3006? The?Journal?of?Clinical?Investigation? ? ? http://www.jci.org? ? ? Volume 116? ? ? Number 11? ? ? November 2006
Virus-induced type I IFN stimulates
generation of immunoproteasomes
at the site of infection
Eui-Cheol Shin,1,2 Ulrike Seifert,3 Takanobu Kato,2 Charles M. Rice,4 Stephen M. Feinstone,5
Peter-M. Kloetzel,3 and Barbara Rehermann1,2
1Immunology Section and 2Liver Diseases Branch, NIDDK, NIH, Department of Health and Human Services, Bethesda, Maryland, USA.
3Institute of Biochemistry, Charité-Universitaetsmedizin Berlin CCM, Berlin, Germany. 4Center for the Study of Hepatitis C,
Rockefeller University, New York, New York, USA. 5Laboratory of Hepatitis Viruses,
Center for Biologics Evaluation and Research, FDA, Bethesda, Maryland, USA.
In the effector phase of a virus-specific adaptive immune response,
primed CD8 T cells migrate to the infected organ, where they rec-
ognize and lyse infected cells that display viral peptides in the
context of MHC class I molecules. Most of these viral peptides are
generated from proteins and larger polypeptides by the 26S pro-
teasome, a major cytosolic antigen-processing complex (1).
IFN-γ, a major cytokine in many viral infections, is known to alter
the composition and function of the proteasome complex (2, 3). In
the absence of IFN-γ, the 26S proteasome complex contains a 20S
catalytic core, arranged as 2 heptameric outer rings with 7 α sub-
units (α1–α7) each and 2 heptameric inner rings with 7 β subunits
(β1–β7) each (reviewed in refs. 2, 3). IFN-γ induces the transcription
and translation of the 3 immunoproteasome subunits β1i (LMP2),
β2i (MECL-1), and β5i (LMP7), which replace their constitutive
counterparts, β1, β2, and β5, respectively, during de novo assem-
bly of proteasomes (4). The presence of TNF-α exerts a synergistic
effect (5, 6). The resulting immunoproteasomes differ from con-
stitutive proteasomes in qualitative and quantitative aspects of
their proteolytic activity (2, 3, 7). Mice that lack the immunopro-
teasome subunits β1i (7, 8) or β5i (9) have been shown to be unable
to efficiently process and present certain CD8 T cell epitopes. One
of the best examples for a strictly immunoproteasome-dependent
CD8 T cell epitope in humans is the HBV core peptide 141–151
(HBcore141–151), which requires the presence of β5i (10).
Replacement of constitutive proteasomes by immunoprotea-
somes in the virus-infected organ has been confirmed in a murine
model of lymphocytic choriomeningitis virus (LCMV) infection.
Within 7 days of LCMV infection, constitutive proteasomes were
almost completely replaced by immunoproteasomes at the site of
infection, the liver (11). This replacement appeared to depend on
IFN-γ, as it was markedly reduced in IFN-γ–/– mice (11). An impor-
tant role of IFN-γ in the induction of immunoproteasomes was
also demonstrated in a murine model of fungal infection (12). In
contrast, type I IFN did not appear to play any role. Immunopro-
teasome induction in LCMV infection was not affected in mice
that lacked the IFN-α/β receptor (IFN-α/βR) (11). Likewise, in
vitro studies on melanoma cells described that only IFN-γ and not
IFN-α induced immunoproteasome subunits and generated func-
tional immunoproteasomes (13).
Since many viruses induce a vigorous type I IFN response much
earlier than an IFN-γ response, we here investigated the role of type I
IFN in the induction of immunoproteasomes. After demonstrating
that type I IFN did indeed trigger induction, assembly, and proteo-
lytic activity of immunoproteasomes in human hepatoma cells and
primary hepatocytes in vitro and that this mechanism could be initi-
ated by intracellular double-stranded RNA (dsRNA), poly(I:C), and
HCV RNA, we then analyzed the differential contribution of type I
IFN and IFN-γ to the induction of immunoproteasomes in HCV-
infected chimpanzees. HCV, a single-stranded RNA virus has infect-
ed more than 4 million people in the United States and is the main
reason for liver transplantation in Western countries (14). Because
a vigorous innate immune response occurs within 1–3 weeks and
an adaptive immune response becomes detectable much later, i.e.,
6–12 weeks after infection (15, 16), we considered HCV infection a
suitable model to differentiate between induction of immunopro-
teasomes by innate and adaptive immune responses.
Nonstandard?abbreviations?used: ALT, alanine aminotransferase; CXCL9, CXC
chemokine ligand 9; dsRNA, double-stranded RNA; HBcore, HBV core; IFN-α–con1,
consensus sequence IFN-α; IFN-α/βR, IFN-α/β receptor; LCMV, lymphocytic cho-
riomeningitis virus; 2,5-OAS-1, 2,5-oligoadenylate synthetase-1; RIG-I, retinoic acid–
inducible gene I; VV B18R, vaccinia virus–encoded B18 receptor protein.
Conflict?of?interest: The authors have declared that no conflict of interest exists.
Citation?for?this?article: J. Clin. Invest. 116:3006–3014 (2006). doi:10.1172/JCI29832.
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 116 Number 11 November 2006
We now report that the induction of immunoproteasome sub-
units in this important infection occurred much earlier than
the intrahepatic IFN-γ response, thereby challenging the current
dogma that IFN-γ is the initial and primary inducer of immu-
noproteasomes during viral infections. Instead, immunoprotea-
somes were induced in hepatocytes much earlier, in response to
virus-induced type I IFN. Thus, rather than being passive targets,
virus-infected hepatocytes actively prepare their antigen-process-
ing machinery for optimal presentation even prior to the arrival of
liver-infiltrating T cells.
Type I IFN induces the expression of immunoproteasome subunits. To
investigate a role of type I IFN in the induction of immunoprotea-
somes, we studied human hepatoma cells and primary hepatocytes,
which are targets of many viral infections in humans. Because the
group of type I IFNs is composed of 13 different IFN-α subtypes
and of IFN-β, we first studied the effect of consensus sequence
IFN-α (IFN-α–con1) (17).
As shown in Figure 1, IFN-α–con1 induced the expression of all
3 immunoproteasome subunits both at the mRNA level (Figure
1A) and at the protein level (Figure 1B) in Huh-7 hepatoma cells.
In contrast, the mRNA level of the proteasome β7 subunit (Figure
1A) and the protein level of the proteasome α4 subunit (Figure 1B),
which were analyzed as controls and are not inducible by cytokines,
remained stable. Type I IFN also induced the expression of immu-
nosubunits in primary human hepatocytes (Figure 1C). In total,
hepatocytes from 6 different donors were tested, and in each case,
type I IFN exerted the same effect on hepatocytes as IFN-γ (data not
shown). Finally, immunosubunits were not only induced by IFN-α–
con1, but also by individual, natural members of the type I IFN
family such as IFN-α2a and IFN-β, in Huh-7 cells (Figure 1D).
Type I IFN–induced immunoproteasomes exhibit the typical structure and
function of IFN-γ–induced immunoproteasomes. As increased expression
of immunoproteasome subunits does not necessarily indicate cor-
rect incorporation into the proteasome complex and generation
of functional immunoproteasomes, we isolated 20S proteasomes
from IFN-α–treated Huh-7 cells and studied their subunit com-
Type I IFN induces the expres-
sion of immunoproteasome
subunits in vitro. (A) Huh-7 cells
were treated with 3 ng/ml IFN-α–
con1 (squares) or 10 ng/ml
(200 U/ml) IFN-γ (triangles)
for the indicated time periods,
after which the mRNA levels of
were quantified by real-time
PCR. mRNA levels were nor-
malized to endogenous refer-
ences (GAPDH and β-actin) and
expressed as fold increase over
pretreatment levels. The β7 sub-
unit was measured as a control.
(B) Huh-7 cells were treated with
the indicated doses of IFN-α–
con1 or IFN-γ for 48 hours, and
Western blot analysis was per-
formed to detect immunoprotea-
some subunits. The α4 subunit
was analyzed as a control. (C)
Primary human hepatocytes
were treated with 3 ng/ml IFN-α–
con1 or 10 ng/ml IFN-γ for the
indicated time periods prior to
Western blot analysis. (D) Huh-7
cells were treated with 1.8 ng/ml
(500 U/ml) IFN-α2a, 3 ng/ml
IFN-α–con1, 3 ng/ml IFN-β, or
10 ng/ml IFN-γ for the indicat-
ed time periods, and Western
blot analysis was performed to
detect immunoproteasome sub-
units. The density of the West-
ern blot bands was quantified
and expressed as percent of the
density of the α4 control band.
3008? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 116 Number 11 November 2006
position by 2-dimensional gel electropheresis. As shown in Figure
2A, 2-dimensional gel electrophoresis revealed the presence of both
β1i and β5i in 20S proteasomes from IFN-α–and IFN-γ–treated
Huh-7 cells, but these were only marginally observed in protea-
somes from untreated Huh-7 cells. These results were confirmed
by immunoprecipitation and 2-dimensional gel electrophoresis of
metabolically labeled 20S proteasomes (Figure 2B).
Next, we studied the function of IFN-α–induced 20S proteasomes
and compared it with that of IFN-γ–induced 20S proteasomes.
Because specific CD8 T cell epitopes that are generated only by
immunoproteasomes and not by constitutive proteasomes are not
yet known for HCV, we studied the generation of the well-described
HBcore141–151 epitope from HBV, which is strictly immunopro-
teasome-dependent (i.e., β5i-dependent), as demonstrated in previ-
ous studies using mass spectrometry and recognition by peptide-
specific cytotoxic T cells as readout (10). The substrate polypeptide
HBcore131–162 was in vitro digested with 20S proteasomes isolated
from either IFN-γ–treated, IFN-α–treated, or untreated Huh-7 cells,
and the digest products were then analyzed for the presence of the
immunoproteasome-dependent peptide HBcore141–151 by mass
spectrometry (Figure 2C). As a control, the presence of the immu-
noproteasome-independent HBcore131–140 was also analyzed.
Importantly, IFN-α–induced proteasome complexes generated
HBcore141–151 with the same kinetics as IFN-γ–induced immuno-
proteasomes, while less HBcore141–151 was detectable when diges-
tion was performed with constitutive proteasomes (Figure 2C). The
immunoproteasome-independent control HBcore131–140 was gen-
erated with the same kinetics by IFN-α– and IFN-γ–induced immu-
noproteasomes and by constitutive proteasomes (Figure 2C).
Collectively, these data demonstrate that type I IFN not only
induced the expression of immunoproteasome subunits at the
mRNA level, but also their translation, correct processing, and
incorporation into the proteasome complex, resulting in the
formation of fully functional immunoproteasomes. Thus, type I
IFN–induced immunoproteasomes were indistinguishable from
IFN-γ–induced immunoproteasomes not only in their subunit
composition, but also in their function.
Intracellular dsRNA induces the expression of immunoproteasome sub-
units in a type I IFN–dependent manner. In the above-described in vitro
studies, hepatocytes and hepatoma cells were treated with exoge-
nous type I IFN to induce immunoproteasomes. Next, we asked
whether endogenous, viral RNA–induced IFN generates immuno-
proteasomes in hepatoma cells. Sensing of intracellular dsRNA by
retinoic acid–inducible gene I (RIG-I) results in the phosphoryla-
tion and activation of IRF-3 and subsequent production of IFN-β.
Secreted IFN-β binds to the IFN-α/βR in an autocrine and para-
crine manner, resulting in activation of the JAK/STAT signaling
pathway, transcription of IFN-stimulated genes, and, via produc-
tion of the IFN-stimulated gene IRF-7, production of various IFN-α
subtypes (reviewed in ref. 18). Because Huh-7 cells respond well to
intracellular dsRNA via activation of RIG-I, but not to extracellular
dsRNA due to the lack of TLR3 (19), Huh-7 cells were transfected
with the dsRNA poly(I:C) to simulate the presence of viral dsRNA.
Extracellular addition of poly(I:C) or transfection with DNA served
as negative controls in this experiment.
As shown in Figure 3, transfection of Huh-7 cells with poly(I:C)
induced IFN-β and the downstream response of 2,5-oligoadenylate
synthetase (2,5-OAS-1) (Figure 3A) and increased the mRNA levels
of all 3 immunoproteasome subunits (Figure 3B) as well as their
protein levels (Figure 3D). In contrast, neither extracellular addi-
tion of poly(I:C) (Figure 3, B and C) nor transfection of Huh-7 cells
with DNA (Figure 3, B and D) had any effect. Collectively, these
data demonstrate that immunoproteasome subunits are not only
induced by exogenous type I IFN (Figure 1), but also by endoge-
nously produced type I IFN, in response to intracellular dsRNA. To
analyze which of the type I IFNs was dominant in this endogenous
induction pathway, we added neutralizing antibodies to IFN-α
and/or IFN-β to the culture. In separate experiments, we also added
the vaccinia virus–encoded B18 receptor protein (VV B18R), which
competes with the IFN-α/βR for IFN binding (20). As shown in
Figure 3E, anti–IFN-β Ab abrogated the induction of immunopro-
teasome subunits by intracellular dsRNA. Likewise, the addition of
VV B18R abrogated the induction of immunoproteasome subunits.
In contrast, neutralization of IFN-α, which is a later step in the
endogenous IFN response cascade (18), did not block the induction
of immunoproteasome subunits. These results prove that induc-
Type I IFN–induced immunoproteasomes exhibit the typical structure
and function of IFN-γ–induced immunoproteasomes. (A) Huh-7 cells
were treated with 3 ng/ml IFN-α–con1 or 10 ng/ml IFN-γ for 36 hours,
and the 20S proteasome complex was biochemically isolated from cell
lysates. Isolated 20S proteasomes were analyzed by 2-dimensional
gel electropheresis and Coomassie blue staining. The locations of β1i
and β5i are indicated in the insets to the right (magnification, ×2). (B)
Huh-7 cells were treated with 3 ng/ml IFN-α–con1 or 10 ng/ml IFN-γ for
36 hours and labeled with 35S-methionine. The 20S proteasome com-
plex was immunoprecipitated and analyzed by 2-dimensional gel elec-
tropheresis and autoradiography. The locations of β1, β5, β1i, and β5i
are indicated. (C) Biochemically isolated 20S proteasome complexes
as shown in A were subsequently incubated with the precursor sub-
strate HBcore131–162 for the indicated time periods. In vitro digests
were analyzed by HPLC and mass spectrometry at the indicated time
points for the presence of the β5i-dependent HBcore141–151 and the
β5i-independent HBcore131–140. Type I IFN–induced immunoprotea-
somes displayed β5i-dependent proteolytic activity.
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 116 Number 11 November 2006
tion of immunoproteasome subunits in response to intracellular
dsRNA is indeed mediated by endogenously produced type I IFN.
In addition, they demonstrate that early steps in the endogenous
type I IFN response cascade, i.e., either neutralization of IFN-β or
prevention of binding to the IFN-α/βR, need to be blocked to abro-
gate the induction of immunoproteasome subunits.
HCV RNA induces the expression of immunoproteasome subunits. To
test whether HCV RNA can induce type I IFN and immunopro-
teasomes, we transfected Huh-7 cells with HCV RNA. As shown in
Figure 4, transfection of Huh-7 cells with HCV RNA induced IFN-β
production and the downstream response of 2,5-OAS-1 (Figure 4A)
and increased mRNA levels of all 3 immunoproteasome subunits
(Figure 4B). These data demonstrate that immunoproteasome
subunits can be induced not only by artificial dsRNA [poly(I:C)],
but also by HCV RNA itself.
mRNA levels of immunoproteasome subunits increase early during acute
HCV infection and are temporally related to the type I IFN response. To
investigate the differential contribution of type I IFN and IFN-γ to
the induction of immunoproteasomes in HCV infection, we pro-
spectively studied 5 chimpanzees with acute HCV infection (Figure
5). Serum alanine aminotransferase (ALT), a marker of liver injury,
peaked in all chimpanzees 8–10 weeks after infection. This peak
Intracellular dsRNA induces the
expression of immunoproteasome
subunits by the secretion of type I
IFN. Huh-7 cells were transfected
with 10 μg poly(I:C) (squares) or 10
μg pcDNA3.1 plasmid DNA (invert-
ed triangles), treated with 10 μg/ml
extracellular poly(I:C) (triangles),
or were mock transfected (circles).
(A) Quantitation of IFN-β and
2,5-OAS-1 mRNA by real-time PCR.
Bars (indicating secreted IFN-β)
are shown only for the poly(I:C)-
transfected Huh-7 cells because no
secreted IFN-β was detectable in
the control cultures of DNA-trans-
fected Huh-7 cells or Huh-7 cells
treated extracellularly with poly(I:C).
(B) Quantitation of mRNA levels of
immunoproteasome subunits β1i,
β5i, β2i and constitutive subunit β7
by real-time PCR. (C–E) Detection
of immunoproteasome subunits β1i,
β5i, and β2i by Western blot analy-
sis. (C and D) Huh-7 cells were
treated with IFN-α and IFN-γ as
well as (C) treated with extracellular
DNA and poly(I:C) or (D) transfect-
ed with DNA and poly(I:C). (E) Four
hours after poly(I:C) transfection of
Huh-7 cells, 5,000 U/ml neutralizing
anti–IFN-α and 2,000 U/ml neutral-
izing anti–IFN-β, alone or in combi-
nation, as well as 1 μg/ml VV B18R
protein were added into culture and
incubated for 48 hours, and West-
ern blot was performed to detect
immunoproteasome subunits. Anti–
IFN-α/β, combination of IFN-α– and
IFN-β–neutralizing antibodies. The
density of the Western blot bands
was quantified and expressed as
percent of the density of the α4
3010? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 116 Number 11 November 2006
was followed by HCV clearance in chimpanzees with a self-limited
course of infection (Ch6455, Ch6461, and Ch1606) or by a partial,
2–3 log10 (i.e., >99%) decrease of serum HCV RNA titers in chimpan-
zees with a chronically evolving course of infection (Ch6475 and
Ch6411) (Figure 5). To study the induction of immunoproteasome
subunits at the site of infection, we quantified mRNA levels of the 3
immunoproteasome subunits, β1i, β5i, and β2i, in serial liver biop-
sies by real-time PCR. As shown in Figure 5, B and C, mRNA levels
of all 3 immunoproteasome subunits started to increase as early as
2 weeks after infection and peaked 5–8 weeks after infection. The
mRNA level of β7, a nonreplaceable subunit of the proteasome, was
studied as a control and remained constant in all chimpanzees at
all study time points (Figure 5B).
Next, the kinetics of immunoproteasome subunit induction
were compared with the expression levels of IFN-γ and TNF-α,
which are well-known inducers of immunoproteasome subunits.
Surprisingly, neither IFN-γ nor TNF-α mRNA levels increased
substantially (greater than 2-fold) in the liver prior to the increase
in immunoproteasome subunit mRNA (Figure 5, D and E). The
lack of an early IFN-γ response prior to the induction of immu-
noproteasome subunits was confirmed by the absent induction of
CXC chemokine ligand 9 (CXCL9; also referred to as MIG) mRNA
(Figure 5D), which is strictly IFN-γ dependent (Supplemental
Figure 1; supplemental material available online with this article;
Thus, the increase in immunoproteasome subunit mRNA
occurred prior to any increase in IFN-γ and TNF-α mRNA levels
(dashed vertical lines in Figure 5) in all chimpanzees. Because this
temporal relation rendered a role of IFN-γ or TNF-α in the induc-
tion of immunoproteasomes highly unlikely, we next examined
the intrahepatic type I IFN response. As downstream marker of the
type I IFN response, we quantitated 2,5-OAS-1 mRNA. As shown
in Figure 5F, an increase of 2,5-OAS-1 mRNA levels was detectable
as early as 2 weeks after infection. Its expres-
sion kinetics correlated well with those of the
3 immunoproteasome subunits and preceded
the increase in IFN-γ mRNA in all chimpan-
zees. In parallel to the 2,5-OAS-1 response, an
increase of mRNA levels of IFN-β mRNA and
several IFN-α subtypes (IFN-α2, IFN-α14, and
IFN-α21) was detectable (Figure 5G). Collec-
tively, these results indicated a role of type I
IFN in the induction of immunoproteasomes
during acute HCV infection.
Induction of immunoproteasome subunits in the
liver precedes infiltration of CD8 T cells. Whereas
mRNA levels of all 3 immunoproteasome sub-
units started to increase as early as 2 weeks
after infection, CD8β mRNA levels increased
no earlier than 5–8 weeks after infection and
peaked 11–16 weeks after infection. As shown
in Figure 6, intrahepatic CD8β levels corre-
lated closely with intrahepatic IFN-γ levels,
suggesting that liver-infiltrating CD8 T cells
produced most of the detectable IFN-γ. The
IFN-γ–producing CD8 T cell population
included HCV-specific T cells, which targeted
multiple epitopes in all structural and non-
structural HCV proteins (data not shown).
Induction of immunoproteasome subunits
during acute HCV infection therefore occurred much earlier than
the intrahepatic IFN-γ response. Indeed, immunoproteasomes were
induced in the liver at the same time as type I IFN. Thus, rather
than being passive targets, virus-infected hepatocytes actively pre-
pare their antigen-processing machinery for optimal presentation
even prior to the arrival of liver-infiltrating T cells.
The present study demonstrates the role of type I IFN in the induc-
tion of functional immunoproteasomes in the target organ of the
CD8 T cell response during a viral infection. Not only exogenous
IFN-α and IFN-β, but also endogenous production of type I IFN
by intracellular HCV RNA or poly(I:C), stimulated induction of
functionally active immunoproteasomes in vitro (Figures 1–4).
Prospective analysis of liver biopsies from HCV-infected chimpan-
zees demonstrated that this mechanism was also operative in vivo
during the early weeks of HCV infection (Figure 5). Collectively,
these results challenge the current dogma of IFN-γ being the sole
inducer of immunoproteasomes during a viral infection.
Our findings may represent a more general antiviral response,
because dsRNA-induced, RIG-I–mediated type I IFN responses in
virus-infected cells have also been described for other RNA viruses
such as Newcastle disease virus (21), vesicular stomatitis virus
(22), and paramyxovirus (23). Type I IFN–mediated induction
of immunoproteasomes was especially evident in HCV infection,
where intrahepatic expression of IFN-γ was detectable very late,
i.e., not earlier than 5–8 weeks after infection (Figure 5). Thus, the
innate type I IFN response not only exerts antiviral functions but
also prepares the infected target organ for the adaptive immune
response, in particular by immunoproteasome-dependent antigen
processing. This mechanism complements a similar role of the
innate immune response by type I IFN–induced upregulation of
MHC molecules that has been described previously (24, 25). Both
HCV RNA induces the expression of immunoproteasome subunits. Huh-7 cells were trans-
fected with 2 μg in vitro transcribed H77 HCV RNA (squares) or were mock transfected
(circles). (A) mRNA levels of IFN-β and 2,5-OAS-1 were determined by real-time PCR.
Secreted IFN-β (bars in left panel) was detectable by ELISA only in the supernatants of
HCV RNA-transfected Huh-7 cells, not in supernatants of mock-transfected Huh-7 cells (not
shown). (B) mRNA levels of immunoproteasome subunits β1i, β5i, and β2i and the constitu-
tive subunit β7 as determined by real-time PCR.
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 116 Number 11 November 2006
mechanisms may increase recognition of infected cells by incom-
ing CD8 T cells and elevate virus-infected cells from passive targets
to active facilitators of the adaptive immune response.
The type I IFN–mediated induction of immunoproteasomes
described herein differed from the results of a previous publica-
tion that described normal induction of immunoproteasomes in
LCMV-infected mice that lack the IFN-α/βR (11). In addition to
virus and host differences between both models, several additional
factors may explain why a type I IFN effect was not observed in the
LCMV model. First, the effect of type I IFN may have been masked
by an early and vigorous IFN-γ response in the LCMV model.
The temporal dissociation between early type I IFN response and
late IFN-γ response in the HCV model was essential to recognize
the additional and specific effect of type I IFN. Second, human
hepatocytes differ from murine hepatocytes in regard to IFN-α/βR
expression and responsiveness to type I IFN (26). Whereas human
hepatocytes express functional IFN-α/βR 2c, murine hepatocytes
predominantly express IFN-α/βR 2a, which exerts inhibitory func-
tions and blunts the effect of type I IFN on murine hepatocytes
(26). Third, the new class of IFN-λ1 (IL-29), IFN-λ2 (IL-28A), and
IFN-λ3 (IL-28B) (27, 28), which activates the same JAK/STAT sig-
naling pathway as type I IFN, may have induced immunoprotea-
somes in IFN-α/βR–/– mice during LCMV infection.
Our findings are supported by microarray studies of gene expres-
sion patterns in acute hepatitis C, which also detect early expres-
sion of immunoproteasome subunits (29, 30). In addition, they are
indirectly supported by data from acute HBV infection (31). In con-
trast to acute HCV infection, acute HBV infection does not induce
any detectable type I IFN response in the liver, as determined by
lack of 2,5-OAS-1 mRNA induction in serial liver biopsies of HBV-
mRNA levels increase early
in acute HCV infection and in
temporal relation to type I IFN
responses. Five chimpanzees
(Ch6455, Ch6461, Ch1606,
Ch6475, and Ch6411) were
studied prospectively during
acute HCV infection. (A) Serum
ALT levels (open squares) and
serum HCV RNA titers (black
diamonds) have previously
been reported (44) and are pre-
sented for reference purposes.
(B–G) Serial liver biopsies were
analyzed for mRNA levels of
(B) β1i (squares) and β7 (trian-
gles); (C) β5i (squares) and β2i
(triangles); (D) IFN-γ (squares)
and CXCL9 (triangles); (E)
TNF-α; (F) 2,5-OAS-1; and (G)
IFN-α2 (black line), IFN-α14
(dashed line), IFN-α21 dotted
line), and IFN-β (gray line).
mRNA levels were normal-
ized to endogenous referenc-
es (GAPDH and β-actin) and
expressed as fold increase
over preinfection levels. In
Ch1606, the relative mRNA
level of IFN-α21 was 36.9 at
week 4. Vertical dashed lines
separate the time intervals
prior to the first major (greater
than 2-fold) increase of IFN-γ
or TNF-α mRNA levels. NT,
not tested due to the shortage
3012? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 116 Number 11 November 2006
infected chimpanzees (31). Consistent with the absence of a type I
IFN response, no increased expression of immunoproteasome sub-
units was observed during the early phase of acute HBV infection
(31). In fact, immunoproteasome subunits were only induced in
the later phase of HBV infection, concomitant with an increase in
intrahepatic IFN-γ mRNA levels (31).
What role does the induction of immunoproteasomes by type I
IFN play in the course of acute HCV infection? Both the kinetics and
the peak levels of intrahepatic immunoproteasome subunit mRNA
expression were comparable in all 5 chimpanzees in our study (Fig-
ure 5). Likewise, kinetics and peak levels of intrahepatic 2,5-OAS-1
and type I IFN mRNA levels were comparable in all 5 chimpanzees
(Figure 5). These findings suggest that differences in the outcome
of HCV infection do not solely depend on the presence or absence
of type I IFN–mediated induction of immunoproteasomes. How-
ever, the timing of type I IFN–mediated induction of immunopro-
teasomes in relation to the adaptive CD8 T cell response in the liver
may be important. An effective CD8 T cell response is an important
factor for the outcome of HCV infection, as previously shown by us
(32–34) and others (15, 35–43). An increasing gap between transient
early type I IFN–mediated induction of immunoproteasomes and
late CD8 T cell infiltration in the liver may render the antigen rec-
ognition by intrahepatic HCV-specific T cells suboptimal. Interest-
ingly, the timing of the intrahepatic increase of IFN-γ mRNA levels
as indicated by the dashed vertical lines in Figure 5 (which correlates
closely with intrahepatic CD8β mRNA levels; Figure 6), occurs later
in chimpanzees with a chronically evolving course of HCV infection.
This is consistent with a previously described delay in ALT peak,
another correlate of CD8 T cell activity in the liver, in a larger group
of chimpanzees with a chronic outcome of infection (44).
This timely dissociation between early transient immunoprotea-
some expression in the liver and late infiltration of CD8 T cells may
be overcome by repeated exogenous administration of high doses
of type I IFN, as impressively shown by the high (95%) HCV clear-
ance rates if recombinant IFN-α is administered during the first 6
months of HCV infection (45–48). The high treatment response rate
cannot readily be explained by IFN’s antiviral effects, because clear-
ance rates are much lower in chronic HCV infection (46, 49), when
immune escape mechanisms are already established (16, 41) and the
HCV-specific T cell response is exhausted (15, 33, 35, 39, 40, 42).
Thus, it is possible that IFN-α–mediated induction of immuno-
proteasomes and better antigen recognition by liver-infiltrating
T cells contribute to the high effectiveness of type I IFN–based ther-
apies (95%) if administered early during HCV infection. Practically,
however, it is very difficult to prove this hypothesis in a clinical set-
ting, because it would require multiple liver biopsies per patient
during the acute phase of hepatitis C. Chimpanzees are not an
appropriate model for this purpose because their HCV titers do not
decrease during therapy with recombinant human IFN-α (50, 51).
We conclude that intrahepatic induction of immunoproteasomes
precedes the intrahepatic expression of IFN-γ in this clinically rel-
evant model of viral infection in humans. Type I IFN secreted in
response to dsRNA in virus-infected hepatocytes induced function-
al immunoproteasomes with characteristic proteolytic activity.
Chimpanzees. Chimpanzees were housed under standard conditions for
humane care and in compliance with NIH guidelines at an AALAC-accredited
facility and were studied under protocols approved by the Animal Care and
Use Committee (Center for Biologics Evaluation and Research) and the Pub-
lic Health Service Interagency Model Committee (NIH). Four chimpanzees
(Ch6455, Ch6461, Ch6475, and Ch6411) were intravenously inoculated with
100 chimpanzee infectious dose 50 (CID50) of monoclonal (H77 p90, geno-
type 1a) HCV RNA–positive and anti-HCV–negative plasma from Ch1536 (44,
52). Ch1606 was intrahepatically inoculated with in vitro–transcribed clonal
HCV RNA (H77 p90, genotype 1a) (44). Serum HCV RNA was quantified
by real-time RT-PCR with a sensitivity of 200 RNA copies/ml as previously
reported (44). Serial liver biopsies were snap-frozen for RNA extraction.
Extraction of RNA, cDNA synthesis, and TaqMan real-time PCR. Total RNA
was isolated from IFN-treated cells and from snap-frozen and mechanically
homogenized liver biopsies using the RNeasy Mini Kit (QIAGEN) with an on-
column DNase digestion step. RNA (200–400 ng) was reverse transcribed with
the First-Strand cDNA Synthesis Kit (Marligen Biosciences) according to the
manufacturer’s instructions. TaqMan real-time PCR was performed in dupli-
cates to determine the mRNA levels of β1i, β5i, β2i, β7, CD8β, IFN-γ, CXCL9,
TNF-α, 2,5-OAS-1, IFN-α2, IFN-α14, IFN-α21, and IFN-β using primers
and probes of TaqMan Gene Expression Assays from Applied Biosystems
according to the manufacturer’s instructions. Due to the limited amount of
liver biopsy tissue, we could not study more than 4 type I IFN subtypes. The
amount of specific mRNA was calculated with Sequence Detector software
(version 1.6.3; Applied Biosystems), normalized to endogenous references
(GAPDH and β-actin), and expressed as fold increase over the corresponding
mRNA levels in preinfection biopsies or untreated cells. The baseline mRNA
levels in preinfection liver biopsies represent the mean value obtained from
up to 8 chimpanzee preinfection biopsies.
Cell culture. Huh-7 hepatoma cell lines were grown in DMEM containing 5%
FCS, 4.5 g/l glucose, 50 μg/ml streptomycin, 50 IU/ml penicillin, and 2 mM
l-glutamine (complete DMEM). Primary human hepatocytes were obtained
from BD Biosciences or through the Liver Tissue Procurement and Distri-
bution System, which was funded by NIH contract no. N01-DK-9-2310.
Induction of immunoproteasome subunits in the liver precedes infiltration of CD8 T cells. CD8β mRNA levels were quantified in serial liver biop-
sies of chimpanzees with acute HCV infection. CD8β mRNA amounts (squares) correlated closely with IFN-γ mRNA amounts (gray lines; values
reproduced from Figure 5D for reference).
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 116 Number 11 November 2006
Hepatocytes were grown in Hepatocyte Maintenance Medium (Cambrex).
All primary human hepatocytes were from HBV-negative and HCV-negative
donors. The percentage of contaminating cells in the primary hepatocyte
cultures was evaluated by fluorescence-activated cell sorting analysis using
anti-CD45 (BD Biosciences) for cells of hematopoietic origin, anti-CD4
(BD Biosciences) for liver sinusoidal endothelial cells (53), and anti-CD14
(BD Biosciences) for Kupffer cells. After exclusion of dead cells by ethidium
monoazide staining, the purity of hepatocytes was 86%–98.8%.
IFN treatment and RNA transfection. Hepatoma cells and primary human
hepatocytes were treated with IFN-α−con1 (kindly provided by L. Blatt, Inter-
Mune Inc., Brisbane, California, USA), human IFN-α2a (Research Diagnos-
tics), human IFN-β1b (Betaseron; Chiron), or human IFN-γ (PeproTech).
Huh-7 cells were transfected by DMRIE-C reagent (Invitrogen) and
Opti-MEM medium (Invitrogen) using either 10 μg poly(I:C) (Sigma-
Aldrich) or 10 μg pcDNA3.1 plasmid DNA in a 6-well culture plate or
2 μg proteinase K–treated H77 HCV RNA in vitro transcribed from pHCV-
H77 (kindly provided by J. Bukh, National Institute of Allergy and Infec-
tious Diseases, Bethesda, Maryland, USA) in a 48-well culture plate. Four
hours after transfection, the culture medium was replaced by complete
DMEM. The production of IFN-β was measured in culture supernatant
using IFN-β ELISA kit (Biosource International). If blocking experiments
were performed, neutralizing IFN-α Ab (R&D Systems) and/or IFN-β Ab
(R&D Systems) or VV B18R (eBiosciences) (20) were added when the cul-
ture medium was replaced. As controls, 10 μg/ml poly(I:C) were added into
Huh-7 cultures without transfection or Huh-7 cells were transfected with
10 μg pcDNA3.1 plasmid DNA.
Western blot analysis. Cell lysates of 50 μg (for the detection of β5i) or
80 μg (for the detection of β1i, β2i, and α4) were separated on 15% SDS-
polyacrylamide gels (54). Immunoblots were probed with human β1i, β5i,
β2i, or α4 proteasome subunit–specific polyclonal rabbit antisera (kindly
provided by M. Groettrup, University of Konstanz, Konstanz, Germany) or
β-tubulin monoclonal Ab (CRP Inc.), and signals were detected with HRP-
conjugated anti-rabbit IgG (Jackson ImmunoResearch Laboratories Inc.)
or anti-mouse IgG (Seramun Diagnostica GmbH) and chemiluminescence
reagent (54). The density of the Western blot bands was quantified using a
GS-800 Calibrated Densitometer (Bio-Rad).
Proteasome purification. 20S proteasomes were isolated from Huh-7
hepatoma cells that had been treated either with 3 ng/ml IFN-α–con1 or
with 10 ng/ml IFN-γ for 36 hours or, as a control, had been left untreated.
Frozen cells were homogenized in lysis buffer containing 10 mM HEPES,
pH 7.2; 80 mM KAc; 5 mM MgAc2; and 0,1% Triton X-100. Lysates were
applied onto DEAE Sephacel (Amersham Biosciences) and washed with 80
mM KAc until no protein was detected by Ponceau staining. Proteasomes
were eluted with 500 mM KAc buffer (10 mM HEPES, pH 7.2; 500 mM
KAc; and 5 mM MgAc2) and concentrated by ammonium sulphate precipi-
tation. Protein fractions were separated by ultra centrifugation on sucrose
gradients (10%–40%) and ultracentrifuged at 200,000 g for 16 hours using
a Beckman Coulter SW 40 rotor. Fractions containing proteasomes were
pooled, applied to a Mono Q column (Pharmacia FPLC),?and eluted with a
gradient of 100–500 mM NaCl in TEAD (20 mM Tris, pH 7.2; 1 mM EDTA;
1 mM NaN3; and 1 mM DTT).
Metabolic labeling and immunoprecipitation of proteasome. Huh-7 hepatoma
cells (1 × 106) were seeded in flasks and cultured in complete DMEM with
3 ng/ml IFN-α–con1 or 10 ng/ml IFN-γ or without any IFN for 36 hours.
During the final 12 hours, cells were labeled with translabel 35S-methio-
nine (100 μCi/ml), washed twice, chased for 6 hours in medium with or
without IFN, and then lysed in lysis buffer (1% NP40; 20 mM Tris-Cl, pH
7.5; 10 mM EDTA; and 100 mM NaCl with protease inhibitors). After
centrifugation (5 min, 20,000 g?at 4°C), the lysates were precleared with
25 μl protein A-Sepharose (50% slurry) and 1 mg/ml BSA overnight at 4°C.
20S proteasomes were immunoprecipitated with 50 μl protein A-Sepha-
rose and 5 μl polyclonal 20S proteasome-specific rabbit antiserum for
4 hours at 4°C. Sepharose beads were washed with lysis buffer contain-
ing 0.5% NP40, resuspended in sample buffer for nonequilibrium pH gel
elctrophoresis (NEPHGE), and subjected to 2-dimensional NEPHGE. Gels
were exposed for autoradiography.
Two-dimensional gel electrophoresis. For separation of 20S proteasomal pro-
teins, isoelectric focusing by carrier ampholytes was combined with SDS-
PAGE (54). Protein (25 μg) was applied to a carrier ampholyte isoelectric
focussing gel. In the second dimension, proteins were loaded on a 1.5-mm-
thick SDS-PAGE (7 × 8 cm) and stained with Coomassie Brilliant Blue G250.
Proteasomal subunits were identified on the basis of their migrational behav-
ior in comparison to reference electrophoreses of 20S proteasomes (55).
Peptide digestion and analysis of digests. Twenty micrograms of the HBV
32-mer polypeptide AYRPPNAPILSTLPETTVVRRRGRSPRRRTPS
(HBcore131–162) and 3 μg of purified proteasomes were incubated in
300 μl of assay buffer (20 mM HEPES/KOH, pH 7.8; 2 mM MgAc2; and
1 mM dithiothreitol) at 37°C for the time periods indicated in Figure
2C. The reaction was terminated by the addition of 0.1% trifluoroace-
tic acid (TFA). Digest products (30 μl) were separated by reverse-phase
HPLC (SMART system equipped with a μRPC C2/C18 SC 2.1/10 column;
Amersham Biosciences) under the following specific conditions: eluent A,
0.05% TFA; eluent B, 70% acetonitrile containing 0.045% TFA; gradient,
10%–95% eluent B in 15 minutes; flow rate, 70 μl/min. Analysis was per-
formed online using a tandem quadrupol mass spectrometer equipped
with an electronspray ion source (LCQ Finnigan MAT; Thermo Electron
Corp.). Each scan was acquired over the range m/z = 200–1,400 in 3 sec-
onds. Peptides were identified by their molecular masses and sequence in
mass spectrometry/mass spectrometry experiments. Immunoproteasome-
dependent generation of HBcore141–151 has also been demonstrated pre-
viously using peptide-specific cytotoxic T cells as readout (10).
The authors thank K. Textoris-Taube for mass spectrometry; U.
Zimny-Arndt for 2-dimensional gel electrophoresis; P. Henklein
for peptide synthesis; M. Groettrup for antibodies; and T.J. Liang,
J.-H. Park, and N. Grandvaux for helpful and stimulating discus-
sion. This study was supported by NIDDK, NIH, and Center for
Biologics Evaluation and Research, FDA, intramural research pro-
grams; by grant SFB 421 from the Deutsche Forschungsgemein-
schaft to U. Seifert and P.-M. Kloetzel; and by U.S. Public Health
Service grant CA85883-01 from the NIH to C.M. Rice.
Received for publication July 24, 2006, and accepted in revised
form August 29, 2006.
Address correspondence to: B. Rehermann, Immunology Section,
Liver Diseases Branch, NIDDK, NIH, DHHS, 10 Center Drive,
Building 10 Room 9B16, Bethesda, Maryland 20892, USA. Phone:
(301) 402-7144; Fax: (301) 402-0491; E-mail: Rehermann@nih.gov.
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