Generation of Infectious Hepatitis C Virus in Immo
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病菌学杂志 2006年第9期
Departments of Pathology Internal Medicine Molecular Microbiology and Immunology, Saint Louis University, St. Louis, Missouri 63110
Department of Microbiology, Tokyo Metropolitan Institute for Neuroscience, Tokyo 183-8526, Japan
ABSTRACT
Progress in understanding hepatitis C virus (HCV) biology has remained a challenge due to the lack of an efficient cell culture system for virus growth. In this study, we examined HCV core protein-mediated immortalized human hepatocytes (IHH) for growth of HCV. In vitro-transcribed full-length RNA from HCV genotype 1a (clone H77) was introduced into IHH by electroporation. Reverse transcription-PCR of cellular RNA isolated from HCV genome-transfected IHH suggested that viral RNA replication occurred. IHH transfected with the full-length HCV genome also displayed viral protein expression by indirect immunofluorescence. In contrast, cells transfected with polymerase-defective HCV (H77/GND) RNA as a negative control did not exhibit expression of the viral genome. Immunogold labeling demonstrated localization of E1 protein in the rough endoplasmic reticulum of RNA-transfected IHH. Virus-like particles of 50 nm were observed in the cytoplasm. After being inoculated with culture media of cells transfected with the full-length HCV genome, nave IHH displayed NS5a protein expression in a dilution-dependent manner, but expression of NS5a was inhibited by prior incubation of culture medium with HCV-infected patient sera. NS5a-positive immunofluorescence of cell culture media of IHH transfected with full-length H77 RNA yielded 4.5 x 104 to 1 x 105 focus-forming units/ml. A similar level of virus growth was observed upon transfection of RNA from HCV genotype 2a (JFH1) into IHH. Taken together, our results suggest that IHH support HCV genome replication and virus assembly.
TEXT
Hepatitis C virus (HCV) is an important cause of morbidity and mortality worldwide. The most important feature of HCV infection is the development of chronic hepatitis in a significant number of infected individuals, with the potential for disease progression to cirrhosis and hepatocellular carcinoma (6, 7, 11, 27). At present, the only approved therapies for chronic HCV infection are alpha interferon (IFN-) with or without ribavirin (9, 21), but these fail to clear HCV from a significant number of patients (22). A number of HCV genomes have been cloned, and sequence divergence indicates that there are several genotypes and a series of subtypes of this virus (28). In the United States, HCV genotypes 1a and 1b are predominant in patients with chronic hepatitis C (32). Progress in understanding HCV biology has remained challenging due to the lack of an efficient cell culture system for virus growth. Establishment of self-replicating full-length HCV genomic replicons from genotypes 1a and 1b in human hepatoma (Huh-7) cells has provided an important tool for the study of HCV replication mechanisms (3, 10, 23). Recently, different groups have reported the generation of infectious virus from transfection of genomic RNA of HCV genotype 2a into Huh-7 cells or its derivatives (5, 15, 29, 33). However, generation of infectious HCV genotype 1a has not been successful to date.
We and others have shown that HCV core protein transcriptionally regulates a number of cellular genes (26). We previously described the generation of immortalized human hepatocytes (IHH) by transfection of the HCV core genomic region from genotype 1a (2, 25). IHH exhibited a weak level of HCV core protein expression, albumin secretion, glucose phosphatase activity, and absence of smooth-muscle actin. IHH also displayed focal cytoplasmic and membrane staining for carcinoembryonic antigen (CEA), biliary glycoprotein (BGP1/CEACAM1), and nonspecific cross-reacting antigen (NCA/CEACAM6) and expression of hepato-biliary transport marker genes (MRP, LST1, and NTCP) (unpublished observations). Together, these results suggested that IHH are well differentiated. HCV core protein selectively degrades STAT1, reduces phosphorylated STAT1 accumulation in the nucleus in a proteasome-dependent manner, and impairs IFN--induced signal transduction via expression of suppressor of cytokine signaling-3 (1, 4, 16, 18). HCV core protein is competent to partially rescue growth of a genetically engineered influenza A virus lacking its own IFN antagonist (4). The core protein can modulate interferon regulatory factor, Jak-STAT, and inducible nitric oxide synthetase pathways, which suggests that there are mechanisms by which the core could affect HCV persistence and pathogenesis (20). Since HCV core protein transcriptionally regulates several cellular genes involved in cell growth, apoptosis, and defense mechanisms, we hypothesize that IHH may set the stage for HCV genome replication and assembly.
(Part of this study was presented at the International Congress of Virology, IUMS, San Francisco, Calif., 2005, and the 12th International Symposium on Hepatitis C Virus and Related Viruses, Montreal, Canada, 2005.)
Replication of the HCV genome and virus protein expression. We investigated whether IHH confer HCV genome replication and generation of infectious virus particles. For this purpose, full-length RNAs from HCV genotype 1a (clone H77) (13) were used. The clone H77 contains a 5' untranslated region (5'UTR), a coding sequence, and a 3'UTR, which are suggested to be necessary for replication (14, 30). In vitro-transcribed full-length HCV RNA from clone H77 was used for transfection of IHH by electroporation. H77/GND (polymerase-defective) RNA was used similarly as a negative control. Briefly, H77 cDNA was linearized by digestion with XbaI, and gel-purified DNA was used for in vitro transcription by T7 RNA polymerase (Promega, Madison, Wis.). In vitro-transcribed RNA (1 to 2 μg) was introduced by electroporation (950 μF and 270 V) into 5 x 106 IHH, using a Bio-Rad Gene Pulser Xcell system (Hercules, Calif.). The transfected cells were plated on collagen-coated plastic dishes and maintained in culture for HCV replication. Total cellular RNA was extracted 5 days posttransfection. To detect the HCV genome, total cellular RNA and random hexamers were used for cDNA synthesis with a SuperScriptIII first-strand-synthesis system (Invitrogen), following the supplier's protocol. PCR amplification was performed with cDNA as a template, using sense (5'-CACTCCCCTGTGAGGAACTACTGTCT-3') and antisense (5'-TGGTGCACGGTCTACGAGACCTCCC-3') primers from 5'UTR at 94°C for 30 s, annealing at 55°C for 60 s, and extension at 72°C for 90 s. Glyceraldehyde-3-phosphate dehydrogenase (GPDH) was used as an internal control, using specific primers (17). Reverse transcription-PCR (RT-PCR) analyses suggested amplification of sequence from the 5'UTR (Fig. 1A). In contrast, cells transfected with H77/GND RNA did not exhibit the presence of the HCV genomic sequence. To rule out the integration of H77 plasmid DNA into IHH, genomic DNA from the cell lines was isolated and examined for the HCV genome by PCR. Our results suggested that the HCV sequence was absent, indicating HCV genomic RNA replication occurred in the cytoplasm of IHH (data not shown). Filtered culture supernatant was also treated with RNaseA prior to isolation of viral RNA. RT-PCR was performed for the NS5A region (17), and we observed amplification of a specific RNA sequence.
Western blot analysis using specific antibodies was performed to analyze the expression of core and NS3 proteins in control and experimental cells. Equal amounts of proteins from whole-cell lysates in sample buffer were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins were transferred onto nitrocellulose, incubated with specific antibodies, and detected by chemiluminescence (Amersham, Piscataway, N.J.). HCV core protein was detected by a specific rabbit antiserum, and NS3 was detected by a specific mouse monoclonal antibody (ViroGen, Watertown, Mass.). The blots were stripped and reprobed, using a mouse monoclonal antibody to actin (Oncogene Science, Cambridge, Mass.). IHH that supported HCV genome replication displayed the presence of core (21 kDa) and NS3 (63 kDa) proteins (Fig. 1B and C). On the other hand, IHH transfected with H77/GND RNA did not show a detectable level of core or NS3 proteins. A weak level of core protein was detected in this set of IHH for immortalization by HCV core protein (Fig. 1B). IHH transfected with HCV full-length RNA were passaged at 4- or 5-day intervals. HCV RNA and protein expression were detected in cell cultures for up to 12 days, and the cultures were discontinued for lack of growth after 2 weeks.
To further examine intracellular expression of HCV protein, IHH transfected with H77 RNA were fixed with 3.7% formaldehyde and incubated at room temperature for 1 h with monoclonal antibodies to NS5a (Biogenesis, Kingstone, N.H.). Cells were washed three times with phosphate-buffered saline (PBS), stained with anti-mouse immunoglobulin (Ig) conjugated with Alexa 568 (Molecular Probes, Eugene, Oreg.), and mounted for fluorescence microscopy. Primary antibodies and secondary antibody-fluorochrome conjugates were titrated for use of optimum dilutions where there was no background fluorescence. We observed cytoplasmic expression of NS5a (Fig. 2A) in 60% IHH after 5 days of transfection. HCV genotype 2a (clone JFH1) has been shown to grow in Huh-7 cells or its derivatives (5, 12, 15, 29, 33). In vitro-transcribed RNA from clone JFH1 was used for transfection of IHH to determine if the immortalized hepatocyte cell line supports HCV growth. Intracellular localization of NS3 protein from JFH1 RNA-transfected IHH was detected by immunofluorescence (Fig. 2C). We have also used Huh-7.5 cells transfected with JFH1 RNA as positive controls (29) and observed NS3 expression by indirect immunofluorescence (data not shown). On the other hand, IHH similarly transfected with RNA from H77/GND or the JFH1/GND clone did not display virus protein expression by immunofluorescence (Fig. 2B and D).
Immunogold localization of virus-like particles. Phase-contrast microscopy suggested that HCV genome-transfected IHH were swollen with large vacuoles in the cytoplasm, whereas the negative controls did not show any detectable changes. We also looked for cellular changes, using electron microscopy, and at the ultrastructural level some of these vacuoles appeared to be empty (Fig. 3A and E). Others contained lipid (Fig. 3E) or material isolated for degradation (Fig. 3D). Ultrastructural changes also included increased polymorphism of the nuclei (Fig. 3E). Immunogold labeling was performed for localization of HCV-like particles in transfected IHH. For this, transfected IHH (4 days in culture) were detached from collagen-coated petri dishes by a brief trypsin treatment, pelleted in a microcentrifuge, and fixed in 4% paraformaldehyde and 1% glutaraldehyde in PBS for 16 h at 4°C. After being washed with PBS, the cells were washed in distilled water, dehydrated in ethanol, and infiltrated with LR White resin (London Resin Company, Berkshire, United Kingdom). The cell pellets were polymerized in BEEM capsules (Ted Pella, Inc., Redding, Calif.) at –20°C under UV light. Thin sections were cut from blocks, collected on Formvar-coated nickel grids, and blocked with 1% fish gelatin and 1% bovine serum albumin (BSA) in PBS for 10 min. Sections were incubated for 2 h in a 1:100 dilution (titrated beforehand for best results) of monoclonal antibody to E1 glycoprotein (305/C3) or normal mouse IgG in PBS containing 0.1% BSA, washed in PBS containing 0.1% BSA, and incubated for 1 h in protein A-10-nm colloidal gold (CG) diluted at a ratio of 1:200 in PBS containing 0.1% BSA. After being washed with PBS, the grids were fixed for 3 min in glutaraldehyde, washed in distilled water, stained with uranyl acetate and lead citrate, and photographed with a JEOL 100 CX electron microscope. No clusters of CG particles were observed in the controls, which were stained with normal mouse IgG without the primary antibody or were mock-transfected and stained with the 305/C3 monoclonal antibody. Several hundred cells were evaluated in each case. Immunogold labeling with E1-specific monoclonal antibody demonstrated the presence of HCV-like particles and E1 protein in IHH. Numerous labeled virus-like particles were observed in the cytoplasm (Fig. 3A and E) and near the plasma membrane (Fig. 3C) of H77 RNA-transfected IHH. The labeled particles were 50 nm in diameter. Extensive labeling was also associated with the rough endoplasmic reticulum, consistent with the synthesis of E1 viral protein (Fig. 3B). In addition, we observed cytoplasmic autophagic vacuoles which contained gold-labeled virus-like particles (Fig. 3D).
Processing of cells into LR White resin for immunogold localization omits the conventional osmium tetroxide fixation step to preserve antigenicity but results in reduced tissue contrast. In addition, the identification of virus particles by immunogold labeling at the ultrastructural level can be tricky. For this, we carried out a series of control experiments to ensure labeling specificity. First, we observed clusters of CG on virus-like particles and single CG particles in the endoplasmic reticulum in several independent anti-E1-labeling experiments. Second, H77/GND RNA-transfected IHH (negative controls) showed no such clusters of CG in the cytoplasm or single CG particles localized along the endoplasmic reticulum or membranes (Fig. 3F). Third, incubation of sections of HCV genome-transfected IHH with normal mouse IgG at IgG concentrations similar to those used for the anti-E1 antibody did not result in any specific immunogold labeling. Fourth, omitting anti-E1 antibody did not result in any specific immunogold labeling. Finally, CG particles in the anti-E1-labeling experiments were primarily confined to cells and were not observed to any degree in the spaces around cells, again suggesting the labeling was specific for E1 protein in cells. Thus, the appearance of virus-like particles in RNA-transfected IHH indicated that HCV 1a replicates and assembles as virus particles.
Infection of IHH by HCV from culture medium. We next examined the presence of HCV in IHH cell culture medium. On different days after transfection, culture medium was filtered through a 0.45-μm cellulose acetate membrane (Millipore, Bedford, Mass.), concentrated to 10- to 20-fold by Millipore ultrafiltration (100-kDa cut off), and used for detection of the HCV genomic sequence by RT-PCR (Fig. 4A). The presence of HCV 5'UTR was detected in culture medium from HCV genome-transfected IHH but not from polymerase-defective HCV RNA-transfected IHH. We obtained 1.1 x 108 genome copies/ml of culture medium using real-time RT-PCR, as described recently (33). Culture supernatant collected for up to 7 days suggested that the peak HCV genome copy number occurred between 4 and 5 days after transfection.
Next, we determined whether the culture medium contained infectious HCV. For this, culture media was serially twofold diluted and inoculated into nave IHH. Cells were incubated for 4 h, washed, and incubated with fresh media for 3 days before indirect immunofluorescence was performed to determine the number of focus-forming units (FFU)/ml of NS5a (H77 clone) or NS3 (JFH1 clone), as recently described (33). Nuclear staining was performed using TO-PRO3-iodide (Molecular Probes), and cells were mounted for confocal laser scanning microscopy (model 1024; Bio-Rad). Figure 4B shows infection of IHH by H77 or JFH1 and is representative of our results. Fluorescent cells were counted, and the counts were correlated with the number of dilutions of cell culture media to determine FFU/ml of H77 and JFH1 clones. We observed 4.5 x 104 to 1 x 105 FFU/ml of H77 and JFH1 clones in the cell culture media 5 days after transfection.
We transfected in vitro-transcribed H77 or JFH1 RNA into IHH and isolated the RNA from the transfected cells. Culture supernatant was also collected for isolation of RNA and determination of infectivity (FFU/ml). Real-time PCR suggested that maximal HCV RNA accumulation from H77 occurred at the intracellular level on day 2 and declined on day 5 (Fig. 4C). We observed a higher genome copy number and infectious virus titer on day 4. Similarly, JFH1 RNA-transfected IHH supernatant displayed a peak genome copy number per ml of 108 and infectivity of 7 x 104 FFU/ml on day 4.
HCV-infected patient serum (OP1843) displaying neutralizing activity against the vesicular stomatitis virus/HCV pseudotype (19) was used in determining neutralization of cell culture-grown HCV. Serum from a healthy volunteer was used as a negative control in HCV neutralization assays. A twofold serial dilution of heat-inactivated serum was incubated with 100 FFU of HCV generated from the H77 clone at 37°C for 30 min. The virus-serum mixture was added to nave IHH and incubated for 3 days. Neutralization was determined by measuring inhibition of NS5a protein expression by immunofluorescence. The results are shown as the percent inhibition based on focus-forming units per milliliter (Fig. 4D). Infectivity (60%) was inhibited by prior incubation of HCV in culture medium with the patient serum at a 1/50 dilution. Inhibition was also observed at different dilutions of sera from three other HCV-infected patients. In contrast, sera from four healthy individuals did not inhibit infectivity at a 1/10 dilution. These results suggest that infectious HCV particles released in the culture medium are neutralized by specific antibodies.
HCV RNA is directly translated, and the precursor viral polypeptide is cleaved proteolytically to form individual proteins. The replicase complex amplifies the RNA via a minus-strand intermediate. Plus-strand RNA progeny are packaged into virus particles and acquire their envelopes probably by budding into the lumen of the endoplasmic reticulum. HCV particles are likely to be exported via the constitutive secretory pathway. Based on this working principle, we have shown in this report that IHH support HCV genome replication and protein expression from genotype 1a. Immunogold labeling using a monoclonal antibody demonstrated localization of HCV E1 glycoprotein in the rough endoplasmic reticulum and the formation of virus-like particles. We transferred culture media of HCV-replicating cells into nave IHH, and HCV infection was detected by RT-PCR and indirect immunofluorescence. We have also observed JFH1 replication and virus growth in IHH. The infectious units appeared to be similar for JFH1 grown in Huh-7 cells and in its derivatives. JFH1 may replicate with a higher efficiency than H77 at the RNA level in Huh-7 cells or its derivatives. However, we focused on determining the generation of infectious HCV from H77 and JFH1 in IHH. In our experimental system, we observed that virus genome copies of H77 and JFH1 were at similar levels in H77 and JFH1 RNA-transfected culture supernatant. The number of focus-forming units per milliliter of H77 and JFH1 was also similar. We did not purify virus particles for negative staining due to the relatively low number of infectious units in the culture media. Three different groups of investigators have reported different densities of HCV genotype 2a particles. Zhong et al. (33) observed peak infectivity at an apparent density of 1.105 gm/ml, and Wakita et al. (29) observed peak infectivity at a density of 1.15 gm/ml. Lidenbach et al. (15) observed a broad distribution of virus infectivity over a range of 1.01 to 1.12 gm/ml. A similar finding suggesting variation of between 1.06 and 1.16 gm/ml in buoyant density of cell culture-grown HCV genotype 2a was reported by Cai et al. (5). HCV is known to associate with serum immunoglobulin and lipoproteins (24). We have observed HCV infectivity within a density range of 1.09 to 1.12 in sucrose gradients, which did not correlate with highest copy number of virus genomic RNA (data not shown).
Recently, HCV production from a HCV-ribozyme construct of genotype 1a (clone H77) in Huh-7 cells was reported, although the infectivity of the virus was not determined (8). Virus genome replication and assembly are multistep processes and are influenced by the intracellular milieu. Inhibition of host cell growth and induction of cytokines, such as interferons, may have an impact on virus replication (3). Our study supports proof of the concept of HCV replication and assembly of genotype 1a in IHH. To our knowledge, this is the first report describing the generation of cell culture-grown HCV from genotype 1a. We speculate that cellular defense mechanisms against HCV infection are attenuated or compromised in IHH. Further studies may help to unravel the specific mechanisms for growth of HCV in IHH and to address important biological questions about the life cycle of HCV. Studies are in progress to determine the factors influencing virus growth, such as serial passage for adaptation in IHH, mutations at specific sites on the HCV genome, and selection of cell populations for attenuated protective mechanisms. We will also characterize the biophysical properties of cell culture-grown HCV and its infectivity in available animal models in the near future.
ADDENDUM While our manuscript was under revision, Yi et al. (31) reported the growth of H77-S in Huh-7.5 cells.
ACKNOWLEDGMENTS
We are grateful to Charles M. Rice for providing the full-length H77 clone, Michael Houghton for the E1 monoclonal antibody, Arvind Patel for antiserum to core protein, George Luo for monoclonal antibody to NS3 protein, and Leonard E. Grosso for detection of HCV RNA. We appreciate the helpful suggestions of Richard W. Compans concerning electron microscopy and Francis V. Chisari in determining virus infectivity. We thank Lin Cowick for preparation of the manuscript.
This work was supported by research grants AI45144 (R.B.R.) and CA85486 (R.R.) from the National Institutes of Health. T.W. was partly supported by grants from the Ministry of Health, Labor and Welfare of Japan, the Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (NIBIO), and Research on Health Sciences focusing on Drug Innovation from the Japan Health Sciences Foundation.
T.K. and A.B. contributed equally to this study.
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Department of Microbiology, Tokyo Metropolitan Institute for Neuroscience, Tokyo 183-8526, Japan
ABSTRACT
Progress in understanding hepatitis C virus (HCV) biology has remained a challenge due to the lack of an efficient cell culture system for virus growth. In this study, we examined HCV core protein-mediated immortalized human hepatocytes (IHH) for growth of HCV. In vitro-transcribed full-length RNA from HCV genotype 1a (clone H77) was introduced into IHH by electroporation. Reverse transcription-PCR of cellular RNA isolated from HCV genome-transfected IHH suggested that viral RNA replication occurred. IHH transfected with the full-length HCV genome also displayed viral protein expression by indirect immunofluorescence. In contrast, cells transfected with polymerase-defective HCV (H77/GND) RNA as a negative control did not exhibit expression of the viral genome. Immunogold labeling demonstrated localization of E1 protein in the rough endoplasmic reticulum of RNA-transfected IHH. Virus-like particles of 50 nm were observed in the cytoplasm. After being inoculated with culture media of cells transfected with the full-length HCV genome, nave IHH displayed NS5a protein expression in a dilution-dependent manner, but expression of NS5a was inhibited by prior incubation of culture medium with HCV-infected patient sera. NS5a-positive immunofluorescence of cell culture media of IHH transfected with full-length H77 RNA yielded 4.5 x 104 to 1 x 105 focus-forming units/ml. A similar level of virus growth was observed upon transfection of RNA from HCV genotype 2a (JFH1) into IHH. Taken together, our results suggest that IHH support HCV genome replication and virus assembly.
TEXT
Hepatitis C virus (HCV) is an important cause of morbidity and mortality worldwide. The most important feature of HCV infection is the development of chronic hepatitis in a significant number of infected individuals, with the potential for disease progression to cirrhosis and hepatocellular carcinoma (6, 7, 11, 27). At present, the only approved therapies for chronic HCV infection are alpha interferon (IFN-) with or without ribavirin (9, 21), but these fail to clear HCV from a significant number of patients (22). A number of HCV genomes have been cloned, and sequence divergence indicates that there are several genotypes and a series of subtypes of this virus (28). In the United States, HCV genotypes 1a and 1b are predominant in patients with chronic hepatitis C (32). Progress in understanding HCV biology has remained challenging due to the lack of an efficient cell culture system for virus growth. Establishment of self-replicating full-length HCV genomic replicons from genotypes 1a and 1b in human hepatoma (Huh-7) cells has provided an important tool for the study of HCV replication mechanisms (3, 10, 23). Recently, different groups have reported the generation of infectious virus from transfection of genomic RNA of HCV genotype 2a into Huh-7 cells or its derivatives (5, 15, 29, 33). However, generation of infectious HCV genotype 1a has not been successful to date.
We and others have shown that HCV core protein transcriptionally regulates a number of cellular genes (26). We previously described the generation of immortalized human hepatocytes (IHH) by transfection of the HCV core genomic region from genotype 1a (2, 25). IHH exhibited a weak level of HCV core protein expression, albumin secretion, glucose phosphatase activity, and absence of smooth-muscle actin. IHH also displayed focal cytoplasmic and membrane staining for carcinoembryonic antigen (CEA), biliary glycoprotein (BGP1/CEACAM1), and nonspecific cross-reacting antigen (NCA/CEACAM6) and expression of hepato-biliary transport marker genes (MRP, LST1, and NTCP) (unpublished observations). Together, these results suggested that IHH are well differentiated. HCV core protein selectively degrades STAT1, reduces phosphorylated STAT1 accumulation in the nucleus in a proteasome-dependent manner, and impairs IFN--induced signal transduction via expression of suppressor of cytokine signaling-3 (1, 4, 16, 18). HCV core protein is competent to partially rescue growth of a genetically engineered influenza A virus lacking its own IFN antagonist (4). The core protein can modulate interferon regulatory factor, Jak-STAT, and inducible nitric oxide synthetase pathways, which suggests that there are mechanisms by which the core could affect HCV persistence and pathogenesis (20). Since HCV core protein transcriptionally regulates several cellular genes involved in cell growth, apoptosis, and defense mechanisms, we hypothesize that IHH may set the stage for HCV genome replication and assembly.
(Part of this study was presented at the International Congress of Virology, IUMS, San Francisco, Calif., 2005, and the 12th International Symposium on Hepatitis C Virus and Related Viruses, Montreal, Canada, 2005.)
Replication of the HCV genome and virus protein expression. We investigated whether IHH confer HCV genome replication and generation of infectious virus particles. For this purpose, full-length RNAs from HCV genotype 1a (clone H77) (13) were used. The clone H77 contains a 5' untranslated region (5'UTR), a coding sequence, and a 3'UTR, which are suggested to be necessary for replication (14, 30). In vitro-transcribed full-length HCV RNA from clone H77 was used for transfection of IHH by electroporation. H77/GND (polymerase-defective) RNA was used similarly as a negative control. Briefly, H77 cDNA was linearized by digestion with XbaI, and gel-purified DNA was used for in vitro transcription by T7 RNA polymerase (Promega, Madison, Wis.). In vitro-transcribed RNA (1 to 2 μg) was introduced by electroporation (950 μF and 270 V) into 5 x 106 IHH, using a Bio-Rad Gene Pulser Xcell system (Hercules, Calif.). The transfected cells were plated on collagen-coated plastic dishes and maintained in culture for HCV replication. Total cellular RNA was extracted 5 days posttransfection. To detect the HCV genome, total cellular RNA and random hexamers were used for cDNA synthesis with a SuperScriptIII first-strand-synthesis system (Invitrogen), following the supplier's protocol. PCR amplification was performed with cDNA as a template, using sense (5'-CACTCCCCTGTGAGGAACTACTGTCT-3') and antisense (5'-TGGTGCACGGTCTACGAGACCTCCC-3') primers from 5'UTR at 94°C for 30 s, annealing at 55°C for 60 s, and extension at 72°C for 90 s. Glyceraldehyde-3-phosphate dehydrogenase (GPDH) was used as an internal control, using specific primers (17). Reverse transcription-PCR (RT-PCR) analyses suggested amplification of sequence from the 5'UTR (Fig. 1A). In contrast, cells transfected with H77/GND RNA did not exhibit the presence of the HCV genomic sequence. To rule out the integration of H77 plasmid DNA into IHH, genomic DNA from the cell lines was isolated and examined for the HCV genome by PCR. Our results suggested that the HCV sequence was absent, indicating HCV genomic RNA replication occurred in the cytoplasm of IHH (data not shown). Filtered culture supernatant was also treated with RNaseA prior to isolation of viral RNA. RT-PCR was performed for the NS5A region (17), and we observed amplification of a specific RNA sequence.
Western blot analysis using specific antibodies was performed to analyze the expression of core and NS3 proteins in control and experimental cells. Equal amounts of proteins from whole-cell lysates in sample buffer were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins were transferred onto nitrocellulose, incubated with specific antibodies, and detected by chemiluminescence (Amersham, Piscataway, N.J.). HCV core protein was detected by a specific rabbit antiserum, and NS3 was detected by a specific mouse monoclonal antibody (ViroGen, Watertown, Mass.). The blots were stripped and reprobed, using a mouse monoclonal antibody to actin (Oncogene Science, Cambridge, Mass.). IHH that supported HCV genome replication displayed the presence of core (21 kDa) and NS3 (63 kDa) proteins (Fig. 1B and C). On the other hand, IHH transfected with H77/GND RNA did not show a detectable level of core or NS3 proteins. A weak level of core protein was detected in this set of IHH for immortalization by HCV core protein (Fig. 1B). IHH transfected with HCV full-length RNA were passaged at 4- or 5-day intervals. HCV RNA and protein expression were detected in cell cultures for up to 12 days, and the cultures were discontinued for lack of growth after 2 weeks.
To further examine intracellular expression of HCV protein, IHH transfected with H77 RNA were fixed with 3.7% formaldehyde and incubated at room temperature for 1 h with monoclonal antibodies to NS5a (Biogenesis, Kingstone, N.H.). Cells were washed three times with phosphate-buffered saline (PBS), stained with anti-mouse immunoglobulin (Ig) conjugated with Alexa 568 (Molecular Probes, Eugene, Oreg.), and mounted for fluorescence microscopy. Primary antibodies and secondary antibody-fluorochrome conjugates were titrated for use of optimum dilutions where there was no background fluorescence. We observed cytoplasmic expression of NS5a (Fig. 2A) in 60% IHH after 5 days of transfection. HCV genotype 2a (clone JFH1) has been shown to grow in Huh-7 cells or its derivatives (5, 12, 15, 29, 33). In vitro-transcribed RNA from clone JFH1 was used for transfection of IHH to determine if the immortalized hepatocyte cell line supports HCV growth. Intracellular localization of NS3 protein from JFH1 RNA-transfected IHH was detected by immunofluorescence (Fig. 2C). We have also used Huh-7.5 cells transfected with JFH1 RNA as positive controls (29) and observed NS3 expression by indirect immunofluorescence (data not shown). On the other hand, IHH similarly transfected with RNA from H77/GND or the JFH1/GND clone did not display virus protein expression by immunofluorescence (Fig. 2B and D).
Immunogold localization of virus-like particles. Phase-contrast microscopy suggested that HCV genome-transfected IHH were swollen with large vacuoles in the cytoplasm, whereas the negative controls did not show any detectable changes. We also looked for cellular changes, using electron microscopy, and at the ultrastructural level some of these vacuoles appeared to be empty (Fig. 3A and E). Others contained lipid (Fig. 3E) or material isolated for degradation (Fig. 3D). Ultrastructural changes also included increased polymorphism of the nuclei (Fig. 3E). Immunogold labeling was performed for localization of HCV-like particles in transfected IHH. For this, transfected IHH (4 days in culture) were detached from collagen-coated petri dishes by a brief trypsin treatment, pelleted in a microcentrifuge, and fixed in 4% paraformaldehyde and 1% glutaraldehyde in PBS for 16 h at 4°C. After being washed with PBS, the cells were washed in distilled water, dehydrated in ethanol, and infiltrated with LR White resin (London Resin Company, Berkshire, United Kingdom). The cell pellets were polymerized in BEEM capsules (Ted Pella, Inc., Redding, Calif.) at –20°C under UV light. Thin sections were cut from blocks, collected on Formvar-coated nickel grids, and blocked with 1% fish gelatin and 1% bovine serum albumin (BSA) in PBS for 10 min. Sections were incubated for 2 h in a 1:100 dilution (titrated beforehand for best results) of monoclonal antibody to E1 glycoprotein (305/C3) or normal mouse IgG in PBS containing 0.1% BSA, washed in PBS containing 0.1% BSA, and incubated for 1 h in protein A-10-nm colloidal gold (CG) diluted at a ratio of 1:200 in PBS containing 0.1% BSA. After being washed with PBS, the grids were fixed for 3 min in glutaraldehyde, washed in distilled water, stained with uranyl acetate and lead citrate, and photographed with a JEOL 100 CX electron microscope. No clusters of CG particles were observed in the controls, which were stained with normal mouse IgG without the primary antibody or were mock-transfected and stained with the 305/C3 monoclonal antibody. Several hundred cells were evaluated in each case. Immunogold labeling with E1-specific monoclonal antibody demonstrated the presence of HCV-like particles and E1 protein in IHH. Numerous labeled virus-like particles were observed in the cytoplasm (Fig. 3A and E) and near the plasma membrane (Fig. 3C) of H77 RNA-transfected IHH. The labeled particles were 50 nm in diameter. Extensive labeling was also associated with the rough endoplasmic reticulum, consistent with the synthesis of E1 viral protein (Fig. 3B). In addition, we observed cytoplasmic autophagic vacuoles which contained gold-labeled virus-like particles (Fig. 3D).
Processing of cells into LR White resin for immunogold localization omits the conventional osmium tetroxide fixation step to preserve antigenicity but results in reduced tissue contrast. In addition, the identification of virus particles by immunogold labeling at the ultrastructural level can be tricky. For this, we carried out a series of control experiments to ensure labeling specificity. First, we observed clusters of CG on virus-like particles and single CG particles in the endoplasmic reticulum in several independent anti-E1-labeling experiments. Second, H77/GND RNA-transfected IHH (negative controls) showed no such clusters of CG in the cytoplasm or single CG particles localized along the endoplasmic reticulum or membranes (Fig. 3F). Third, incubation of sections of HCV genome-transfected IHH with normal mouse IgG at IgG concentrations similar to those used for the anti-E1 antibody did not result in any specific immunogold labeling. Fourth, omitting anti-E1 antibody did not result in any specific immunogold labeling. Finally, CG particles in the anti-E1-labeling experiments were primarily confined to cells and were not observed to any degree in the spaces around cells, again suggesting the labeling was specific for E1 protein in cells. Thus, the appearance of virus-like particles in RNA-transfected IHH indicated that HCV 1a replicates and assembles as virus particles.
Infection of IHH by HCV from culture medium. We next examined the presence of HCV in IHH cell culture medium. On different days after transfection, culture medium was filtered through a 0.45-μm cellulose acetate membrane (Millipore, Bedford, Mass.), concentrated to 10- to 20-fold by Millipore ultrafiltration (100-kDa cut off), and used for detection of the HCV genomic sequence by RT-PCR (Fig. 4A). The presence of HCV 5'UTR was detected in culture medium from HCV genome-transfected IHH but not from polymerase-defective HCV RNA-transfected IHH. We obtained 1.1 x 108 genome copies/ml of culture medium using real-time RT-PCR, as described recently (33). Culture supernatant collected for up to 7 days suggested that the peak HCV genome copy number occurred between 4 and 5 days after transfection.
Next, we determined whether the culture medium contained infectious HCV. For this, culture media was serially twofold diluted and inoculated into nave IHH. Cells were incubated for 4 h, washed, and incubated with fresh media for 3 days before indirect immunofluorescence was performed to determine the number of focus-forming units (FFU)/ml of NS5a (H77 clone) or NS3 (JFH1 clone), as recently described (33). Nuclear staining was performed using TO-PRO3-iodide (Molecular Probes), and cells were mounted for confocal laser scanning microscopy (model 1024; Bio-Rad). Figure 4B shows infection of IHH by H77 or JFH1 and is representative of our results. Fluorescent cells were counted, and the counts were correlated with the number of dilutions of cell culture media to determine FFU/ml of H77 and JFH1 clones. We observed 4.5 x 104 to 1 x 105 FFU/ml of H77 and JFH1 clones in the cell culture media 5 days after transfection.
We transfected in vitro-transcribed H77 or JFH1 RNA into IHH and isolated the RNA from the transfected cells. Culture supernatant was also collected for isolation of RNA and determination of infectivity (FFU/ml). Real-time PCR suggested that maximal HCV RNA accumulation from H77 occurred at the intracellular level on day 2 and declined on day 5 (Fig. 4C). We observed a higher genome copy number and infectious virus titer on day 4. Similarly, JFH1 RNA-transfected IHH supernatant displayed a peak genome copy number per ml of 108 and infectivity of 7 x 104 FFU/ml on day 4.
HCV-infected patient serum (OP1843) displaying neutralizing activity against the vesicular stomatitis virus/HCV pseudotype (19) was used in determining neutralization of cell culture-grown HCV. Serum from a healthy volunteer was used as a negative control in HCV neutralization assays. A twofold serial dilution of heat-inactivated serum was incubated with 100 FFU of HCV generated from the H77 clone at 37°C for 30 min. The virus-serum mixture was added to nave IHH and incubated for 3 days. Neutralization was determined by measuring inhibition of NS5a protein expression by immunofluorescence. The results are shown as the percent inhibition based on focus-forming units per milliliter (Fig. 4D). Infectivity (60%) was inhibited by prior incubation of HCV in culture medium with the patient serum at a 1/50 dilution. Inhibition was also observed at different dilutions of sera from three other HCV-infected patients. In contrast, sera from four healthy individuals did not inhibit infectivity at a 1/10 dilution. These results suggest that infectious HCV particles released in the culture medium are neutralized by specific antibodies.
HCV RNA is directly translated, and the precursor viral polypeptide is cleaved proteolytically to form individual proteins. The replicase complex amplifies the RNA via a minus-strand intermediate. Plus-strand RNA progeny are packaged into virus particles and acquire their envelopes probably by budding into the lumen of the endoplasmic reticulum. HCV particles are likely to be exported via the constitutive secretory pathway. Based on this working principle, we have shown in this report that IHH support HCV genome replication and protein expression from genotype 1a. Immunogold labeling using a monoclonal antibody demonstrated localization of HCV E1 glycoprotein in the rough endoplasmic reticulum and the formation of virus-like particles. We transferred culture media of HCV-replicating cells into nave IHH, and HCV infection was detected by RT-PCR and indirect immunofluorescence. We have also observed JFH1 replication and virus growth in IHH. The infectious units appeared to be similar for JFH1 grown in Huh-7 cells and in its derivatives. JFH1 may replicate with a higher efficiency than H77 at the RNA level in Huh-7 cells or its derivatives. However, we focused on determining the generation of infectious HCV from H77 and JFH1 in IHH. In our experimental system, we observed that virus genome copies of H77 and JFH1 were at similar levels in H77 and JFH1 RNA-transfected culture supernatant. The number of focus-forming units per milliliter of H77 and JFH1 was also similar. We did not purify virus particles for negative staining due to the relatively low number of infectious units in the culture media. Three different groups of investigators have reported different densities of HCV genotype 2a particles. Zhong et al. (33) observed peak infectivity at an apparent density of 1.105 gm/ml, and Wakita et al. (29) observed peak infectivity at a density of 1.15 gm/ml. Lidenbach et al. (15) observed a broad distribution of virus infectivity over a range of 1.01 to 1.12 gm/ml. A similar finding suggesting variation of between 1.06 and 1.16 gm/ml in buoyant density of cell culture-grown HCV genotype 2a was reported by Cai et al. (5). HCV is known to associate with serum immunoglobulin and lipoproteins (24). We have observed HCV infectivity within a density range of 1.09 to 1.12 in sucrose gradients, which did not correlate with highest copy number of virus genomic RNA (data not shown).
Recently, HCV production from a HCV-ribozyme construct of genotype 1a (clone H77) in Huh-7 cells was reported, although the infectivity of the virus was not determined (8). Virus genome replication and assembly are multistep processes and are influenced by the intracellular milieu. Inhibition of host cell growth and induction of cytokines, such as interferons, may have an impact on virus replication (3). Our study supports proof of the concept of HCV replication and assembly of genotype 1a in IHH. To our knowledge, this is the first report describing the generation of cell culture-grown HCV from genotype 1a. We speculate that cellular defense mechanisms against HCV infection are attenuated or compromised in IHH. Further studies may help to unravel the specific mechanisms for growth of HCV in IHH and to address important biological questions about the life cycle of HCV. Studies are in progress to determine the factors influencing virus growth, such as serial passage for adaptation in IHH, mutations at specific sites on the HCV genome, and selection of cell populations for attenuated protective mechanisms. We will also characterize the biophysical properties of cell culture-grown HCV and its infectivity in available animal models in the near future.
ADDENDUM While our manuscript was under revision, Yi et al. (31) reported the growth of H77-S in Huh-7.5 cells.
ACKNOWLEDGMENTS
We are grateful to Charles M. Rice for providing the full-length H77 clone, Michael Houghton for the E1 monoclonal antibody, Arvind Patel for antiserum to core protein, George Luo for monoclonal antibody to NS3 protein, and Leonard E. Grosso for detection of HCV RNA. We appreciate the helpful suggestions of Richard W. Compans concerning electron microscopy and Francis V. Chisari in determining virus infectivity. We thank Lin Cowick for preparation of the manuscript.
This work was supported by research grants AI45144 (R.B.R.) and CA85486 (R.R.) from the National Institutes of Health. T.W. was partly supported by grants from the Ministry of Health, Labor and Welfare of Japan, the Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (NIBIO), and Research on Health Sciences focusing on Drug Innovation from the Japan Health Sciences Foundation.
T.K. and A.B. contributed equally to this study.
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