33). We therefore
for antiviral host defense of mammalian cells. In the sought to identify the viperin segments responsible
present study, we aimed to more comprehensively for interaction with the CIA factors. We used FLP-
characterize the roles of the various CIA targeting IN T Rex cells inducibly expressing FLAG-tagged
factors in viperin interaction and assembly of the versions of full-length viperin, N- or C-terminally
radical SAM Fe/S cluster. To this end, we truncated versions (TN50 or TC20 that lack of the
performed co-immunoprecipitation and 55Fe N-terminal 50 or C-terminal 20 residues,
radiolabeling experiments to define the relative respectively), or the Fe/S cluster-deficient mutant
importance of the CIA factors in viperin binding protein M1 in which the three cofactor-coordinating
and maturation. Overall, our studies suggest the cysteine residues of the radical SAM domain were
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existence of a unique CIA targeting pathway that exchanged to alanine (6). IP of these four viperin o
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uses CIA1 but not the other targeting factors for versions revealed that the C-terminal region of the lo
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Fe/S cluster insertion into viperin. protein was important for interaction with CIA1- e
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CIA2B-MMS19, because no co-IP of these CIA fro
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Results targeting factors was observed with TC20 (Fig. http
Viperin interacts with CIA1, MMS19, 2A). Notably, the CIA interaction appeared to be ://w
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CIA2A, and CIA2B ̶ In order to define the relative strongest for the cluster-deficient viperin mutant w
roles of the CIA targeting factors CIA2A, CIA2B, M1, suggesting a preferential binding to the .jbc
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and MMS19 in the maturation of viperin, we first apoform of viperin. The finding that the CIA rg
b/
performed co-immunoprecipitation (co-IP) targeting complex proteins interact with the C y g
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experiments to analyze their potential interaction. terminus of viperin was further supported by co-IP es
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HEK293T cells were transiently co-transfected experiments performed with plasmid-encoded, n A
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with plasmids encoding non-tagged wild-type FLAG-tagged viperin fragments encompassing the ril 1
viperin and either CIA1-FLAG, CIA2B-HA, C-terminal 100 (C100; residues 261-361) or 70 , 2
0
1
CIA2A-Myc, or MMS19-FLAG. Anti-viperin-IP (C70; 291-361) amino acid residues. C70 fragment 9
followed by immunoblotting revealed that each of bound to the CIA targeting complex similarly as
the four CIA targeting factors interacted with wild-type and TN50 viperin (Fig. 2B). Although
viperin (Fig. 1A-D). In a reverse approach, an C100 is larger the binding of C100 to the CIA
antivirally active FLAG-tagged viperin, expressed targeting complex was weaker compared to C70.
in FLP-IN T Rex cells (6), recovered all three One explanation might be improper folding of
endogenous CIA targeting complex components C100. This data suggests that the viperin C terminus
(CIA1-CIA2B-MMS19) in anti-FLAG-IP (Fig. acts as the necessary and sufficient interaction site
1E). Since no antibody against CIA2A is available, for the CIA1-CIA2B-MMS19 targeting complex.
we could not confirm the interaction of viperin with A strikingly different result was obtained for
endogenous CIA2A. Together, our co-IP the viperin interaction with CIA2A. Co-IP and
experiments reveal that viperin specifically immunoblotting revealed that viperin interacted
interacts with all four CIA targeting factors. with plasmid-expressed CIA2A-Myc through its N
rather than C terminus, because wild-type and
TC20 viperin bound CIA2A-Myc efficiently,
3
Iron-sulfur cluster insertion into viperin 

whereas neither the TN50 mutant protein nor the 11) and correlated with elevated CIA2B protein
C100 and C70 fragments interacted (Fig. 2C). In levels present in the input lysate (lanes 8 and 11,
contrast, the latter three viperin variants recovered grey bars). In contrast, expression of MMS19
endogenous CIA1-CIA2B-MMS19, confirming the altered neither the cellular CIA2B protein level nor
structural integrity of the C-terminal CIA targeting the interaction between CIA2B and viperin (Fig. 3A
complex interaction site. Taken together, our results and D, lane 10). Likewise, co-expression of CIA1
demonstrate that viperin associates with the two or CIA2B along with FLAG-viperin hardly
CIA2 isoforms at separate domains. influenced protein level or recovery of MMS19
(Fig. 3A and C; lanes 9 and 10). Notably,
CIA1 and CIA2B promote the interaction of expression of the entire CIA1-CIA2B-MMS19
MMS19 with viperin ̶ Previous functional analyses targeting complex substantially improved the
of individual CIA targeting complex components interaction of each of the complex constituents by
have suggested that unique combinations of these FLAG-viperin, including MMS19 (Fig. 3A-D; lane
constituents specifically assist the maturation of 11).
different apoprotein subsets, a finding mirrored by We next compared the behavior of CIA2A to
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different target Fe/S protein interaction patterns that of CIA2B. Co-expression of CIA2A and CIA1 o
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(33). The co-purification of the CIA targeting together with viperin improved the recovery of lo
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complex constituents CIA1-CIA2B-MMS19 CIA1 by FLAG-viperin (Fig. 3E and F; lane 8), e
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together with viperin raised the question whether all similar to the situation of CIA2B (Fig. 3A and B; fro
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of these maturation factors directly contact the lane 8). In turn, the binding of CIA2A by viperin http
radical SAM enzyme or whether there is a binding only slightly increased in the presence of CIA1 ://w
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hierarchy. First, we determined how the expression (Fig. 3E and H; lane 8), although co-expression of w
of individual CIA targeting factors might influence CIA1 and CIA2A led to elevated cellular protein .jbc
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viperin binding to the remaining components. levels of both factors (Fig. 3F and H; lane 8). The rg
b/
HEK293T cells were transiently co-transfected increased CIA1 levels were likely caused by y g
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with a plasmid encoding FLAG-tagged wild-type stabilizing effects of CIA2A and/or CIA2B (33), es
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viperin and with varying combinations of plasmids yet could only partially explain the high recovery of n A
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encoding the CIA targeting factors. Protein-protein CIA1 by viperin (Fig. 3B and F, lane 8, black vs. ril 1
interactions were analyzed by anti-FLAG affinity grey bars). The results argue for an additional , 2
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purification and immunoblotting (Fig. 3A). For supportive function of the CIA2 proteins in the 9
quantitative analysis, antigen-associated CIA1-viperin interaction. Similar to CIA1, also
chemiluminescence signals were recorded and then MMS19 hardly influenced the interaction of
normalized to the amount of immunoprecipitated CIA2A with viperin (Fig. 3E; lane 10). Taken
viperin which was normalized to both the amount together, our co-immunoprecipitation results
of tubulin and viperin in the total cell lysate (Fig. suggest that the main interactors of viperin are
3B-D, black bars). For comparison, levels of CIA1 and both CIA2 isoforms, whereas MMS19
plasmid-encoded proteins within the input lysates associates with viperin only indirectly via CIA1 and
were also normalized to tubulin (Fig. 3B-D, grey CIA2B.
bars). The densitometric analysis revealed that the
interaction of viperin with CIA1 was enhanced in The interaction between viperin and the CIA
the presence of CIA2B (Fig. 3A and B; lanes 8 and targeting complex is mediated mainly by CIA1 ̶ In
11), but not by MMS19 (lane 9). Conversely, order to better understand which of the CIA
viperin interaction with CIA2B was enhanced by targeting factors are in direct physical contact with
co-expression of CIA1 (Fig. 3A and D; lanes 8 and viperin, we depleted individual CIA targeting
4
Iron-sulfur cluster insertion into viperin 

complex constituents by RNAi technology and then binding was enhanced by co-expression of CIA1
measured the viperin interaction of the residual CIA (lanes 4, 5 and 9). We also observed that CIA1 can
factors. Knock-down efficiencies of the CIA weakly associate in the absence of the C terminus
mRNAs (10-20 % of control cells) were estimated of viperin, probably via CIA2A, but only in the
by quantitative real-time PCR (qPCR; Fig. S1A-D), overexpression system (Fig. S2). When we
and the steady-state protein levels of each CIA analyzed the behavior of MMS19, this protein
factor were assessed by immunoblotting (Fig. S1E). appeared to indirectly bind to viperin, because their
In line with previous observations (33) individual co-purification required over-expression of both
depletion of CIA1, CIA2B, or MMS19 decreased CIA1 and CIA2B (Fig. 4A and C, lanes 2 and 6).
the steady-state protein levels of the remaining CIA The remaining weak interaction between MMS19
targeting complex components, despite the fact that and viperin in the absence of CIA1 or CIA2B (Fig
the mRNA levels of these proteins were not 4A, lanes 3, 5, 7, 8, and 11) is best explained by
affected (Fig. S1). This observation is consistent incomplete RNAi knock-down. This observation
with the mutual stabilization of the CIA targeting indicates that MMS19 associates with viperin by
complex components (33) (c.f. Fig. 2). Since the binding to the CIA1-CIA2B complex (see also Fig.
D
decreased levels of the remaining CIA factors may 3). Conversely, MMS19 depletion had no effect on o
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complicate the interpretation of subsequent the viperin interaction with CIA1, CIA2B or lo
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interaction studies, we improved the RNAi CIA2A confirming their direct association with e
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approach by additional plasmid-based expression viperin (Fig. 4A to E; lanes 4, 9 and 10). In fro
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of the remaining CIA constituents. This maintained summary, our results indicate that the major viperin http
their high abundance as revealed by binding partner is CIA1, whose interaction is ://w
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immunoblotting of cell extracts (Fig. 4A; Input). enhanced by both CIA2A and CIA2B, mainly by w
The only exception was the low level of CIA2A in exerting a stabilization of CIA1. While CIA2B .jbc
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the absence of CIA1 (Fig. 4A; lanes 3 and 7), binds to viperin in a CIA1-dependent fashion at the rg
b/
consistent with the mutual stabilization of these C terminus, CIA2A can associate independently y g
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CIA factors. with the N terminus of viperin. MMS19 interacts es
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With this improvement of the RNAi with viperin only via CIA1 and CIA2B as part of n A
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depletion approach, we analyzed the interaction of the CIA targeting complex. ril 1
plasmid-encoded FLAG-tagged viperin with the , 2
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1
different CIA targeting factors by Fe/S cluster insertion into viperin is not 9
immunoprecipitation as in Fig. 3. The interaction dependent on CIA2A, CIA2B, or MMS19 ̶ The
was optimal when all three CIA targeting complex binding hierarchy observed for viperin interaction
components, i.e. CIA1, CIA2B, and MMS19, were with the CIA1-CIA2B-MMS19 complex and
co-expressed (Fig. 4A-D; lanes 2 and 6). In samples CIA2A raised the question which individual CIA
containing low levels of both CIA2 isoforms, i.e. constituents contribute to Fe/S cluster insertion into
upon depletion of CIA2A with no ectopic viperin. We directly examined the maturation
expression of CIA2B and vice versa (Fig. 4A, lanes process by following the in vivo incorporation of
11,12), the association of viperin with CIA1 was 55Fe into viperin as a measure of Fe/S cluster
severely impaired, suggesting that each CIA2 assembly at the radical SAM domain (6). To this
protein can stabilize the viperin-CIA1 interaction. end, FLP-IN T Rex cells expressing FLAG-tagged
Inversely, CIA2B bound to viperin only when CIA1 viperin were transiently transfected with siRNAs
was abundant (Fig. 4A and D; lanes 2,6,10), while directed against the CIA targeting complex
CIA2A associated with viperin also in the absence components and against the general CIA factor
of CIA1 (Fig. 4A and E; lanes 3 and 7), even though IOP1, a protein which is of critical importance for
5
Iron-sulfur cluster insertion into viperin 

maturation of all cytosolic-nuclear Fe/S proteins relative to control cells reassuring that only CIA1
and thus thought to act upstream of the CIA of the CIA targeting complex is essential for viperin
targeting complex (35). After two successive maturation. Interestingly, the combined depletion
rounds of siRNA transfection at a three-day of CIA2A and CIA2B slightly impaired maturation
interval, cells were supplemented with 55Fe-loaded of viperin by 40%. This effect was still less
transferrin, and viperin expression was induced for pronounced than depletion of CIA1 alone, and
48 h. Cells were harvested, and the 55Fe content of likely is best explained by the destabilization of
the cell lysate (indicative of total cellular iron CIA1 in the absence of its two CIA2 binding
uptake; Fig. 5A) and of immunoprecipitated viperin partners (Fig. S3C), again suggesting that CIA1
(Fig. 5B) were determined by scintillation counting. depletion rather than the CIA2 proteins themselves
The efficient depletion of the individual CIA were causative of this maturation decline. Taken
proteins was verified by immunoblot analysis (Fig. together, these results suggest a direct important
S3A and B). IRP1 was used as an indicator for function of CIA1 in the maturation of viperin,
CIA2A knockdown efficiency, as IRP1 stability is whereas CIA2B, MMS19, and CIA2A appear to
dependent on CIA2A (31). The strongest play dispensable roles despite their association with
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impairment of viperin maturation was observed viperin. Their function therefore might be restricted o
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upon depletion of IOP1 (Fig. 5B), resulting in a to the stabilization of CIA1 thus assuring its optimal lo
a
fourfold decrease in 55Fe/S cluster insertion. This binding to viperin. de
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result is consistent with the central role of IOP1 in fro
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cytosolic-nuclear Fe/S protein assembly (36-38). Discussion http
Among the CIA targeting complex components This study identified critical cell biological ://w
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only depletion of CIA1 resulted in a significant requirements for [4Fe-4S] cluster assembly of the w
effect (55% decrease) on 55Fe incorporation into antiviral radical SAM protein viperin. Since this .jbc
.o
viperin, while iron uptake remained normal (Fig. radical SAM Fe/S cluster has been found to be rg
b/
5A and B). In contrast, individual depletion of essential for the antiviral function of viperin, y g
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MMS19, CIA2B, or CIA2A did not result in detailed knowledge about its maturation es
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pronounced effects on cellular 55Fe uptake or mechanism is crucial to understand the n A
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viperin 55Fe/S cofactor maturation, even though the physiological basis of viperin activation. ril 1
expected substantial effects on other cytosolic and Previously, we have defined an essential role of the , 2
0
1
nuclear Fe/S proteins such as IRP1 (CIA2A), CIA targeting factor CIA1 in Fe/S cluster insertion 9
GPAT (CIA1), and POLD1 (IOP1, CIA1 and into the radical SAM domain of viperin. However,
MMS19) were observed (Fig. S3A-D; c.f. (31,33)). the potential roles of the CIA1-interacting proteins
Since depletion of individual CIA targeting MMS19, CIA2B, and CIA2A have not yet been
factors apart from CIA1 showed no detectable addressed (31,33). Likewise, the assembly of a
effect on viperin maturation, we tested whether radical SAM-type of Fe/S cluster in general has not
combined depletion of two or three CIA targeting been investigated so far. Here, we identified the
factors might impair this process. When CIA1 was CIA proteins MMS19, CIA2B, and CIA2A as
among the depleted proteins, we consistently interaction partners of viperin, in addition to CIA1
observed a 2-3-fold decrease in 55Fe/S cluster (6). Binding of each of the four CIA targeting
incorporation into viperin, yet no diminution in factors was independent on the ability of viperin to
cellular 55Fe uptake (Fig. 5C and D, Fig. S3E), bind a Fe/S cluster because mutation of the three
similar to the single knockdown of CIA1. In conserved cysteine ligands (mutant M1)
contrast, the combined depletion of CIA2B and strengthened rather than weakened the interaction.
MMS19 did not affect the maturation of viperin This finding suggests that the CIA targeting factors
6
Iron-sulfur cluster insertion into viperin 

interact with the apoform of viperin, a mechanism CIA2B-MMS19 complex tightly interacts with the
expected for bona fide maturation factors and C terminus of viperin, yet the primary binding

consistent with an earlier report for the assembly of partner is CIA1.
the nuclear Fe/S protein XPD (39). The binding CIA2A binding to the N-terminal region of
events therefore may be expected to be tightly viperin occurred independently of the other CIA
connected to the Fe/S cluster maturation process targeting factors, as their depletion did not affect the
(discussed below). CIA2A-viperin binding efficiency. This was
It was surprising to find that both isoforms of particularly true for the depletion of CIA1, but may
human CIA2 bound to viperin, because previously not immediately be evident (33). CIA1 knockdown
we have reported radically different CIA2A and resulted in lower cellular levels of CIA2A because
CIA2B interactomes with hardly any overlap for of the missing stabilizing effect of CIA1.
the two proteins (33). A molecular explanation for Nevertheless, the amount of viperin-bound CIA2A
viperin binding to both CIA2 isoforms came from per total cellular CIA2A remained high, clearly
the mapping of the precise interaction domains on indicating that CIA2A can associate with viperin
viperin for the four CIA targeting proteins by without the other three CIA factors. Conversely,
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performing co-immunoprecipitation experiments depletion of CIA2A did not affect the extent of o
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with viperin mutants lacking the N or C termini and viperin binding to the CIA targeting complex (Fig. lo
a
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with C-terminal viperin fragments (Fig. 2). These 4) suggesting that the binding events at the viperin e
d
studies showed that CIA2B (in complex with CIA1 termini are independent. In fact, CIA2A binding to fro
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and MMS19) and CIA2A bind to the C and N viperin is even increased under CIA2B or MMS19 http
termini, respectively, of viperin (Fig. 6A). Co- depletion conditions. This effect may have been ://w
w
immunoprecipitation studies performed after RNAi caused indirectly by multiple reasons including i) w
depletion of individual CIA factors suggested a the stabilization of CIA2A by viperin and/or CIA1 .jbc
.o
hierarchy for binding of CIA1, CIA2B, and binding, and ii) a competition of CIA2A with rg
b/
MMS19. At the C terminus, the major viperin CIA2B for complex formation with CIA1 (Fig. 6A y g
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interaction partner appears to be CIA1, because its and B). Taken together, viperin can interact with es
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depletion substantially decreased the association of CIA2A and the CIA targeting complex CIA1- n A
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both CIA2B and MMS19, indicating that the CIA2B-MMS19 at opposing termini in independent ril 1
viperin association of the latter two proteins is binding events. The interaction network between , 2
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1
mediated by CIA1 (Fig. 4, left). The sum of our the CIA factors and viperin largely contributes to 9
interaction analysis data is best interpreted by the the stability of the CIA factors in the cytosol.
view that viperin, via its C terminus, directly The preferential binding of CIA1, CIA2B,
associates with CIA1 which recruits CIA2B, and and MMS19 to the C terminus of viperin and the
this complex facilitates binding of MMS19 (Fig. CIA2A interaction with viperin’s N terminus raised
6A). This interpretation is supported by the finding the interesting question of what the contribution of
that MMS19 depletion does not affect CIA1 each of these independent binding events to Fe/S
association to viperin. The fact that in this case the cluster assembly may be. This fundamental
CIA2B interaction with viperin is weakened, is prerequisite for viperin activation could be directly
readily explained by the stabilizing role of MMS19 addressed by radiolabeling and
for CIA1 and CIA2B (31). Overall, the most immunoprecipitation experiments employing the
efficient interaction was formed, when all three 55Fe isotope to estimate Fe/S cluster assembly in
CIA targeting complex proteins were present. This vivo. Depletion of the general CIA component IOP1
is consistent with their mutual stabilization. strongly deceased the radiolabeling of viperin by a
Collectively, our data suggest that the CIA1- factor of four, highlighting the essential
7
Iron-sulfur cluster insertion into viperin 

requirement of the CIA system for Fe/S cluster CIA2A binding to viperin raises the question
assembly on viperin (Fig. 5). In contrast, the of the functional meaning of this association. A
knockdown of the other CIA targeting factors, with direct role in Fe/S protein maturation seems
the exception of CIA1 (6), did not significantly unlikely from our experiments because i) CIA2A
decrease 55Fe incorporation into viperin, even depletion does not hamper 55Fe/S association with
though other cytosolic and nuclear Fe/S proteins are viperin, and ii) CIA2A binding to the viperin N
impaired under this condition (31,33). This result terminus cannot substitute for the complete
clearly demonstrates that CIA1 not only directly abrogation of Fe/S cluster insertion observed upon
associates with viperin but also performs the deletion of the viperin C terminus including the C-
predominant if not exclusive role as a targeting terminal conserved tryptophan as the binding site of
factor in Fe/S cluster insertion. Viperin is the first the CIA targeting complex (6). Currently, the
known cytosolic Fe/S protein whose maturation physiological meaning of the CIA2A-viperin
solely depends on the early, general part of the CIA interaction is not understood, requiring future
machinery and on CIA1, but not on the other three studies to examine the implications of this complex
CIA targeting factors. The Fe/S cluster maturation formation on viperin function in the antiviral
D
mechanism of viperin thus represents a novel response in an infected cell. o
w
n
branch of the late phase of the CIA pathway (Fig. lo
a
d
6B). Experimental Procedures e
d
The importance of CIA1 for viperin Plasmids and siRNAs - Eukaryotic fro
m
maturation was also evident from double and triple expression vector pI.18 was kindly provided by Jim http
RNAi depletion approaches for the CIA factors Robertson (National Institute for Biological ://w
w
(Fig. 5D). Whenever CIA1 was depleted, 55Fe/S Standards and Control, Hertfordshire, United w
cluster formation was severely impaired. In Kingdom) and used for the expression of N- .jbc
.o
contrast, double knockdown of CIA2B and MMS19 terminally FLAG-tagged wild-type viperin, TN50- rg
b/
yielded wild-type efficiencies of Fe/S cluster viperin (residues 51-361), and viperin M1 (Cys to y g
u
insertion into viperin. As an exception, depletion of Ala exchange of Fe/S cluster ligating residues) (6). es
t o
both CIA2A and CIA2B slightly (40%) diminished TC20-viperin (residues 1-341), C100-viperin n A
p
55Fe radiolabeling of viperin. At first glimpse, this (residues 261-361), C70-viperin (residues 291-361) ril 1
result may suggest that at least one of the two CIA2 as well as CIA1, CIA2B, and MMS19 (without or , 2
0
1
isoforms is required for an efficient maturation with C-terminal FLAG- and HA-tags, respectively) 9
process. However, this effect of the simultaneous were cloned into pI.18 using standard PCR cloning
depletion of the two CIA2 isoforms on viperin Fe/S methods. KOD Hot Start polymerase (Novagen),
cluster assembly may be better explained by their restriction enzymes and T4 DNA ligase
strong stabilizing effect on CIA1 (see above). In the (Fermentas) were used according to the
absence of the two CIA2 proteins, CIA1 strongly manufacturers’ recommendations. All plasmids
vanishes (Fig. S3C), and this may then indirectly were DNA sequenced to verify correctness, and
lead to the observed maturation defect. Together, oligonucleotide primer sequences are available
these findings suggest that the diminution of the upon request.
viperin Fe/S cluster assembly efficiency in the The following siRNAs were purchased
absence of both CIA2A and CIA2B is caused by the from Ambion: IOP1 (s34746, s34747, s34748),
concomitant depletion of the key maturation factor CIA1 (s17970, s17971), MMS19 (s34553, s34552),
CIA1 which functions without CIA2B and CIA2A (s38636, ss38638), CIA2B (s28462,
MMS19. s28461), and Negative control (Negative control #
1).
8
Iron-sulfur cluster insertion into viperin 

Tissue culture, transfection, co- Antibodies - Mouse (ms) α-viperin
immunoprecipitation and 55Fe incorporation - HEK (dilution (d) 1:1000), rabbit (rb) α-viperin (d
293T cells were grown in Dulbecco’s modified 1:5000), rb α-CIA1 (d 1:500), rb α-CIA2B (d
Eagle’s medium (DMEM) supplemented with 5% 1:1500), rb α-MMS19 (d 1:500), rb α-tubulin (d
fetal calf serum (FCS) and passaged in ratio of 1:5 1:4000) rb α-HA epitope (d 1:4000), rb α-myc
every 3 or 4 days. HEK 293T FLP-IN T Rex cells epitope (d 1:2500), and rb α-FLAG epitope (d
inducibly expressing N-terminally FLAG-tagged 1:5000) were distributed by Abcam. Ms α-FLAG
viperin and viperin mutants (2) were propagated in epitope (d 1:2500) was obtained from Stratagene.
DMEM supplemented with 5% Tet-free FCS Ms α-IRP1 was a kind gift of R. Eisenstein
(PAA) and passaged similar to normal HEK cells. (Wisconsin, USA). Rb α-IOP1 was raised against
Protein expression was induced by 1 µg/ml of purified recombinant full-length protein in the
tetracycline (Sigma) for 24 to 48 h. HEK293T cells laboratory of RL. Horseradish peroxidase-
were seeded a day before plasmid transfection. The conjugated secondary antibody was obtained from
concentration of DNA and the ratio of transfection (Pierce) and antigen detection was performed using
D
reagent (Genejuice) were used as suggested by the the SuperSignal West Pico or Femto kit (Pierce). o
w
n
manufacturer (Merck Millipore). lo
a
d
For siRNA transfection, HEK293T cells RNA extraction and real-time RT-PCR - Total e
d
were seeded into 6-well-plates. One day later, a cellular RNA was extracted using the Nucleospin fro
m
final concentration of 10 nM siRNA were RNA II kit (Macherey-Nagel) according to the http
transfected using the tranfection reagent manufacturer’s recommendations. Aliquots of 600 ://w
w
(jetPRIME) according to the manufacturer’s ng or 1 µg of RNA were used to synthesize cDNA w
(Polypus) recommendations, followed by medium with the Quantitect reverse transcription (RT) kit .jbc
.o
exchange after 24 h. The following day, transfected (Qiagen). All real-time RT-PCR reactions were rg
b/
cells were reseeded and the transfection procedure performed with 7900HT fast-time PCR system y g
u
was repeated. In some experiments protein- (Applied Biosystems). In the subsequent PCR es
t o
encoding plasmids were transfected 24 h after the reactions, the γ-actin, CIA1, CIA2B, MMS19 and n A
p
second round of siRNA application using the CIA2A mRNA was detected with QuantiTect ril 1
jetPRIME transfection reagent. Knock-down primers QT00996415, QT00004158, QT00003724, , 2
0
1
efficiency was determined by PCR and QT00078330 and QT01027348 respectively using 9
immunoblotting at day 5 after siRNA transfection. the QuantiTect SYBR Green RT-PCR Kit
Co-IP and 55Fe incorporation studies was (Qiagen).
performed as previously described (6).

Acknowledgement: We thank Karin Edlund at Clinical Microbiology, Virology, Umeå University for
excellent technical assistance and her help with cloning. This work was supported by the Laboratory for
Molecular Medicine Sweden (MIMS), the Umeå Center for Microbial Research (UCMR), Swedish
Research Council (2011-2795) and The Swedish Foundation for Strategic Research (ICA10-0059, FFL12-
0089) (A.K.Ö.), EMBO short term fellowship (ASTF444-2015)(A.S.U.). R.L. acknowledges generous
financial support from Deutsche Forschungsgemeinschaft (LI 415/6, SPP 1710, and SPP 1927).
Conflict of interest: The authors declare that they have no conflicts of interest with the contents of this
article.
9
Iron-sulfur cluster insertion into viperin 

Author contributions: ASU, OS and CP designed and performed the experiments, analyzed, compiled
and interpreted the data, and wrote the manuscript. RR performed experiments. AKÖ and RL designed,
interpreted, compiled the data, wrote the manuscript and provided funding. All authors analyzed the
results and approved the final version of the manuscript.
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