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Improved culture system for hepatitis C virus infection
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A University of California, San Diego School of Medicine researcher has developed the first tissue culture of normal, human liver cells that can model infection with the Hepatitis C virus (HCV) and provide a realistic environment to evaluate possible treatments. The novel cell line, described in the July 16 issue of PLoS ONE, will allow pharmaceutical companies to effectively test new drug candidates or possible vaccines for the HCV infection, which afflicts about 170 million people worldwide. Currently, there is no animal model that is effective for testing such therapies.
"....Clinical Implications
This study suggests that the human hepatocyte culture system described here will complement the Huh-7 virions system in understanding the HCV life cycle, it effects on the hepatocyte, the natural host cell, as well as the possible development of novel therapeutics and vaccines. The human hepatocyte culture system may facilitate studies of the role of insulin resistance and fatty liver on HCV infection since these conditions can be mimicked in the human hepatocytes. Similarly, the mechanisms by which African Americans are refractory to HCV treatment could possibly be analyzed by infecting human hepatocytes from different ethnic backgrounds and studying their response to treatments..... To our knowledge, this is the first report of a physiologically significant amplification of HCV infection (up to 7 log10 ) with naturally occurring genotypes 1, 2 , 3, and 4 in a normal human hepatocyte culture system, suggesting that research with this physiological system may complement that available with the current replicon systems."
Assistant Professor of Medicine Martina Buck, Ph.D., researcher at UC San Diego's Department of Medicine and Moores UCSD Cancer Center developed the novel culture system, which mimics the biology of HCV infection in humans.
"This is the first efficient and consistent model system for HCV to be developed," said Buck, adding that it will now enable researchers not only to conduct mechanistic experiments in culture, such as blocking the virus pathways, but also to more effectively screen possible therapies for HCV. "There is a need for new treatments, and for development of a possible vaccine for HCV. Now we have a model system to support work by investigators in this area."
Currently, there is only a single treatment for HCV, PEG- interferon--β . The drug combination has an average response rate of about 50 percent in HCV cases, but it is much lower than that, closer to 20 percent, in individuals with liver cirrhosis. It can also cause severe flu-like side effects. Approximately 10,000 deaths due to cirrhosis of the liver and several thousand more from liver cancer are attributed to HCV infection in the United States each year.
The HCV life cycle is only partially understood because, until now, it has not been possible to efficiently infect normal human hepatocytes, or liver cells, in culture. According to Buck, the valuable Huh-7 system currently in use to test HCV uses cloned, synthetic HCV RNA expressed from liver tumor cells. These cells cannot be infected with naturally occurring HCV obtained from infected patients.
In contrast, the culture developed by the UCSD scientists allows direct infection with HCV genotypes 1, 2, 3 and 4 from the blood of HCV-infected patients. This system will enable researchers to study the complete viral lifecycle in its normal host cell, providing novel scientific opportunities. The study reports that the system has been tested using over 30 virus donors as well as multiple donors of hepatocytes, with the production of infectious HCV for all genotypes tested.
Published 16 Jul 2008 PLoS one
Direct Infection and Replication of Naturally Occurring Hepatitis C Virus Genotypes 1, 2, 3 and 4 in Normal Human Hepatocyte Cultures
Martina Buck1,2*
1 Department of Medicine and Moores Cancer Center, University of California, La Jolla, California, United States of America, 2 Department of Medicine, VA Healthcare Center, San Diego, California, United States of America
Abstract
Background
Hepatitis C virus (HCV) infection afflicts about 170 million individuals worldwide. However, the HCV life cycle is only partially understood because it has not been possible to infect normal human hepatocytes in culture. The current Huh-7 systems use cloned, synthetic HCV RNA expressed in hepatocellular carcinoma cells to produce virions, but these cells cannot be infected with naturally occurring HCV obtained from infected patients.
Methodology/Principal Findings
Here, we describe a human hepatocyte culture permissible to the direct infection with naturally occurring HCV genotypes 1, 2, 3 and 4 in the blood of HCV-infected patients. The culture system mimics the biology and kinetics of HCV infection in humans, and produces infectious virions that can infect naive human hepatocytes.
Conclusions/Significance
This culture system should complement the existing systems, and may facilitate the understanding of the HCV life cycle, its effects in the natural host cell, the hepatocyte, as well as the development of novel therapeutics and vaccines.
Discussion
The differentiated normal human hepatocyte cell culture described here is a system suitable for investigations of the HCV life cycle, of naturally occurring genotypes 1, 2, 3 and 4 obtained from HCV-infected patients. The infection of normal human hepatocytes was robust for at least 3 weeks and consistent, since normal human hepatocytes from 29 different liver donors were infected with sera from at least one of 33 HCV-infected patients. The HCV amplification achieved in these experiments was up to 7 log10 and comparable to that detected in HCV-infected human livers.
HCV infection was validated by seven different approaches: i) time-dependent amplification of newly synthesized HCV RNA (as detected by Northern blot and RT-QPCR ); ii) time-dependent amplification of HCV proteins in cell layer and media (as detected by immunopurification); iii) production of HCV virions that were infectious to naive human hepatocyte cultures ( as detected by RT-PCR for HCV RNA; HCV E-2 de novo synthesis with [35S]-methionine; and E-2 amplification by immunopurification); iv) production of HCV virions with densities below 1.09 g/cm3, consistent with highly infectious virions [50]-[51]; v] blockade of HCV infection with antibodies specific to HCV E-2 and CD-81, or cholesterol depletion with M_CD [40]-[42]; vi) IFN-β and methyl cytidine , inhibitors of HCV replication [7], [47] prevented HCV amplification; and vii] induction of a physiological response of interferon-related genes to the HCV infection.
Valuable studies by Fournier, Molina and their coworkers have allowed the culture of HCV in primary human hepatocytes [25]-[27]. However, this culture hepatocyte system had limited efficiency since less than 15% of the sera were infectious [25], the amplification was about 1 log10 [25]-[26], the infection declined after 8 days , it was detectable only in the cell layers until day-14, and there was no evidence of the production of infectious virions [25]-[27] . The main apparent differences between the system reported by Fournier, Molina and co-workers [25]-[27], and our system are the following: i] our cellular viability was higher (95% vs. 70-90%) and more stringent (apoptosis and ALT assessment); ii] our cell attachment was induced with higher concentration of fetal calf serum ( 20% vs. 5%) for longer (up to 18 hr vs. 4 hr); iii] in our system, the matrix was rat-tail collagen; iv ] in our system, the collagen matrix was freshly prepared within 24hr of hepatocyte plating, at a concentration of 50 μg/ml or greater; v] in our system, the culture plates were coated with polylysine; and vi] our density plating was higher (1.8 million vs. 0.14 million/60 cm2 plate) [25] .
The current Huh-7-derived HCV virions system is simple, but allows replication of only synthetic RNA expressed from selected cloned genomic or subgenomic HCV. Although naive Huh-7-derived cells can be infected with virions produced by Huh-7 cells transfected with HCV RNA from specific clones, this system cannot be infected with naturally occurring HCV, indicating another major departure of the Huh-7 system from the biology of human HCV infection. In contrast, the normal human hepatocyte system is permissible to infection with naturally occurring HCV genotypes from most patients tested to date, reproducing the high susceptibility of humans to HCV infection of all genotypes [4].
In addition blockers of cell entry and inhibitors of HCV replication [7], [41], [42], [47] prevented HCV replication in the human hepatocyte culture system. Collectively, these results suggest that the normal human hepatocyte culture system mimics some relevant aspects of the infection of hepatocytes by HCV in patients. Therefore, the normal human hepatocyte system may allow high throughput testing of patients' HCV susceptibility to novel drugs, and identification of putative molecular mechanisms within hepatocytes that may explain patients' genetic or acquired resistance to a treatment. For example, hepatocytes cell cultures and HCV could be from donors with various ethnic and genetic backgrounds such as those refractory to HCV treatment (e.g., African Americans). In addition, this hepatocyte system may facilitate early identification of unanticipated cellular targets of novel HCV inhibitors in the context of an HCV infection, possibly, preventing drug-induced liver injury in clinical trials [52]. Further, clinically relevant conditions for HCV infection, such as insulin resistance, fatty liver and iron overload [53]-[54] can be mimicked in these cultures (MB, unpublished observations). In contrast, these physiologically important culture conditions are unlikely to be mimicked in the Huh-7 hepatocellular carcinoma system.
In addition, the human hepatocyte culture system had a physiological response to the HCV infection since many of the 83 interferon-related genes were substantially affected, as reported in the livers of HCV-infected patients [55]. These findings suggest that the human hepatocyte system for HCV infection may contribute to the understanding of the relationship between HCV viral proteins and cellular anti-viral mechanisms, including IFN induction and IFN signaling [48].
The Huh-7 system is capable of generating infectious HCV virions; however, the repertoire is limited with only several HCV chimeric clones available at present [7]. Both the Huh-7 and human hepatocyte systems have comparable high-level replication for a few weeks. While only ~2% of the replicon Huh-7-derived cells were NS5-positive on day-5, reaching 100% infection on day-24 [8], about 95% of the human hepatocytes were infected with naturally occurring HCV at 24 hr, as indicated by the expression of HCV E-2 and core by confocal scanning laser microscopy. Thus, the human hepatocyte culture system is infected with naturally occurring HCV, at least as rapidly as the Huh-7 cells, mimicking the kinetics of HCV infection in humans. In support of our human hepatocyte HCV infection system, primary hepatocytes from the tree shrew Tupaia belangeri, specie susceptible to infection by hepatitis viruses, were also susceptible to HCV infection with sera from HCV-infected patients [56].
Current research uses readily available Huh7-derived hepatocellular carcinoma cells, while the HPV-18/E6E7-human hepatocyte system uses immortalized cells. However, unlike the normal human hepatocytes described in this study, the Huh-7- derived cells are de-differentiated and characterized by abnormal proliferation, deregulated gene expression, dysfunctional mitochondria and aberrant endocytosis and signaling pathways [15]-[18], [20]-[24]. Similarly, the HPV-18/E6E7 immortalized human hepatocytes have an ectopic expression of hTERT [13], which results in aberrant proliferation and could be tumorigenic [24]. It is unknown whether the HPV-18/E6E7 immortalized human hepatocytes are producing infectious HCV virions.
The HCV infection in patients induces proliferation of HCV-infected hepatocytes as determined in liver biopsies by nuclear staining of PCNA (M Buck, unpublished observations). In contrast, the HCV infection system occurs in Huh-7 cancer cells with a high baseline proliferation rate, and does not affect the degree of cell proliferation (M Buck, unpublished observations). Therefore, the differentiated, HCV-infected normal human hepatocyte culture system may help identifying the effects of HCV viral proteins on hepatocyte proliferation and carcinogenesis, signaling pathways, endocytosis, gene expression, and mitochondrial function [14].
The HCV infection of the hepatocyte culture system with naturally occurring HCV virions mimics the biology and kinetics of HCV infection in humans, and produces infectious virions that can infect naive human hepatocytes. We also determined that most of the HCV virions for genotypes 1, 2, and 3 produced by the primary and secondary human hepatocyte cultures, have a density consistent with those of infectious HCV virions, as proposed previously by Hijikata, Yi and their coworkers [50]-[51]. It remains to be determined by future investigations whether these HCV virions are infectious to chimpanzees and to mice containing human liver grafts as it has been documented with the Huh-7 generated virions [9], [12].
Clinical Implications
This study suggests that the human hepatocyte culture system described here will complement the Huh-7 virions system in understanding the HCV life cycle, it effects on the hepatocyte, the natural host cell, as well as the possible development of novel therapeutics and vaccines. The human hepatocyte culture system may facilitate studies of the role of insulin resistance and fatty liver on HCV infection since these conditions can be mimicked in the human hepatocytes. Similarly, the mechanisms by which African Americans are refractory to HCV treatment could possibly be analyzed by infecting human hepatocytes from different ethnic backgrounds and studying their response to treatments.
Introduction
An estimated 170 million individuals have chronic hepatitis C virus (HCV) infection worldwide [1]. About 70% of infected individuals develop a chronic infection; for some, this includes fibrosis, cirrhosis, and hepatocellular carcinoma [2], [3]. Approximately, 10,000 deaths due to cirrhosis and several thousand more deaths due to hepatocellular carcinoma are attributed to HCV infection in the United States each year [4]. Unfortunately, there is no vaccine available and the current treatment for HCV infection, PEG-interferon--β in combination with ribavirin, achieves sustained responses only in ~50% of treated patients [5].
The mechanisms responsible for the HCV life cycle in the liver of infected individuals are only partially understood because it has not been possible to infect normal human hepatocytes in culture with naturally occurring HCV obtained from HCV-infected patients [6], and because HCV is known to infect only humans and chimpanzees [4].
Recently, Lindenbach, Zhong , Wakita , Yi , Murakami and their coworkers [7]-[11], were able to replicate synthetic HCV RNA in hepatocellular carcinoma Huh-7-derived cells with the efficient production of HCV virions that were infectious to cultured Huh-7-derived cells [7]-[11] chimpanzees [9], [12] , and mice containing human liver grafts [12]. Importantly, virus recovered from these animals was highly infectious in cell culture [12]. Also, Aly and coworkers have developed a human hepatocyte cell line immortalized with human papilloma virus (HPV) 18/E6E7 susceptible to HCV infection [13]. The cell line's susceptibility to HCV infection was further increased by inhibiting the interferon regulatory factor-7 (IRF-7) [13].
The current Huh-7-derived HCV virions system uses non-naturally occurring, cloned HCV genotype 2a strain (JFH-1) [7], cloned HCV genotype 1a ( H77-S) containing five adaptive mutations [10] , or cloned HCV genotype 1b [11]. Limitations of this method are the use of cloned HCV, and the failure to infect these cells with naturally occurring HCV obtained from infected patients [14]
Further, hepatocellular carcinoma cell lines depart from normal human hepatocytes since they have abnormal proliferation, deregulated gene expression, dysfunctional mitochondria, and aberrant signaling and endocytosis pathways [15]-[20]. Relevant abnormalities of the Huh-7-derived cell lines include an absence of caveolin-1 and caveolin-2 [17], a mutated p53 (Y220C) [21], overexpression of the pituitary tumor transforming gene (PTTG) [22], cell cycle-independent expression of human telomerase reverse transcriptase (hTERT) [23], higher expression of glucose metabolism enzymes (glucose-6-phosphate 1-dehydrogenase and isocitrate dehydrogenase) and of a mitochondrial protein (dicarboxylate carrier) [24] . In addition, Huh-7 cells expressed the highest level of _-fetoprotein, a marker for hepatocellular carcinoma and de-differentiation, among 25 hepatocellular carcinoma cell lines tested [24]. Although the HPV-18/E6E7 immortalized human hepatocytes can be infected with serum-derived HCV, albeit at lower levels, it also over expresses hTERT [13], and like Huh-7-derived cells, its proliferation behavior should be that of tumor cells [24]. Consequently, any perturbation of these normal hepatocyte functions by the HCV infection cannot be studied completely and/or accurately in the Huh-7-derived HCV virions system or the HPV-18/E6E7 immortalized human hepatocytes [14].
Valuable studies by Fournier, Molina and their coworkers have allowed the culture of HCV in primary human hepatocytes [25]-[27]. However, this culture hepatocyte system had limited efficiency since less than 15% of the sera were infectious [25], the amplification was less than 1 log10 [25], [26], the infection declined after 8 days , it was detectable only in the cell layers until day-14, and there was no evidence of the production of infectious virions [25]-[27] Thus, there are still no effective means for directly culturing and significantly amplifying HCV from typical clinical specimens using differentiated normal human hepatocytes [4]. Here, we report the development of a normal human hepatocyte culture system permissible to the infection with, and physiologically significant amplification of, naturally occurring HCV.
Results
Infection of Primary Human Hepatocyte Cultures with Naturally Occurring HCV
We used primary human hepatocytes that were isolated from normal liver explants, and sera from chronically HCV-infected patients with high viral titers, in an attempt to infect human hepatocytes. Some stringent conditions of the culture system were required for achieving a successful HCV infection of human hepatocytes (see Methods).
Using these conditions, we attained a robust infection of human hepatocyte cultures with naturally occurring HCV obtained from 33 of 36 consecutive HCV-infected patients, with 3 of 36 failures. HCV infection was achieved in human hepatocyte cultures from 29 different liver donors. Cells were cultured at a high density on a three-dimensional specific collagen type 1 matrix, and in a defined medium without serum, and with liver sinusoidal cells, conditions that allowed hepatocytes to become highly differentiated, recapitulating the physiology of hepatocytes within the liver as we reported previously [14], [18], [28]. On day-5, the human hepatocyte culture system was composed of approximately 95% hepatocytes, and 5% liver sinusoidal endothelial cells and hepatic stellate cells, mimicking the hepatocyte organoid rodent cell culture [29]. As determined by RT-PCR , these uninfected human hepatocyte cultures expressed glial fibrillary acidic protein (GFAP), CD-34, complement receptor-1 (CR1), hepatocyte growth factor (HGF) and CD-68, genes also expressed in a normal liver by hepatic stellate cells, endothelial cells, smooth muscle cells and macrophages [19], [30]-[33]. The RNA values were compared to those of a normal uninfected human liver (Figure 1A). This may be relevant since these stellate cells and sinusoidal endothelial cells produce HGF, a critical factor for hepatocyte differentiation and survival [31], [34]-[35]. In turn, HGF activates the CCAAT/enhancer binding protein-β _ (C/EBPβ) [36] which upon phosphorylation induces hepatocyte survival [28].
Naturally occurring HCV genotype 1 infection of human hepatocyte cultures developed rapidly, as reflected by the intense expression of HCV E-2, core and NS3 proteins in the cell layers, after a 24-hr exposure, on laser scanning confocal microscopy using specific antibodies [37] (Figure 1B and 1C). The HCV E-2 and core proteins were co-localized in the perinuclear region of the human hepatocytes infected with HCV genotype 1, while uninfected control hepatocytes had only background fluorescence (Figure 1B). This perinuclear localization resembles the previously reported HCV virions in the liver of HCV-infected patients and chimpanzees [38]-[39]. In the HCV-infected hepatocytes, the TO-PRO3 stain for nucleic acids, detected hepatocyte nuclear DNA but also nucleic acids in the cytoplasm of hepatocytes in a 'salt and pepper' pattern (Figure 1B). Because this nucleic acid is co-localized with HCV proteins, such as E-2 and core, and it is not expressed either in uninfected control human hepatocyte cultures (Figure 1B), or in the same hepatocytes cultured in the presence of control human serum, it is not spurious patients' RNA or DNA contaminating the cells or the inoculum, and most likely represents HCV RNA. Moreover, the scanning confocal laser microscopy technique eliminates the possibility of detecting nucleic acids attached to the hepatocyte cell membrane. Further the expression of HCV E-2 and core as well as the cytoplasm nucleic acid was also observed in the liver of HCV-infected patients, but not in control livers (Figure 1B). Another HCV protein, NS3, was also detected in the human hepatocyte cultures infected with HCV genotype 1 after a 24-hr exposure, but not in uninfected control human hepatocyte cultures (Figure 1C). The expression of HCV NS3 was also observed in the liver of HCV-infected patients, but not in control livers (Figure 1C). A similar expression of HCV E-2, core and NS3 proteins was also observed in human hepatocyte cultures after a 24-hr infection with HCV genotypes 2 or 3 (Figure S1).
HCV Amplification in the Human Hepatocyte Culture System
Under the conditions that we described, human hepatocyte cultures remained infected for at least 3 weeks. The amplification of HCV genotype 1 infection was analyzed by immunopurifying HCV virions from the medium through HCV E-2 affinity chromatography. The HCV amplification was robust judging by the increased HCV core and E-2 in the medium from time zero (inoculum) to 72 hr (Figure 2A). Control samples from uninfected hepatocytes lacked detectable HCV core or E-2 proteins.
We evaluated the presence of full-length HCV RNA in the human hepatocyte culture by Northern blot, using specific probes cloned from each donor patient's HCV RNA. The large viral load required for this cloning was obtained from large phlebotomies needed for the treatment of iron overload in patients with Genetic Hemochromatosis that also had HCV infection genotypes 1, 2 and 3. The HCV RNA was of the expected size, and it was detected only in HCV-infected human hepatocyte cultures, but not in uninfected human hepatocyte cultures (Figure 2B). We also assessed the infection-replication cascade by determining HCV viral particles in the hepatocyte cultures from time zero to 72 hr and from zero to week-3. As detected by quantitative RT-PCR, using modified standard clinical COBAS amplification primers, the HCV RNA increased exponentially up to 5 log10 , 72 hr after a HCV genotype 1 infection (P<0.01) (Figure 2C) , and remained at a steady-state between 5 log10 and 6 log10 for up to week-3 for HCV genotypes 1, 3 and 4 (P<0.001) (Figure 2D). Using the same assay protocol, the HCV RNA, corrected by total RNA, was comparable in human hepatocyte cultures after day-2 and in the liver of HCV-infected patients (Figure 2C and 2D). These data further support the validity of the human hepatocyte culture system to study HCV infection. Although the half-life of HCV virions is less than 5 hours in HCV-infected patients [40], it remains to be determined whether the half-life of HCV virions in the hepatocyte culture is similar in the absence of a fully competent immune system.
Moreover, a similar amplification was detected for HCV proteins E-2, and core up to 21 days in human hepatocytes infected with HCV genotype 1 (P<0.05) (Figure 3A), obtained from a patient chronically infected with HCV. Also, we detected a rapid amplification of HCV genotypes 1, 2, 3 and 4, twenty-four hours after HCV infection judging by E-2 immunopurification (Figure 3B). Uninfected, control human hepatocyte cultures did not express either HCV E-2 or core proteins (Figure 3A and 3B). In addition, expression of HCV NS3 and NS5a was also amplified in human hepatocytes infected with HCV genotypes 1, 2 and 3 (Figure 3C and 3D). Collectively, these data indicate a robust HCV infection of the normal human hepatocyte culture system for up to 3 weeks (Figures 2 and 3).
The HCV infection of the normal human hepatocyte system was consistent. Sera from 33 of 36 HCV-infected patients successfully infected the normal human hepatocyte culture system. Instability of the HCV in the sera might have negatively affected infection of the human hepatocytes in 3 cases. None of the sera from the uninfected control subjects induced any false positive parameter of HCV infection in the normal human hepatocyte system. The subject population included individuals with chronic HCV infection, viral load >700,000 IU/ml and genotypes 1 (n: 21), 2 (n: 5), 3 (n: 6), or 4 (n: 4), but negative for Hepatitis A and B, and HIV. Control sera were obtained from 3 subjects negative for Hepatitis A, B and C, and HIV.
HCV-Infection of Normal Human Hepatocytes is Dependent on CD-81 and HCV E-2
The mechanisms of hepatitis C viral entry have been extensively investigated. It is known that E-2 dimerizes with E-1, and associates with the cellular CD-81 and the SR-BI receptors, which has proven to be critical for entry of HCV virions [41], [42]. It has also been documented that HCV entry mechanisms involve cholesterol content of the plasma membrane [42]-[45]. Therefore, we assessed whether these mechanisms also are required for HCV infection in normal human hepatocyte cultures. We found that HCV infection of the human hepatocyte culture system with genotype 1 can be blocked with antibodies specific to CD-81 or HCV E-2 (Figure 4A), or by cholesterol depletion with M_CD (Figure 4B) (P<0.05 for all treatments), as reported previously for Huh-7 cells [7] [42], [46]. Further, the HCV E-2 antibodies also inhibited HCV infection with genotypes 2, 3 and 4 (P<0.05 for all treatments). Control human IgG did not affect HCV infection (Figure S2).
As expected, the effects of M_CD were rescued by reconstituting cholesterol (Figure 4B), as reported previously for Huh-7 cells [42]. In addition, HCV replication was inhibited by methyl cytidine (P<0.05) (Figure 4C), as reported previously for Huh-7 cells [47]. The effects of these interventions were determined by measuring HCV RNA. The significant effects of these blockers and inhibitors on HCV infection suggest that the human hepatocyte culture system is physiologically relevant to the study of HCV infection, as previously documented for the Huh-7 cell system.
The HCV Infection Modulates Interferon-Related Genes in the Human Hepatocyte Culture System
Because IFN induction and IFN signaling are important responses to HCV infection [48], we investigated these pathways in the hepatocyte culture system before and after HCV infection. Using a microarray assay to assess expression of 83 interferon-related genes in the human hepatocyte culture system, 72-hr after infection with HCV genotypes 1, 2 or 3, we found a substantially altered gene expression, when compared to uninfected, control human hepatocytes (Figure 5A and Table S1). Consistent increases were observed after a 72-hr HCV infection for the following genes : i] IL-15 ( # 48); ii] IFN-R 1 (# 33) ; iii] IRF-3 (# 69); iv] IRF-7 ( # 73) ; v] IRF-8 ( # 74) ; vi] IRF-2 binding protein 1 ( # 67) ; vii] IRF-2 binding protein 2 ( # 68); viii] IRF-2 (# 66) ; ix] Adenosine deaminase (# 1) ; and x] Oligoadenylate synthase (# 79) (Table S1 ). In contrast, consistent decreases were observed after a 72-hr HCV infection for the following genes : i] IFN-induced Protein 44-like ( # 17); ii] IFN-β 2 (# 27) ; iii] IRF-α 4 (# 29); iv] IRF-α 14 ( # 26) ; v] IRF-α 8 ( # 32) ; vi] IRF-α 6 ( # 31); vii] IRF-α 1 ( # 25); viii] IRF-related Development Regulator 1 (# 41) ; ix] IL-21R (# 50) ; x] IL-22R _ ( # 51); xi] IL-28A ( # 52); and xii] Chemokine ligand 10 ( # 6) (Table S1 ).
Treatment of human hepatocytes with interferon 18hr before HCV infection, inhibited HCV amplification as measured by RT-PCR for HCV RNA 72 hr later (P<0.05) (Figure 5B). Treatment of human hepatocytes with interferon for 72 hr , 24-hr after the HCV infection with genotypes 1, 2, 3 or 4, inhibited HCV amplification by ~1 log10 (from ~7 log10 ), when compared to untreated HCV-infected human hepatocytes, as measured by RT-PCR for HCV RNA (P<0.05) (Figure 5C). These results further emphasize the physiological relevance of the HCV-infected hepatocyte culture system.
The Normal Human Hepatocyte Culture System Produces Infectious HCV
HCV virions produced by the primary HCV-infected human hepatocyte cultures were infectious to naive normal human hepatocyte cultures. The infectivity of naturally occurring HCV virions from HCV-infected patients (into the 'primary' culture) and HCV virions produced by normal human hepatocyte cultures (into the 'secondary' culture) was comparable (Figure 6). This was judged by the viral amplification as determined by immunopurification of HCV E-2, and quantified either on a Kodak 4000 Imaging Station and software as described [31] (P<0.05) (Figure 6, upper panel), or by the incorporation of [35S]-methionine into the newly synthesized HCV E-2 protein in the secondary infection (P<0.01) (Figure 6, lower panel).
Because it has been suggested that infectious HCV virions and infectious Huh-7 produced particles represent a subset banding at a density slightly lower than that of most virions and particles [49]-[51], we isolated HCV virions by isopycnic ultracentrifugation through an iodixanol gradient as described by Yi and coworkers [51]. As described in Table 1 for HCV genotype 1, the density of the naturally occurring HCV in the serum of the patient fluctuated between 0.979 g/cm3 and 1.258 g/cm3. Similarly, the densities of the HCV virions in primary and secondary human hepatocyte infection fluctuated between 0.987 g/cm3 and 1.298 g/cm3, and 0.990 g/cm3 and 1.259 g/cm3, respectively (Table 1). The highly infectious HCV virions and particles are believed to concentrate in those with densities below 1.09 g/cm3 [50] [51]. In our analysis for HCV genotype 1, approximately 82% of the patient's HCV, 86% of the HCV in the primary infection, and 90% of the HCV in the secondary infection corresponded to those densities, suggesting that they were infectious (Table 1). The higher HCV densities may correspond to other forms of HCV virions. For example, the nucleocapsid of HCV, isolated by different detergent treatments, was estimated to have a buoyant density of 1.25 g/ml in sucrose [49] , and some clinical HCV samples have densities >1.13 g/ml [49]-[50]
A similar pattern was observed for HCV genotypes 2 and 3. The percentage of HCV virions that concentrate in densities below 1.09 g/cm3 , were approximately 82% and 80% for HCV genotype 2, and 85% and 87% for HCV genotype 3 , in the primary infection and in the secondary human hepatocyte culture system , respectively (Table S2)
To our knowledge, this is the first report of a physiologically significant amplification of HCV infection (up to 7 log10 ) with naturally occurring genotypes 1, 2 , 3, and 4 in a normal human hepatocyte culture system, suggesting that research with this physiological system may complement that available with the current replicon systems [7]-[9].
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