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*
Beirne Carter Center for Immunology Research and
Department of Pathology, University of Virginia, Charlottesville, VA 22908
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-2. By using a series of vaccinia/HCV recombinants, we found that
the HCV core protein was sufficient for immunosuppression, prolonged
viremia, and increased mortality. These results suggest that the HCV
core protein plays an important role in the establishment and
maintenance of HCV infection by suppressing host immune responses, in
particular the generation of virus-specific CTLs. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The high incidence of HCV persistence after infection suggests that this virus has evolved a mechanism to evade the host response, probably by inhibiting the immune response necessary for viral clearance during acute infection. One proposed mechanism of immune evasion is the generation of viral variants during infection that could escape from Ab or CTL recognition. This strategy is consistent with the high error rate of the viral polymerase during viral RNA replication and may account for the high frequency of HCV quasispecies detected in HCV-infected patients (6). Alternatively, as observed for many of the large DNA viruses, such as adenovirus and herpes simplex virus (7, 8, 9), HCV may encode one or more products that act to inhibit viral clearance by the host and can lead to progressive or persistent viral infection.
The induction of virus-specific CTLs during infection is a well-established mechanism for virus elimination during infection. In the case of HCV, CTLs have been recovered from both the liver (10) and the periphery (11) of chronically infected patients. However, the frequency of HCV-specific CTL precursor (CTLp) and effectors is much lower than that observed during infection with other persistent viruses, such as hepatitis B virus or HIV (12, 13). It is currently unknown whether this low CTLp in chronically infected patients reflects an immunosuppressive mechanism causally linked to the ability of the HCV to establish and maintain viral persistence after infection.
The HCV RNA genome is approximately 9.5 kb in length and encodes a long polyprotein. Ten discrete proteins are produced from this polyprotein by proteolytic processing. The structural proteins include the core (C) and the E1 and E2 glycoproteins and are found in the N-terminal portion of the polyprotein. The nonstructural (NS) proteins involved in RNA replication are found in the remainder of polyprotein. Among the HCV gene products, only the highly conserved HCV core protein has been suggested to have an immunomodulatory function. This is based on the in vitro demonstration that the core protein binds to the cytoplasmic domain of certain members of the TNF receptor superfamily (14) and can modulate the sensitivity of cells expressing HCV core to TNF-mediated lysis in culture (15).
Since HCV infects only humans and chimpanzees and replicates inefficiently in cell culture, studies aimed at assessing the abilities of individual HCV-encoded proteins to modulate host immune responses during HCV infection have been difficult to perform. To examine this issue in a model system, we have used infection of mice with recombinant vaccinia viruses (VV) expressing HCV gene products as a way to assess possible immunomodulatory effects of specific HCV polypeptides on VV infection. The contributions of specific components of the immune response to VV infection in mice have been well defined, and recovery from VV infection has been demonstrated to be strongly correlated with the generation of a virus-specific CTL response (16, 17).
In the study described here, we examined the virulence of various VV/HCV recombinants in mice and studied the host response induced by recombinant viruses. Surprisingly, we found that a recombinant VV expressing the structural protein of HCV produced a lethal disseminated infection in mice and concomitantly suppressed the VV-specific CTL response and the production of proinflammatory cytokines as well. Using a series of VV recombinants expressing various C-terminally truncated polyproteins, this immunosuppressive effect was mapped to the core protein. These results suggest that expression of HCV core during HCV infection could account for the low frequency of CTLp observed in chronically infected patients and that HCV core may play a critical role in establishing and maintaining persistent HCV infection.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The P815 (H-2d) mastocytoma and BSC40 monkey kidney cell lines were maintained in DMEM supplemented with 10% (v/v) FBS and 1% (w/v) glutamine.
Plasmid constructions and generation of VV recombinants
VV/HCV recombinants are designated by the region of the HCV (H
strain; genotype 1a) polyprotein sequence expressed. Two recombinants,
vHCV-S and vHCV-NS, have been described previously (18, 19). For
mapping studies, a nested set of C-terminal deletions was constructed
using PCR to introduce a stop codon at or near the end of each
individual protein. VV insertion vectors were either pTM3 (20) or pBRTM
(19), and the HCV-specific portions amplified by PCR were verified by
sequence analysis. The resulting plasmids were then used to rescue the
corresponding VV/HCV recombinants by standard methods (21). The salient
features of the six VV constructs used in this study are summarized in
Fig. 1
. The vHCV-S expresses HCV C, E1,
E2, p7, NS2, and a portion of NS3. The vHCV-C/p7, vHCV-C/E2, vHCV-C/E1,
and vHCV-C express the C to p7, C to E2, C to E1, and C proteins,
respectively. The vHCV-NS expresses the majority of NS2 (which begins
at residue 809) through the end of polyprotein (NS5B). VV recombinants
were rescued, plaque purified, and expanded in BSC40 cells. Their
titers were determined by standard plaque assay using BSC40 cells.
Various VV/HCV recombinants were no different in their viral growth
rates (data not shown). The production of the expected HCV-specific
proteins was verified by infection of BSC40 cells, radiolabeling, and
immunoprecipitation with specific antisera (18, 19) (data not shown).
The negative control VV recombinant expressing ß-galactosidase,
vSC11, has been described (22).
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Infection of mice and virus titration
Four- to six-week-old BALB/c mice were purchased from Taconic Farms (Germantown, NY). Mice were inoculated i.p. with various doses of the VV recombinants as indicated in the text and figure legends. On days 1–5 after virus inoculation, mice were sacrificed, and liver tissue was harvested to determine recombinant VV titers. Tissue samples were weighed, homogenized in Iscove’s DMEM containing 10% newborn calf serum, and centrifuged at 500 x g for 10 min, and pellets were resuspended in PBS containing 1% FCS and 1 mM MgCl2. Virus was released from the pellets by three freeze-thaw cycles followed by sonication.
To assay for plaque-forming virus, 200 µl of 10-1–10-3 dilutions were incubated with confluent BSC40 monolayers in six-well plates for 1 h at 37°C. Inocula were removed and replaced with DMEM containing 10 µg/ml penicillin/streptomycin, 10% FCS, and 2 mM L-glutamine followed by incubation for 2 days at 37°C. Monolayers were fixed with 7% formaldehyde and stained with crystal violet. Plaques were counted to determine the viral titer of each liver sample, and the viral titer was calculated as log plaque-forming units (pfu) per gram of tissue.
Measurement of primary VV-specific CTL responses
To determine the primary VV-specific CTL response, mice were infected i.p. with 5 x 107 pfu of a vHCV-S recombinant or control recombinant vHCV-NS. On day 5 postinoculation, mice were sacrificed, and spleens were harvested. A single cell suspension of splenocytes was prepared and purified through Isopaque-Ficoll. Cells were harvested from the layer between the Isopaque-Ficoll and medium, and washed twice with medium to remove the residual Ficoll. To measure VV-specific CTL responses, these purified splenocytes were tested using 51Cr-labeled P815 target cells previously infected with wild-type VV. The percentage of specific 51Cr release was determined by standard procedures (23). Values for 51Cr release are the mean of quadruplicate samples; SDs were typically <5%.
Quantitation of VV-specific primary CTL
To determine CTLp frequency by limiting dilution (24), three
BALB/c mice per group were infected with 5 x 107 pfu
of vHCV-S or vHCV-NS (as a control). At 5 days postinfection, spleen
cells from infected mice were pooled and diluted to 5 x
104 to 4 x 106 cells/well. Diluted
splenocytes were cultured with VV-infected syngeneic splenocytes as
stimulators (105 cells/well). Cells in individual wells
were harvested after 5 days, and CTL activity was measured on
VV-infected P815 target cells. P815 (H-2d) target cells
were infected for 1 h at 37°C with the wild-type VV and were
labeled with 150 µCi of 51Cr for 2 h at 37°C.
After washing to remove free label, target cells were incubated with
VV-specific CTL effectors for 6 h at 37°C, as described
previously (23, 25). Calculations using the Taswell method generate a
frequency estimate (1/f), a corresponding 95% confidence interval, and
the 
2 estimate of probability (p) for
the frequency estimate. In these studies, p > 0.05
indicates that the frequency estimate is statistically acceptable.
Cytokine analysis of bulk cultures
Primary mixed lymphocyte cultures were prepared using spleens
harvested on day 5 from mice infected with vHCV or vSC11 VV
recombinants as described above. Splenocytes were restimulated with
irradiated VV-infected splenocytes. Responder cells were cultured at
4 x 106 cells/ml with stimulators at a 5:1
responder:stimulator ratio in Iscove’s DMEM containing 10 µg/ml
penicillin/streptomycin, 10% heat-inactivated FCS, 2 mM
L-glutamine, and 0.05 mM 2-ME. Cultures were incubated at
37°C, and a portion of each culture (culture supernatant and cell
pellet) was harvested after 24, 48, 72, and 96 h of stimulation.
Culture supernatants were assayed for cytokine levels by ELISA (26, 27). A standard ELISA protocol (PharMingen, San Diego, CA) was used to
measure the quantities of IL-2 and IFN- in culture supernatants at
each time point. For the standard curve to determine the amount of
produced cytokines, IL-2 and IFN- purchased from PharMingen were
serially diluted and used for ELISA assay.
Assay for target cell recognition by VV-specific CTL
VV-specific CTL were generated by a standard method. Two BALB/c mice (6–8 wk old) were infected with 107 pfu of recombinant virus (vSC11) encoding a ß-galactosidase gene. Spleens were harvested from vSC11-primed mice and were stimulated in vitro with vSC11-infected and irradiated (2000 rad) naive splenocytes. The level of target cell lysis was determined by a standard 51Cr release assay using P815 (H-2d) as target cells. P815 (H-2d) cells were infected with 10 multiplicity of infection of vHCV-NS, vHCV-S, or vHCV-C or were mock infected and were labeled with 51Cr. CTL assay was performed as previously described (23). The spontaneous release was <10%. The lysis of 51Cr-labeled VV-infected target cell by VV-specific CTL was inhibited by unlabeled VV-infected target cells (data not shown), suggesting that the VV-specific CTL activity on P815 targets is mediated by class I MHC-restricted CD8+ T cells.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To evaluate the possible effect of expression of HCV gene products
on the virulence of and the host response to VV in the mouse, we
infected BALB/c (H-2d) mice i.p. with 108 pfu
of a recombinant VV (rVV) expressing either the 5' half (vHCV-S) or the
3' half of the HCV genome (vHCV-NS). The vHCV-S virus expresses the HCV
structural proteins, i.e., C, E1, E2, and p7, as well as the
nonstructural protein, NS2, and a portion of the NS3 protein, while the
vHCV-NS expresses the nonstructural proteins of HCV exclusively (Fig. 1
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Mice infected with vHCV-S became clinically moribund by days 1–2
postinfection, and 90% of these mice died by 5–7 days after infection
(Fig. 2A). By contrast, mice
infected with 108 pfu of the HCV-NS virus, which expresses
the HCV nonstructural protein, survived, showed no clinical signs of
infection or mortality, and recovered from infection as efficiently as
mice infected with the control vSC11 virus (Fig. 2A). This
latter virus is a rVV expressing the pSC11 recombination vector that is
used for foreign gene insertion into the VV genome (see Materials
and Methods). These findings suggested that expression of one or
more HCV structural proteins dramatically increased the virulence of
the normally avirulent VV.
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B). In addition, mice receiving
smaller viral inocula, i.e., 107 or 106 pfu,
recovered from infection (Fig. 2B) and showed neither
morbidity nor mortality up to 21 days postinfection (data not shown). To determine the pathological changes associated with lethal infection of vHCV-S-inoculated mice, recipients of 108 pfu of vHCV-S virus were sacrificed on days 4–5 postinfection, and various tissues were harvested for histologic studies. We found histologic evidence of both focal and confluent necrosis in the spleens, livers, and ovaries of these mice (data not shown). These findings suggest progressive lytic infection by the vHCV-S virus in these primary target organs of VV replication.
HCV structural gene expression is associated with sustained VV replication in vivo
In view of the necropsy findings described above, we next
determined if the HCV structural proteins expressed by the vHCV-S virus
were associated with sustained virus replication and elevated virus
titers in vivo. Three mice per group were infected with 108
pfu of vHCV-S, vHCV-NS, or the control recombinant vSC11 virus.
Recipient livers were harvested daily over 5 days, and virus titers in
the infected livers were determined. As shown in Table I, after an initial burst of virus
replication on day 1 postinfection, recipients of the control vSC11
virus and the vHCV-NS virus expressing the HCV nonstructural proteins
rapidly cleared virus from their livers, and liver viral titers from
these mice were below detectable levels by day 4 of infection. This
finding is consistent with the uniform survival of mice infected with
these viruses and suggests a vigorous host response to infection with
these rVV.
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). Suppression of the VV-specific CTL response by HCV protein(s)
The finding that expression of the HCV structural proteins by rVV resulted in the lack of viral clearance suggested that expression of one or more HCV structural proteins drastically inhibited the host response to viral infection. Since virus-specific CTL have been demonstrated to play a critical role in virus clearance and recovery from experimental viral infections, including VV infection (16, 17), it was of interest to determine whether the CTL response to primary VV infection was suppressed in mice infected with vHCV-S virus.
When mice were infected with 5 x 107 pfu of the
vHCV-NS virus, their immune splenocytes, harvested on day 5
postinfection, exhibited significant cytolytic activity on VV-infected
targets in a standard in vitro cytotoxicity assay (Fig. 3A). The magnitude of the in
vivo primary VV-specific CTL response in vHCV-NS infected mice on day 5
postinfection was comparable to that in mice infected with the control
vSC11 virus (data not shown). As expected, infectious virus was also
not detectable in the livers of vHCV-NS-infected mice on day 5
postinfection (Fig. 3B).
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A). A comparable inhibition of in vivo primary CTL activity
was also observed on day 7 postinfection in vHCV-S-infected recipients
that survived to day 7 (data not shown). Commensurate with the low
levels of VV-specific CTL activity in the spleens of vHCV-S-infected
recipients, the livers of vHCV-S-infected mice had high levels of
infectious virus on day 5 postinfection (Fig. 3B). Liver
virus titers were at least 1000-fold greater than those of the
vHCV-NS-infected recipients on day 5 postinfection. These results
suggest that the enhanced virulence of the vHCV-S virus may be due in
part to the inhibition of a VV-specific CTL response in infected
recipients through the action of one or more HCV structural proteins
expressed in vHCV-S-infected cells.
To more precisely determine the degree of suppression of VV-specific
CTL activity induced by the vHCV-S virus, we determined the frequency
of CTLp in spleens of mice on day 5 postinfection with vHCV-S or
vHCV-NS using limiting dilution analysis according to the methods of
Taswell (4). We found that mice infected with the rVV expressing the
HCV structural proteins had approximately a 10-fold lower frequency of
CTLp on day 5 postinfection than recipients infected with virus
expressing the nonstructural proteins (Fig. 4). The frequency of CTLp was 1.6
CTLp/106 immune splenocytes for the virulent vHCV-S virus
and 12.5 CTLp/106 immune splenocytes for the avirulent
vHCV-NS virus. This result is in keeping with the above findings on the
magnitude of the in vivo primary CTL response to these two viruses.
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production by
immune splenocytes from vHCV-S-infected mice in response to antigenic
stimulation in vitro. Again, this finding suggests that one or more HCV
gene products can profoundly alter the host response to VV infection. Identification of HCV core protein as the gene product involved in immune modulation
Since the HCV-S virus expresses four putative HCV structural
proteins, C, E1, E2, and p7, it was important to determine whether the
immune suppression induced by infection with the vHCV-S virus was
mediated by a specific HCV structural gene product. To approach this
question, we constructed a panel of rVV expressing progressively larger
deletions in the cDNA encoding the HCV structural proteins (see Fig. 1). To evaluate the impact of the incremental loss of HCV structural
genes on the host response to infection, we evaluated the in vivo
primary VV-specific CTL responses to each of these deletion mutants, to
vHCV-S virus, and to the vHCV-NS virus on day 5 after infection. As
shown in Fig. 5A, the
immunosuppressive activity exhibited by the vHCV-S virus expressing the
full complement of HCV structural proteins was also demonstrable in
mice infected with a rVV expressing HCV residues 1–192, which encodes
the full length of the HCV core protein exclusively. This result
strongly suggests that the HCV core protein is the viral gene product
that suppresses the host response to infection and enhances the
virulence of VV. In agreement with this cytotoxicity data, we also
found that mice infected with the rVV expressing only the HCV core
protein (vHCV-C) had elevated virus titers in their livers on day 5 of
infection (Fig. 5B) and succumbed to lethal infection (data
not shown). Virus titers in vHCV-C-infected mice were comparable to
those in mice infected with the vHCV-S virus expressing the full
complement of HCV structural proteins.
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As noted above, along with the assessment of in vivo primary CTL
responses to infection, we also compared the in vitro production of
IFN- and IL-2 in response to wild-type vaccinia-infected APCs of
immune splenocytes taken on day 5 after infection with the vHCV-NS
virus or the vHCV-S and vHCV-C viruses. IFN- production by immune
splenocytes from mice infected with the core-expressing vHCV-C virus
was profoundly suppressed (Fig. 6). The
degree of inhibition of IFN- production in vitro was comparable to
that observed in splenocytes of mice infected with the vHCV-S virus
expressing the full complement of HCV structural proteins. Infection
with these core-expressing rVV also resulted in a modest, but
significant, inhibition of Ag-stimulated IL-2 production by immune
splenocytes from vHCV-C- and vHCV-S-infected mice. Immune cells from
mice infected with the vHCV-NS virus produced high levels of both
IFN- and IL-2 (Fig. 6).
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production as
well as the enhanced virulence observed in mice infected with the
vHCV-S virus. Since activated CD8+ T lymphocytes effectors
have been reported to produce high levels of IFN- in response to Ag
stimulation (28, 29, 30), inhibition of the induction of virus-specific CTL
by an HCV core-dependent suppressive mechanism would lead to decreased
numbers of CTL effectors and diminished in vitro IFN- production in
response to viral antigenic stimulation.
One potential mechanism by which HCV core could inhibit CTL induction
is that HCV core may interfere with viral Ag presentation at a step
along the MHC class I processing pathway in cells expressing core
protein. To determine the impact of HCV core on viral Ag presentation
by MHC class I molecules, we examined the capacity of VV-specific
CD8+ CTL generated in response to infection with wild-type
VV to lyse target cells displaying VV peptides after infection with the
core-expressing vHCV-S, vHCV-C virus, or with the core-negative vHCV-NS
virus. The VV-specific CTL do not lyse uninfected P815 target cells,
and the effects of VV-specific CTL are MHC restricted. As shown in Fig. 7, core protein expression had no
significant effect on the recognition of VV epitopes by VV-specific
CTL. This result suggests that core protein does not disrupt Ag
processing and/or presentation of viral Ag to CD8+ T
lymphocytes as shown by adenovirus E3/19K protein (8). Therefore, the
inhibitory effect of core on the host response to viral infection is
unlikely to be at the level of CD8+ T lymphocyte
recognition of virally infected APCs.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in vitro in
response to VV Ags than immunocytes from animals infected with a rVV
expressing vHCV-NS. Cells from these animals also exhibited a modest,
but significant, decrease in in vitro production of IL-2. Using a
series of rVV expressing various deletions in the gene complex encoding
the HCV structural proteins, we identified the HCV core protein as the
structural gene product that both accounted for the suppression of the
host response to infection and enhanced VV virulence. Viruses have evolved a variety of mechanisms to ensure their replication and survival and to circumvent the host immune response (31). One of the best characterized mechanisms is the inhibition of CD8+ CTL response to the virus by the ability of viral gene products to inhibit the processing of viral proteins and/or the presentation of viral peptide/MHC complexes on the surface of virus-infected target cells (32, 33, 34). Indeed, as noted above, expression of the HCV core protein does suppress CTL responses in this model system. However, we could not detect any effect of core protein on the presentation and recognition of processed viral Ag by activated virus-specific CD8+ CTL effectors. Rather, our data from the analysis of the cytolytic activity of primary CD8+ cytolytic effectors taken directly from infected animals as well as from limiting dilution analysis of the frequency of activated CD8+ T lymphocytes giving rise to cytolytic effectors argue for an effect of core protein primarily at the level of CD8+ CTL induction. Therefore, it should be emphasized that one or more HCV gene product(s) may modulate the host response during natural HCV infection for the inhibition of viral Ag processing and presentation by MHC class I molecules. In our murine model, however, we have no evidence that the core protein enhances virulence and suppresses the host response by this mechanism.
Available evidence suggests that core protein has several functions in
HCV replication, including viral RNA encapsidation into viral
nucleocapsid (35, 36). Because of the localization of the core gene at
the 5' end of the HCV polyprotein transcript, the core protein is
likely to be the first viral gene product produced in the
virus-infected cells. If, as our data suggest, core protein expression
may profoundly suppress the host response to virus infection, the
effect of core on the host immune response would be evident in an
earlier phase of virus infection before primary virus-specific
CD8+ T lymphocyte precursors give rise to CTL effectors. In
this connection, it is noteworthy that mice infected with vHCV-C
already demonstrate a 10-fold higher titer of virus in the liver as
early as day 1 postinfection compared with mice comparably infected
with control vaccinia (vSC11) or vHCV-NS (Table I). This finding
re-enforces the view that HCV core protein acts to suppress the host
immune response at an early point in the process of viral infection.
Multiple mechanisms could account for the increase in viral titers
observed within 24 h of infection. Both type 1 IFNs, 
and ß,
have been shown to have important and nonredundant roles in the control
of VV infection (37). NK cells are also significant to the early
control of viral infections, and are greatly stimulated by IL-12 or
IFN- and -ß. IL-12 produced by macrophages or NK cells synergize
with TNF- to stimulate the production of IFN- by NK cells. IL-12
also appears to be critical for the development of Th1 responses, which
can, in turn, augment CTL responses. Therefore, a disruption in the
expression or function of any of these cytokines could explain not only
the increased early viral titers observed, but also the deficiencies in
IL-2, IFN-, and CTL as shown here. Precedent for such a mechanism
has been demonstrated with the inhibition of IL-12 synthesis by measles
virus (38).
The induction of an effective CD8+ CTL response and the
production of IFN- during infection have been implicated as
important factors in resistance to as well as recovery from VV
infection in the mouse (16, 17). Available evidence suggests that an
effective CD8+ CTL response to virus infection can be
mounted in the absence of either IFN- production or a functional
IFN-R (27). Therefore, in the murine model of HCV core-mediated
immune suppression described here, it is unlikely that core protein
acts by inhibiting IFN- production early in infection and thereby
suppresses an IFN--dependent step in CD8+ T lymphocyte
differentiation into activated effectors. Since CD8+ CTL
effectors produce high levels of IFN- in response to Ag (28, 29, 30), it
is more likely that the diminished IFN- production by immune
splenocytes after infection with core expressing rVV reflects a core
protein-dependent inhibition of activation of virus-specific
CD8+ T cells into IFN--producing CTL effectors. One
intriguing mechanism that could account for both the enhanced
replication of core expressing VV early in infection and the
suppression of the virus-specific CTL response reported here is
suggested by the recent report of binding of HCV core protein to the
cytoplasmic tail of the human lymphotoxin-ß receptor (14, 15).
If HCV core can directly interact with the intracellular signaling
domains of one or more members of the murine TNF receptor family in
infected cells and inhibit the function of these receptors, then this
inhibitory effect on TNF receptor signaling might account for the
suppression of both the early innate response to virus infection and
the induction of a specific CTL response observed here.
Although we observed a profound effect of HCV core expression on
IFN- production by immune splenocytes, the effect of core on IL-2
production was less dramatic. One possible explanation for this result
is suggested by the recent finding by Dr. Charles Rice’s laboratory
(unpublished observation) that HCV core can up-regulate transcription
of the IL-2 promoter. It is not yet clear whether this activity
reflects another strategy employed by HCV to dysregulate the host
response, but it does point out the potential for this viral gene
product to alter multiple steps in the host response to infection with
HCV.
In the studies reported here, enhanced virulence of VV was used as a functional readout of HCV core protein-dependent suppression of the host response. Although rVV have been used by other investigators to evaluate the effect of expression of foreign genes on the immune response to VV infection, it was of concern that the immunosuppressive effect of core observed by us in this model system may reflect a suppressive effect dependent upon an interaction between HCV core and one or more VV gene products unique to this class of DNA viruses. Although this possibility cannot as yet be formally excluded, it appears unlikely, since we have been able to demonstrate that HCV core expression enhances the replication and persistence of Sindbis virus (a member of the positive strand RNA alphavirus) in vivo in mice when the core gene is expressed in a recombinant Sindbis virus (Y. S. Hahn, unpublished observation). This result adds support to our view that core protein may likewise have an immunomodulatory effect during HCV infection in humans.
One of the unique features of human HCV infection is the high incidence of development of persistent HCV infection (3). This fact strongly suggests that HCV has evolved one or more strategies to suppress the host response during acute infection and thereby facilitate the development of viral persistence. The results reported here provide compelling evidence that the expression of the HCV core protein may play a critical role in the establishment and possibly the maintenance of persistence during HCV infection in humans.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Young S. Hahn, Beirne Carter Center for Immunology Research, Box MR4–4012, University of Virginia, Charlottesville, VA 22908. E-mail address:
3 Abbreviations used in this paper: HCV, hepatitis C virus; CTLp, precursor of CTL; C, core; NS, nonstructural; VV, vaccinia virus; pfu, plaque-forming units.
Received for publication May 4, 1998. Accepted for publication September 29, 1998.
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 W. Li, J. Li, D. L. J. Tyrrell, and B. Agrawal Expression of hepatitis C virus-derived core or NS3 antigens in human dendritic cells leads to induction of pro-inflammatory cytokines and normal T-cell stimulation capabilities J. Gen. Virol., January 1, 2006; 87(1): 61 - 72. [Abstract] [Full Text] [PDF]
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Z. Q. Yao, S. N. Waggoner, M. W. Cruise, C. Hall, X. Xie, D. W. Oldach, and Y. S. Hahn SOCS1 and SOCS3 Are Targeted by Hepatitis C Virus Core/gC1qR Ligation To Inhibit T-Cell Function J. Virol., December 15, 2005; 79(24): 15417 - 15429. [Abstract] [Full Text] [PDF]
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S. N. Waggoner, M. W. Cruise, R. Kassel, and Y. S. Hahn gC1q Receptor Ligation Selectively Down-Regulates Human IL-12 Production through Activation of the Phosphoinositide 3-Kinase Pathway J. Immunol., October 1, 2005; 175(7): 4706 - 4714. [Abstract] [Full Text] [PDF]
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M. W. Cruise, H. M. Melief, J. Lukens, C. Soguero, and Y. S. Hahn Increased Fas ligand expression of CD4+ T cells by HCV core induces T cell-dependent hepatic inflammation J. Leukoc. Biol., August 1, 2005; 78(2): 412 - 425. [Abstract] [Full Text] [PDF]
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M. Joo, Y. S. Hahn, M. Kwon, R. T. Sadikot, T. S. Blackwell, and J. W. Christman Hepatitis C Virus Core Protein Suppresses NF-{kappa}B Activation and Cyclooxygenase-2 Expression by Direct Interaction with I{kappa}B Kinase {beta} J. Virol., June 15, 2005; 79(12): 7648 - 7657. [Abstract] [Full Text] [PDF]
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 S. Boni, J.-P. Lavergne, S. Boulant, and A. Cahour Hepatitis C Virus Core Protein Acts as a trans-Modulating Factor on Internal Translation Initiation of the Viral RNA J. Biol. Chem., May 6, 2005; 280(18): 17737 - 17748. [Abstract] [Full Text] [PDF]
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S. Sundstrom, S. Ota, L. Y. Dimberg, M. G. Masucci, and A. Bergqvist Hepatitis C Virus Core Protein Induces an Anergic State Characterized by Decreased Interleukin-2 Production and Perturbation of Mitogen-Activated Protein Kinase Responses J. Virol., February 15, 2005; 79(4): 2230 - 2239. [Abstract] [Full Text] [PDF]
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Y. Li, T. Zhang, C. Ho, J. S. Orange, S. D. Douglas, and W.-Z. Ho Natural killer cells inhibit hepatitis C virus expression J. Leukoc. Biol., December 1, 2004; 76(6): 1171 - 1179. [Abstract] [Full Text] [PDF]
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A. L. Eisen-Vandervelde, S. N. Waggoner, Z. Q. Yao, E. M. Cale, C. S. Hahn, and Y. S. Hahn Hepatitis C Virus Core Selectively Suppresses Interleukin-12 Synthesis in Human Macrophages by Interfering with AP-1 Activation J. Biol. Chem., October 15, 2004; 279(42): 43479 - 43486. [Abstract] [Full Text] [PDF]
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Z. Q. Yao, A. Eisen-Vandervelde, S. N. Waggoner, E. M. Cale, and Y. S. Hahn Direct Binding of Hepatitis C Virus Core to gC1qR on CD4+ and CD8+ T Cells Leads to Impaired Activation of Lck and Akt J. Virol., June 15, 2004; 78(12): 6409 - 6419. [Abstract] [Full Text] [PDF]
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P. Maillard, J.-P. Lavergne, S. Siberil, G. Faure, F. Roohvand, S. Petres, J. L. Teillaud, and A. Budkowska Fc{gamma} Receptor-like Activity of Hepatitis C Virus Core Protein J. Biol. Chem., January 23, 2004; 279(4): 2430 - 2437. [Abstract] [Full Text] [PDF]
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J.-W. Youn, S.-H. Park, J. H. Cho, and Y. C. Sung Optimal Induction of T-Cell Responses against Hepatitis C Virus E2 by Antigen Engineering in DNA Immunization J. Virol., November 1, 2003; 77(21): 11596 - 11602. [Abstract] [Full Text] [PDF]
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P. Sarobe, J. J. Lasarte, A. Zabaleta, L. Arribillaga, A. Arina, I. Melero, F. Borras-Cuesta, and J. Prieto Hepatitis C Virus Structural Proteins Impair Dendritic Cell Maturation and Inhibit In Vivo Induction of Cellular Immune Responses J. Virol., October 15, 2003; 77(20): 10862 - 10871. [Abstract] [Full Text] [PDF]
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K. Herzer, C. S. Falk, J. Encke, S. T. Eichhorst, A. Ulsenheimer, B. Seliger, and P. H. Krammer Upregulation of Major Histocompatibility Complex Class I on Liver Cells by Hepatitis C Virus Core Protein via p53 and TAP1 Impairs Natural Killer Cell Cytotoxicity J. Virol., August 1, 2003; 77(15): 8299 - 8309. [Abstract] [Full Text] [PDF]
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A. Bergqvist, S. Sundstrom, L. Y. Dimberg, E. Gylfe, and M. G. Masucci The Hepatitis C Virus Core Protein Modulates T Cell Responses by Inducing Spontaneous and Altering T-cell Receptor-triggered Ca2+ Oscillations J. Biol. Chem., May 23, 2003; 278(21): 18877 - 18883. [Abstract] [Full Text] [PDF]
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Q. Ning, S. Lakatoo, M. Liu, W. Yang, Z. Wang, M. J. Phillips, and G. A. Levy Induction of Prothrombinase fgl2 by the Nucleocapsid Protein of Virulent Mouse Hepatitis Virus Is Dependent on Host Hepatic Nuclear Factor-4alpha J. Biol. Chem., April 25, 2003; 278(18): 15541 - 15549. [Abstract] [Full Text] [PDF]
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J. Ni, E. Hembrador, A. M. Di Bisceglie, I. M. Jacobson, A. H. Talal, D. Butera, C. M. Rice, T. J. Chambers, and L. B. Dustin Accumulation of B Lymphocytes with a Naive, Resting Phenotype in a Subset of Hepatitis C Patients J. Immunol., March 15, 2003; 170(6): 3429 - 3439. [Abstract] [Full Text] [PDF]
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H. Wedemeyer, X.-S. He, M. Nascimbeni, A. R. Davis, H. B. Greenberg, J. H. Hoofnagle, T. J. Liang, H. Alter, and B. Rehermann Impaired Effector Function of Hepatitis C Virus-Specific CD8+ T Cells in Chronic Hepatitis C Virus Infection J. Immunol., September 15, 2002; 169(6): 3447 - 3458. [Abstract] [Full Text] [PDF]
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S. Mahalingam, J. Meanger, P. S. Foster, and B. A. Lidbury The viral manipulation of the host cellular and immune environments to enhance propagation and survival: a focus on RNA viruses J. Leukoc. Biol., September 1, 2002; 72(3): 429 - 439. [Abstract] [Full Text] [PDF]
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C. Soguero, M. Joo, K. A. Chianese-Bullock, D. T. Nguyen, K. Tung, and Y. S. Hahn Hepatitis C Virus Core Protein Leads to Immune Suppression and Liver Damage in a Transgenic Murine Model J. Virol., August 12, 2002; 76(18): 9345 - 9354. [Abstract] [Full Text] [PDF]
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S. Bichr, R. Rende-Fournier, G. Vona, A.-M. Yamamoto, E. Depla, G. Maertens, and C. Brechot Detection of neutralizing antibodies to hepatitis C virus using a biliary cell infection model J. Gen. Virol., June 1, 2002; 83(7): 1673 - 1678. [Abstract] [Full Text] [PDF]
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A. Varaklioti, N. Vassilaki, U. Georgopoulou, and P. Mavromara Alternate Translation Occurs within the Core Coding Region of the Hepatitis C Viral Genome J. Biol. Chem., May 10, 2002; 277(20): 17713 - 17721. [Abstract] [Full Text] [PDF]
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P. Sarobe, J. J. Lasarte, N. Casares, A. Lopez-Diaz de Cerio, E. Baixeras, P. Labarga, N. Garcia, F. Borras-Cuesta, and J. Prieto Abnormal Priming of CD4+ T Cells by Dendritic Cells Expressing Hepatitis C Virus Core and E1 Proteins J. Virol., April 16, 2002; 76(10): 5062 - 5070. [Abstract] [Full Text] [PDF]
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Z.-X. Liu, H. Nishida, J.-W. He, M. M. C. Lai, N. Feng, and G. Dennert Hepatitis C Virus Genotype 1b Core Protein Does Not Exert Immunomodulatory Effects on Virus-Induced Cellular Immunity J. Virol., February 1, 2002; 76(3): 990 - 997. [Abstract] [Full Text] [PDF]
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 G. PERLEMUTER, A. SABILE, P. LETTERON, G. VONA, A. TOPILCO, Y. CHRETIEN, K. KOIKE, D. PESSAYRE, J. CHAPMAN, G. BARBA, et al. Hepatitis C virus core protein inhibits microsomal triglyceride transfer protein activity and very low density lipoprotein secretion: a model of viral-related steatosis FASEB J, February 1, 2002; 16(2): 185 - 194. [Abstract] [Full Text] [PDF]
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 C.-T. K. Tseng and G. R. Klimpel Binding of the Hepatitis C Virus Envelope Protein E2 to CD81 Inhibits Natural Killer Cell Functions J. Exp. Med., December 31, 2001; 195(1): 43 - 50. [Abstract] [Full Text] [PDF]
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J. Sun, F. Bodola, X. Fan, H. Irshad, L. Soong, S. M. Lemon, and T.-S. Chan Hepatitis C Virus Core and Envelope Proteins Do Not Suppress the Host's Ability To Clear a Hepatic Viral Infection J. Virol., December 15, 2001; 75(24): 11992 - 11998. [Abstract] [Full Text] [PDF]
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Z. Q. Yao, D. T. Nguyen, A. I. Hiotellis, and Y. S. Hahn Hepatitis C Virus Core Protein Inhibits Human T Lymphocyte Responses by a Complement-Dependent Regulatory Pathway J. Immunol., November 1, 2001; 167(9): 5264 - 5272. [Abstract] [Full Text] [PDF]
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P. Maillard, K. Krawczynski, J. Nitkiewicz, C. Bronnert, M. Sidorkiewicz, P. Gounon, J. Dubuisson, G. Faure, R. Crainic, and A. Budkowska Nonenveloped Nucleocapsids of Hepatitis C Virus in the Serum of Infected Patients J. Virol., September 1, 2001; 75(17): 8240 - 8250. [Abstract] [Full Text] [PDF]
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M. Schweizer and E. Peterhans Noncytopathic Bovine Viral Diarrhea Virus Inhibits Double-Stranded RNA-Induced Apoptosis and Interferon Synthesis J. Virol., May 15, 2001; 75(10): 4692 - 4698. [Abstract] [Full Text]
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A. Bergqvist and C. M. Rice Transcriptional Activation of the Interleukin-2 Promoter by Hepatitis C Virus Core Protein J. Virol., January 15, 2001; 75(2): 772 - 781. [Abstract] [Full Text]
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T.-H. Wang, R. C. A. Rijnbrand, and S. M. Lemon Core Protein-Coding Sequence, but Not Core Protein, Modulates the Efficiency of Cap-Independent Translation Directed by the Internal Ribosome Entry Site of Hepatitis C Virus J. Virol., December 1, 2000; 74(23): 11347 - 11358. [Abstract] [Full Text]
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 J. Matsumoto, S. Kawai, K. Terao, M. Kirinoki, Y. Yasutomi, M. Aikawa, and H. Matsuda Malaria Infection Induces Rapid Elevation of the Soluble Fas Ligand Level in Serum and Subsequent T Lymphocytopenia: Possible Factors Responsible for the Differences in Susceptibility of Two Species of Macaca Monkeys to Plasmodium coatneyi Infection Infect. Immun., March 1, 2000; 68(3): 1183 - 1188. [Abstract] [Full Text] [PDF]
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 T. J. Liang, B. Rehermann, L. B. Seeff, and J. H. Hoofnagle Pathogenesis, Natural History, Treatment, and Prevention of Hepatitis C Ann Intern Med, February 15, 2000; 132(4): 296 - 305. [Abstract] [Full Text] [PDF]
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 T. Arichi, T. Saito, M. E. Major, I. M. Belyakov, M. Shirai, V. H. Engelhard, S. M. Feinstone, and J. A. Berzofsky Prophylactic DNA vaccine for hepatitis C virus (HCV) infection: HCV-specific cytotoxic T lymphocyte induction and protection from HCV-recombinant vaccinia infection in an HLA-A2.1 transgenic mouse model PNAS, January 4, 2000; 97(1): 297 - 302. [Abstract] [Full Text] [PDF]
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T. Shimoike, S. Mimori, H. Tani, Y. Matsuura, and T. Miyamura Interaction of Hepatitis C Virus Core Protein with Viral Sense RNA and Suppression of Its Translation J. Virol., December 1, 1999; 73(12): 9718 - 9725. [Abstract] [Full Text]
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