JOURNAL OF VIROLOGY,0022-538X/01/$04.00�0 DOI: 10.1128/JVI.75.24.11992–11998.2001

Dec. 2001, p. 11992–11998 Vol. 75, No. 24

Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Hepatitis C Virus Core and Envelope Proteins Do Not Suppressthe Host’s Ability To Clear a Hepatic Viral InfectionJIAREN SUN,* FRANCIS BODOLA, XUEGONG FAN, HABIB IRSHAD, LYNN SOONG,

STANLEY M. LEMON, AND TEH-SHENG CHAN

Department of Microbiology and Immunology, University of Texas Medical Branch at Galveston,Galveston, Texas 77555-1070

Received 5 June 2001/Accepted 14 September 2001

Several hepatitis C virus (HCV) proteins have been shown in vitro to interact with host cellular componentsthat are involved in immune regulation. However, there is a paucity of data supporting the relevance of theseobservations to the in vivo situation. To test the hypothesis that such an interaction suppresses immuneresponses, we studied a line of transgenic C57BL/6 mice that express the HCV core and envelope proteins inthe liver. The potential effects of these proteins on the hepatic immune response were evaluated by challengingthese mice with a hepatotropic adenovirus. Both transgenic and nontransgenic mice developed similar coursesof infection and cleared the virus from the liver by 28 days postinfection. Both groups of mice mounted similarimmunoglobulin G (IgG), IgG2a, interleukin-2, and tumor necrosis factor alpha responses against the virus.Additionally, BALB/c mice were able to clear infection with recombinant adenovirus that does or does notexpress the HCV core and envelope 1 proteins in the same manner. These data suggest that HCV core andenvelope proteins do not inhibit the hepatic antiviral mechanisms in these murine experimental systems andthus favor a model in which HCV circumvents host responses through a mechanism that does not involvegeneral suppression of intrahepatic immune responses.

Hepatitis C virus (HCV), a major cause of chronic liverdisease worldwide, currently infects about 1.8% of the U.S.population (1). Most HCV-infected patients become chroni-cally infected, and a substantial proportion of such patientsdevelop clinical manifestations of liver disease. Although thenatural history of the chronic infection is not well understood,the high frequency with which infection progresses to chronic-ity suggests that HCV has evolved specific mechanisms to cir-cumvent the host’s immune responses.

A number of distinct models have been proposed to explainthe phenomenon of persistent HCV infection. Several studieshave described the emergence of viral variants that could es-cape from the immune system. During the course of a chronicinfection, antibodies (Abs) recognizing hypervariable region 1of the viral E2 envelope protein undergo changes in theirepitope specificities. In addition, cytotoxic T lymphocytes(CTL) from a chronically infected chimpanzee failed to recog-nize the HCV quasispecies present at 4 months postinfection(3, 25). Collectively, these studies suggest that, despite activeimmune responses, a viral population may emerge with theselection of variants that possess an enhanced ability to persistin the host.

On the other hand, the establishment of a persistent HCVinfection may not always be due to genetic variation of thevirus (15). It has been suggested that HCV, like adenovirus,herpes simplex virus, and many other viruses, may encode oneor more proteins that act to inhibit viral clearance by the host.Indeed, two viral proteins, NS5A and the envelope glycopro-tein E2, have been shown to repress the host’s double-strand-

ed-RNA-inducible protein kinase R (PKR), resulting in a stateof selective interferon (IFN) resistance (7, 21). When ex-pressed in mammalian cells, NS5A confers IFN resistance onvesicular stomatitis virus, which normally is sensitive to theantiviral actions of IFN (6). In addition, the HCV core proteinhas been shown to bind to the cytoplasmic domains of tumornecrosis factor (TNF) receptor 1, lymphotoxin � receptor, andgC1q receptor (4, 13, 17, 28), resulting in interference withFas/TNF-�-induced apoptosis and T-cell proliferation in vitro(8, 16).

Given the roles of these cellular proteins in the normaldevelopment of peripheral lymphoid organs, transduction ofapoptotic signals, and initiation and resolution of inflammation(18, 24), these data raise the possibility that the HCV structuralproteins (core, E1, and E2) have significant immunomodula-tory functions. However, there is a paucity of data either sup-porting or refuting the relevance of these in vitro observationsto the in vivo situation. To distinguish between the relativecontributions of evasive and suppressive factors in HCV per-sistence in vivo, we took advantage of a recently developedHCV transgenic (tg) mouse (14a) and examined the effectsof the HCV structural proteins on intrahepatic immune re-sponses during a viral infection. Our data suggest that the HCVcore and envelope proteins are not inherently immunosuppres-sive in murine experimental systems and thus favor a model inwhich HCV circumvents immune responses through a mecha-nism that does not require profound immune suppression.

MATERIALS AND METHODS

Mice. Generation of HCV tg mice has been described in detail elsewhere(14a). Briefly, cDNA corresponding to the structural protein coding region (core,E1, and E2/p7) of the genotype 1b HCV-N strain of HCV was subcloned intoplasmid pGEMAlbSVPA under the control of the liver-specific murine albuminpromoter-enhancer. The insert was excised by digestion with XhoI and injected

* Corresponding author. Mailing address: Department of Microbi-ology and Immunology, University of Texas Medical Branch, 301 Uni-versity Blvd., Galveston, TX 77555-1019. Phone: (409) 747-0186. Fax:(409) 747-6869. E-mail: jisun@utmb.edu.

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into F1 hybrid zygotes (C3H/HeJ � C57BL/6J). The offspring were screened forthe presence of tg DNA by Southern blot analysis, and a tg F0 founder (S-N/863)was mated with C57BL/6 mice (H-2b; Jackson Laboratories, Bar Harbor, Maine)to produce F1 and subsequent generations of animals. BALB/c mice (H-2d) werepurchased from Jackson Laboratories. All mice were housed in a specific-patho-gen-free facility. The Institutional Animal Care and Use Committee of theUniversity of Texas Medical Branch approved the study.

Viral infection of the liver. Tg and non-tg mice were infected with a replica-tion-impaired (with E1 deleted) adenovirus (Ad�gal) that expresses bacterial�-galactosidase (�-Gal), as previously reported by Herz and Gerard (9). Ten tgmice, 2 to 3 months of age, were each inoculated intravenously with 3 � 109 PFUof Ad�gal. Equal numbers of age-matched non-tg littermates were infected ascontrols. In separate experiments, 20 2-month-old female BALB/c mice weresimilarly infected with 3 � 108 PFU of Ad�gal and a recombinant adenovirusexpressing the core and E1 segments of the HCV polyprotein (AdCE1) (2). At3 to 6, 14, and 28 days postinfection, two to four mice in each group weresacrificed by CO2 asphyxiation. Liver tissues were snap frozen in liquid nitrogenfor DNA isolation or placed in OCT cryostat-embedding compound (Tissue-Tek,Torrance, Calif.) for morphological analyses. Serum samples were stored at�70°C for subsequent enzyme-linked immunosorbent assays (ELISAs) and ala-nine aminotransferase (ALT) measurements.

Immunohistochemistry and Western blotting. After being fixed in acetone andblocked with normal goat serum and avidin-biotin (Avidin/Biotin blocking kit;Vector Laboratories, Burlingame, Calif.), cryosections were incubated at 4°Covernight with a rabbit anti-peptide antibody specific for the E2 protein ofHCV-N (1:500). The sections were incubated with biotinylated goat anti-rabbitimmunoglobulin G (IgG) followed by avidin-conjugated peroxidase (VectastainElite ABC kit) and developed in 3,3�-diaminobenzidine tetrahydrochloride. De-tection of lymphocytes in tissue samples was carried out as described previously(20). For immunoblotting, 2 �g of protein extracted from the livers of miceinfected with AdCE1 and Ad�gal or from a stably transfected cell line, Huh7/191-20, that expresses HCV core, E1, and E2 proteins were electrophoresed ina sodium dodecyl sulfate–12% polyacrylamide gel (unpublished data). The pro-teins were electrotransferred to a polyvinylidene difluoride membrane (Bio-Rad,Hercules, Calif.) and probed with a mixture of monoclonal Abs (MAbs) specificfor HCV core (Anogen, Mississauga, Canada), �-actin (Sigma, St. Louis, Mo.),and adenovirus hexon protein (Chemicon, Temecula, Calif.), followed by ahorseradish peroxidase-conjugated anti-mouse IgG (Southern BiotechnologyAssociates, Birmingham, Ala.) and enhanced chemiluminescence detection re-agents (Amersham Pharmacia Biotech, Little Chalfont, England).

Detection of Ad�gal in liver cryosections. Following a brief fixation with 0.5%glutaraldehyde, frozen liver sections were incubated with 0.2 mg of X-Gal (5-bromo-4-chloro-3-indolyl-�-D-galactopyranoside)/ml at 37°C for 60 to 120 min.Infected cells expressing �-Gal activity were stained blue, whereas uninfectedcells were counterstained red (neutral red). Five images from two liver sectionswere randomly selected and captured with a Zeiss Axioskop microscopeequipped with a Sony DXC-970MD video camera (10� objectives). Infected anduninfected regions were quantified using the MetaView Imaging System 4.0software (Universal Imaging System, West Chester, Pa.). Thresholds were se-lected with colors representative of the desired color range of the infected oruninfected cells. Infectivity of the hepatocytes was expressed as the average ratioof infected to uninfected cells.

Determination of hepatic viral load. The Ad�gal copy numbers in the liverwere assessed by real-time quantitative PCR. Total DNA was extracted from 50mg of liver tissue from each mouse by using a QIAamp DNA Mini Kit (QiagenInc., Valencia, Calif.). Analysis was carried out on a Roche LightCycler with itscore reagent kit (DNA Master SYBR Green 1). The PCR primers correspondingto the adenovirus hexon gene were 5�-GAGCCAGCATTAAGTTTGATAGCA-3� and 5�-AGATAGTCGTTAAAGGACTGGTCGTT-3�. The extractedDNA samples were diluted 1:100 with Tris-EDTA buffer, and 5-�l aliquots wereplaced in glass capillary cuvettes (Roche) containing 15 �l of PCR master mix.Reaction cycles included denaturation at 94°C for 30 s, annealing at 60°C for 5 s,and extension at 72°C for 10 s. The reaction produced a 126-bp product. Theobserved threshold cycle for a no-template control was typically around 35 cycles.The standard curve of the threshold cycles versus the template abundance waslinear over 5 orders of magnitude, with a regression correlation typically greaterthan 0.98. All data were combined from two independent experiments.

Measurements of serum IgG and cytokines. Serum samples were analyzed foradenovirus-specific IgG by ELISA with a procedure modified from a previousreport by Chirmule et al. (5). Briefly, 96-well Immunolon-IV microtiter plates(Dynatech, Chantilly, Va.) were coated with Ad�gal (108 particles/ml). Serumdilutions (1:50) were added to duplicate wells, and the plates were incubated at4°C overnight. The plates were incubated with biotinylated rabbit anti-mouse

IgG or IgG2a, followed by avidin-conjugated peroxidase (BD PharMingen). Theenzyme activity was determined calorimetrically using O-phenylenediamine di-hydrochloride reagent (Sigma) and read at 492 nm. Interleukin-2 (IL-2) andTNF-� in the serum samples were measured with commercial ELISA kits (BDPharMingen) according to the manufacturer’s recommendations. The pairedMAbs specific for IL-2 and TNF-� were JES6-1A12/JES6-5H4 and G281-2626/MP6-XT3, respectively. Serum samples (1:3) were incubated in the plates at 4°Covernight, followed by incubation with a biotinylated anti-IL-2 or anti-TNF-�MAb. Avidin-conjugated peroxidase was added to the plates, and the enzymeactivity was determined calorimetrically as described above.

TUNEL assay for detection of apoptosis in situ. The terminal deoxynucleoti-dyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay wascarried out on deparaffinized, formalin-fixed liver sections treated with protein-ase K (20 mg/ml). After endogenous peroxidase was blocked by immersing thesections in 3% H2O2 for 30 min, slides were incubated with a buffer solutioncontaining terminal deoxynucleotide transferase and digoxigenin-labeled de-oxynucleotide triphosphates (ApopTag Peroxidase; Intergen Company, Pur-chase, N.Y.) in a humidified chamber at 37°C for 1 h. An antidigoxigenin-peroxidase conjugate was added for 30 min, followed by 3,3�-diaminobenzidinetetrahydrochloride as a chromogen. The slides were scored blindly with respectto the tg status of the animal. One section from each mouse was examined ineach of two independent assays. The total number of apoptotic cells in 10randomly selected microscopic fields (40�) was scored in each section.

Statistical analysis. All statistical evaluations were conducted using analysis ofvariance for two factors, independently and interactively. Tabulations were fa-cilitated by using Abstat 1.90 software (Anderson-Bell Corp., Arvada, Colo.).

RESULTS

Since the intrahepatic immune system has a number ofunique properties, including lymphocyte populations that aredistinct from those of the spleen and lymph nodes (19), it isimportant to examine the potential immunosuppressive effectsof HCV proteins in the context of the hepatic microenviron-ment. To assess the role of HCV structural proteins expressedat levels similar to those encountered in infected humans, weused a tg mouse line with liver-specific expression of HCVcore, E1, and E2/p7 proteins. Immunocompetent mice arecapable of eliminating recombinant adenovirus primarilythrough a cellular immune response (27, 29). Thus, the pro-longed presence of adenovirus in these tg mice would supportthe view that the HCV structural proteins suppress the im-mune system.

First, we evaluated the expression of the HCV transgene inthe tg mice. The E2 protein was expressed in tg liver tissue witha distribution that was cytoplasmic and particularly prominentin the pericentral regions (Fig. 1A). No E2 antigen was presentin non-tg mice. RNA transcripts of the transgene were detect-able in the livers of tg mice by Northern blot analysis (14a).Next, we infected these mice and their non-tg littermates witha recombinant adenovirus (Ad�gal) that expresses a reporter�-Gal. When 3 � 109 PFU of Ad�gal was injected through thetail vein, the hepatotropism of the virus was evidenced by�-Gal staining of the liver but not the spleen, kidneys, or lungs(data not shown). Three groups of mice in each of two exper-iments were sacrificed during the early (days 3 to 6), interme-diate (day 14), and late (day 28) stages of the infection. Both tgand non-tg mice demonstrated a striking hepatic infection dur-ing the early stage (Fig. 1B). The �-Gal activity was easilydetectable in infected hepatocytes, and the excellent contrastbetween �-Gal and neutral red (counterstain) allowed accu-rate enumeration of infected hepatocytes. The results fromthese experiments showed that tg and non-tg mice followedsimilar courses of infection, with the disappearance of mostAd�gal-infected hepatocytes by 28 days postinfection (Fig. 1B

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and 2A). To provide an independent measure of viral clear-ance, we also determined the viral load in the liver tissues by ahighly sensitive real-time quantitative PCR. Consistent withfunctional loss of viral �-Gal activity, there was an average of90 and 94% reduction of the viral genome in tg and non-tgmice, respectively, by 28 days postinfection (Fig. 2B). At about30 viral particles per cell in both groups of animals, these viralloads may draw near the threshold of the lacZ assay (Fig. 2A)and thus prohibit further identification of infected cells beyondthat point. Together, these results demonstrated no impair-ment in the ability of the HCV tg mice to respond to andsuccessfully eliminate an intrahepatic infection with an unre-lated virus.

Since both humoral and cellular immune responses havebeen demonstrated to play critical roles in adenovirus clear-ance and recovery from experimental adenovirus infections(10, 27, 29), we examined whether these responses to the viralinfection were quantitatively affected in tg mice. Despite theexpression of the HCV structural proteins in tg animals, tg andnon-tg mice produced similar quantities of adenovirus-specificIgG, as well as IgG2a, which is generally accepted as an indi-cator of the Th1-mediated immune response (Fig. 3A and B).Immunohistological examinations showed that the two groupsof mice had comparable abilities to recruit CD4 and CD8 Tcells to inflammatory lesions in the liver (Fig. 1C). Also, theserum levels of TNF-� and IL-2 in tg mice were equivalent tothose found in non-tg littermates (Fig. 3C and D), suggestingno apparent compromise in either the humoral or cellulareffector mechanisms.

In immunocompetent mice, adenovirus infection of the liveris accompanied by a transient elevation of serum ALT activity,which is followed by a significant reduction in virus within 3weeks (27). Thus, we determined whether the presence of theHCV structural proteins was associated with exacerbated liverinjury and/or delayed resolution of the biochemical lesion. Thepeak of the chemical liver injury occurred at 14 days postin-fection in both tg and non-tg mice, and ALT levels declinedsignificantly in both groups of animals thereafter (Fig. 4A).The resolution of hepatic inflammation in both groups of micewas also confirmed by measurements of the total liver weight,as well as resolution of acute histopathological abnormalities(data not shown). Since the tg mice demonstrated robustTNF-� production in response to Ad�gal infection (Fig. 3C),we also examined the possibility that expression of the HCVcore protein in the liver could result in an exacerbated TNF-induced apoptosis in the tg mice (4, 8, 17, 28). Using TUNELassays, we found that both tg and non-tg mice had elevatednumbers of apoptotic cells in the liver during the early stagesof infection. However, the number of apoptotic cells declinedin both groups as the infection progressed to the later stages

(Fig. 4B). There were no significant differences between tg andnon-tg animals in these studies.

To determine whether the absence of immunosuppressiondue to HCV structural proteins that we observed in the HCVtg mice could be restricted to the H-2b/k genetic background orthe level of tg expression, we infected BALB/c (H-2d) micewith 3 � 108 PFU of Ad�gal and a recombinant adenovirus(AdCE1) expressing the core and E1 segments of the HCVgenome (2). Such infection resulted in a substantially higherabundance of the intracellular HCV core protein (Fig. 5A).Despite this, we observed a significant reduction of viral load in

FIG. 2. Clearance of intrahepatic Ad�gal infection in mice. (A)Liver sections were stained with X-Gal as described in the legend toFig. 2A. Image analysis was used to assess the extent of Ad�gal infec-tion. The data points represent the average ratio of infected to unin-fected hepatocytes from individual mice. While the percentage ofinfected cells continued to decrease (P � 0.01), the pattern of declinein both groups of mice remained the same (P 0.05). No significantdifference was found between tg (Tg�) and non-tg (Tg�) mice (P 0.05). (B) Ad�gal genome abundance in livers of infected mice asdetermined by real-time quantitative PCR analysis (see Materials andMethods). No difference in viral load, either overall (P 0.05) or atany given date (P 0.05), was found between the two groups of mice.The viral load decreased significantly during the course of infection inboth groups of mice (P � 0.01). In both panels, the data points werederived from two independent experiments.

FIG. 1. Histochemical and immunohistochemical staining of liver samples from HCV tg (Tg�) and non-tg (Tg�) mice. (A) Livers from anon-tg mouse (C57BL/6J) and an HCV tg mouse stained for HCV E2 antigen. Liver sections were stained as described in Materials and Methods.The tg mouse demonstrated cytoplasmic distribution of specific E2 antigen (40� objective). Parallel staining using rabbit antibody specific for anirrelevant antigen (a salivary protein of the sand fly Lutzomyia longipalpis) revealed no positively stained cells in either tg or non-tg mice (data notshown). (B) �-Gal expression in hepatocytes at 3, 14, and 28 days following infection with Ad�gal. Mice were injected with 3 � 109 PFU of Ad�galsuspended in 100 �l of PBS via the tail vein. Liver sections were stained with X-Gal and counterstained with neutral red, staining infectedhepatocytes blue and uninfected hepatocytes red. (C) T-cell recruitment to inflammatory lesions in the livers of Ad�gal-infected mice at 3 dayspostinfection. Sections were incubated at 4°C overnight with a biotinylated MAb specific for mouse CD4 or CD8 (1:500), followed by avidin-conjugated peroxidase. Representative photomicrographs are shown.

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the BALB/c mice infected with AdCE1, which was comparableto that in the mice infected with Ad�gal (Fig. 5B). Both groupsof mice recovered from the infection, as reflected in a decreasein serum ALT activities from early peak values (data notshown). These results lend strong support to the conclusionthat in mice the HCV core protein does not hinder the host’sability to resolve an intrahepatic viral infection. This absenceof immunosuppression is neither mouse strain specific nordependent on the level of HCV core expression.

DISCUSSION

Using a series of vaccinia virus/HCV recombinants, Large etal. have examined the influence of specific HCV gene productson the immune responses to vaccinia virus in a murine model(14). In that study, vaccinia virus recombinants expressing var-ious regions of the HCV polyprotein were inoculated intra-peritoneally into naı̈ve BALB/c mice. The HCV core proteincontributed to a greater pathogenicity of vaccinia virus/HCVrecombinants in BALB/c mice (H-2d), suggesting that it mightcontribute to viral persistence by directly suppressing host im-mune responses, in particular, the generation of virus-specificCTL. In the present study, however, we evaluated the effect ofthe HCV core protein in tg mice that express an abundance ofthe HCV structural proteins that is much closer to that foundin infected human livers. Surprisingly, we found no evidence ofsuppression of intrahepatic viral clearance mechanisms inthese mice (H-2b/k) by the HCV core, E1, or E2 protein. Micefrom a genetically different background (H-2d) also efficientlycleared a recombinant adenovirus that expressed a muchhigher level of the HCV core protein. Mice from both geneticbackgrounds made timely recoveries from the intrahepatic in-fection. The absence of demonstrable immunosuppressioncannot be attributed to the lack of a need for effective immuneresponses to clear a recombinant adenovirus, since persistenceof the virus has been demonstrated in nude mice by Southern

FIG. 3. Production of virus-specific IgG and cytokines following an intrahepatic viral infection. Mice were injected with Ad�gal as for Fig. 2A,and their serum samples were collected at the indicated time points. The error bars represent the standard deviations among individual animals.The relative concentrations of virus-specific IgG (A) and IgG2a (B) are expressed as absorbance, or optical density (O.D.). The figures arerepresentative of two or three experiments with similar results. The levels of circulating TNF-� (C) and IL-2 (D) were determined by ELISA. Tg�,tg mice; Tg�, non-tg mice.

FIG. 4. Recovery from liver injury following Ad�gal infection inHCV tg (Tg�) and non-tg (Tg�) mice. The experiments were carriedout as described in the legend to Fig. 2A. (A) Serum ALT levels areshown in individual animals as a function of time following infection.There was no significant difference in ALT values between tg andnon-tg mice (P 0.05). (B) TUNEL assay for detection of apoptosisin the liver. Shown are the numbers of apoptotic cells present in 10randomly selected fields (magnification, �40) in individual sections.No difference in the number of apoptotic cells, either overall (P 0.05) or at any given time point (P 0.05), was found between twogroups of mice. The number of apoptotic cells decreased significantlyduring the course of infection in both groups of mice (P � 0.01). Theslides were scored blindly for apoptotic cells with respect to the dateand tg status of the animal.

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blot analysis and �-Gal histochemistry for up to 60 days postin-fection (26, 29). In addition, administration of immunosup-pressants, such as FK506, cyclosporine A, dexamethasone, orAb-soluble fusion protein blocking CD40 ligand, has beenshown to significantly prolong the presence of the virus ininfected mouse liver (12, 23, 29). In patients with hepatitis C,HCV RNA is typically detectable only by RT-PCR, and dem-onstration of viral proteins in the liver is often difficult. Thus,the demonstration of HCV mRNA transcripts in the tg micecoupled with evidence of polyprotein expression and steatosis(14a) indicates that the abundance of HCV proteins expressedin tg mice was adequate for the intended purpose of this study.

The difference between our results and those of Large et al.(14) may be in the type of challenge infection. Vaccinia viruscan replicate in the ovaries and spleen in addition to the liverin mice. The greater pathogenicity of the core-expressing vac-

cinia virus was found by Large et al. to be associated withprogressive lytic infection and necrosis in those organs (14). Incontrast, the mice used in our study were challenged with arecombinant adenovirus that is highly hepatotropic when in-oculated through the tail vein. Such an experimental system ismore likely to reflect the situation existing in the HCV-infectedlivers of patients.

Apoptosis has been suggested as a common pathway to virusclearance by host CTL and NK cells. Many virus genomesencode proteins that suppress apoptosis in order to escapeimmune attack by the host (18). Conflicting results have sug-gested that the HCV core protein can either protect trans-fected cells from Fas- and TNF-�-induced apoptotic cell deathor sensitize them to it (8, 16, 28). Our results suggest that HCVcore neither exacerbates nor inhibits apoptosis in vivo in theface of a robust, endogenous TNF-� response. Furthermore, asour tg-mouse experiments have shown, the HCV E2 proteindoes not hamper the host’s ability to mount effective intrahe-patic immune responses, even though it has been suggested tointerfere with and suppress PKR responses (21).

These data clarify previous contradictory reports regardingthe functions of core and E2 proteins, as they are derived froman in vivo system in which the abundances of these proteins aremore reflective of the situation in human patients. While ourdata favor a model in which HCV circumvents the immuneresponses through a mechanism that does not involve generalimmunosuppression, they do not exclude the possibility thatthe virus modulates the complex interplay between the dynam-ics of viral diversity and the early breadth of CTL responses.For instance, viral escape mutants have been shown to give riseto T-cell receptor antagonists capable of inhibiting CTL clonesthat recognize prototypic viral epitopes (3, 11, 22). In addition,the additive or synergistic effects of other HCV proteins notexpressed in these studies (e.g., NS5A) may conceivably con-tribute to viral resistance to the IFN-mediated antiviral mech-anism, leading to persistent infection (7).

ACKNOWLEDGMENTS

We gratefully acknowledge Tony Carroll at Glaxo Wellcome forproviding AdCE1 virus, Kui Li for providing Huh7/191-20 cells, ElbertWhorton for statistical analysis, Shu-Yuan Xiao for evaluating his-topathological sections, Hong Diao for excellent technical assistance,Chiaho Shih for critical reading of the manuscript, and Mardelle Sus-man for assistance with manuscript preparation.

This work was supported by grants from the National Cancer Insti-tute (SBIR-CA88770), the National Institute of Allergy and InfectiousDiseases (U19-AI40035), and the John Sealy Memorial EndowmentFund for Biomedical Research.

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