Infected HepG2 Cells Research Articles

Overexpression of CYP27 in hepatic and extrahepatic cells: role in the regulation of cholesterol homeostasis.

In the liver, sterol 27-hydroxylase (CYP27) participates in the classic and alternative pathways of bile acid biosynthesis from cholesterol (Chol). In extrahepatic tissues, CYP27 converts intracellular Chol to 27-hydroxycholesterol (27OH-Chol), which may regulate the activity of 3-hydroxy-3-methylglutaryl CoA reductase (HMG-CoA-R). This study attempts to better define the role of CYP27 in the maintenance of Chol homeostasis in hepatic and extrahepatic cells by overexpressing CYP27 in Hep G2 cells and Chinese hamster ovary (CHO) cells through infection with a replication-defective recombinant adenovirus encoding for CMV-CYP27. After infection, CYP27 mRNA and protein levels increased dramatically. CYP27 specific activity also increased two- to fourfold in infected cells (P < or = 0.02), with a marked increase in conversion of [(14)C]Chol to [(14)C]27OH-Chol (approximately 150%; P < or = 0.01). Accumulation of 27OH-Chol in CHO cells was associated with a 50% decrease in HMG-CoA-R specific activity (P < or = 0.02). In infected Hep G2 cells, the significant increase in bile acid synthesis (46%; P < or = 0.006), which prevented the accumulation of intracellular 27OH-Chol, resulted in increased HMG-CoA-R activity (183%; P < or = 0.02). Overexpression of CYP27 in Hep G2 cells also increased acyl CoA-cholesterol acyltransferase (71%, P < or = 0.02) and decreased cholesteryl ester hydrolase (55%, P < or = 0.02). In conclusion, CYP27 generates different physiological responses depending on cell type and presence or absence of bile acid biosynthetic pathways.

Limited proteolysis induces woodchuck hepatitis virus infectivity for human HepG2 cells

Previous work from our laboratory has shown that digestion of hepatitis B virus (HBV) with V8 protease rendered the virus infectious for human hepatoblastoma cell line (HepG2). It was hypothesized that the cleavage exposes a 16 amino acid region that includes a consensus ‘fusion’ motif necessary to mediate infectivity. Since woodchuck hepatitis virus (WHV) and HBV possess significant homology in this region of their envelope proteins, including the V8 protease cleavage site, the possibility that WHV infectivity for HepG2 cells could be induced by V8 digestion was explored. WHV isolated from the serum of chronically infected woodchucks, digested with V8 protease, was shown to loose its preS domain. V8 digested WHV eluted from gel filtration columns with a size similar to that of undigested virus, suggesting that digestion with V8 protease did not cause significant changes in virion size. The amount of progeny virus secreted into the culture medium following infection of HepG2 cells with V8 digested WHV reached 2.5 pg/ml, after 8 days. Moreover, WHV DNA replicative intermediates could be detected in the cells following infection with protease digested, but not undigested, viruses. These data suggest that protease modification of WHV, a non-human virus, induced infectivity for human tissue culture cells. These results are consistent with the hypothesis that exposure of an amino acid region of the envelope polypeptide that contains a consensus fusion motif is important in Hepadnavirus entry.

Retention and degradation of N-glycoproteins in the rough endoplasmic reticulum.

Recent studies have shown that newly synthesized proteins and glycoproteins are submitted to a quality control mechanism in the rough endoplasmic reticulum (ER). In this report we present two models: One model will illustrate a transient retention in rough ER leading to a further degradation of glycoproteins in the cytosol, (soluble alkaline phosphatase expressed in Man-P-Dol deficient CHO cells lines). The second model will illustrate a strict retention of glycoproteins in rough ER without degradation nor recycling through the Golgi (E1, E2 glycoproteins of Hepatitis C virus in stably transfected UHCV-11.4 cells and in infected Hep G2 cells). In both cases, oligomannoside structures are markers of these phenomena, either as free soluble released oligomannosides in the case of degradation, or as N-linked oligomannosides for strict retention in rough ER.

Adenovirus-mediated regulable target gene expression in vivo.

To regulate expression of a transferred gene in response to an exogenous compound, we have combined a high capacity adenoviral vector devoid of all viral coding sequences with a regulatory system that can be used to express a target gene in vivo in a selected site and at a desired time. This system uses a chimeric transactivator, GLp65, which consists of a mutated progesterone receptor-ligand binding domain fused to the GAL4 DNA binding domain and part of the activation domain of the human p65 protein, a component of the NF-kappaB complex. In the presence of the antiprogestin mifepristone, this chimeric regulator binds to a target gene containing the 17-mer GAL4 binding site, resulting in an efficient ligand-inducible transactivation of the target gene. We inserted the regulator GLp65 and a regulable human growth hormone target gene containing the 17-mer GAL4 binding site into the same adenoviral vector. To obtain tissue-specific expression of the target gene, we coupled the regulator to a liver-specific promoter. Infection of HepG2 cells and experimental mice with the adenovirus resulted in consistently high induction levels of human growth hormone in the presence of mifepristone whereas the transgene expression was undetectable in the absence of the ligand. Taken together, our regulable adenoviral vector represents an important tool for transgene regulation that can be used for potentially diverse applications, ranging from tissue-specific gene expression in transgenic animals to human gene therapy.

Hepatitis B virus replication in human HepG2 cells mediated by hepatitis B virus recombinant baculovirus.

A novel transient mechanism for studying hepatitis B virus (HBV) gene expression and replication using recombinant HBV baculovirus to deliver the HBV genome to HepG2 cells was generated. In HBV baculovirus infected HepG2 cells, HBV transcripts, and intracellular and secreted HBV antigens are produced; replication occurs as evidenced by the presence of high levels of intracellular replicative intermediates and protected HBV DNA in the medium. Density-gradient analysis of extracellular HBV DNA indicated that the DNA was contained predominantly in enveloped HBV virions. Covalently closed circular (CCC) DNA is present indicating that, in this system, HBV core particles are capable of delivering newly synthesized HBV genomes back into the nuclei of infected cells. HBV gene expression is driven exclusively from endogenous promoters. Levels of HBV gene expression and replication can be achieved in HBV baculovirus-infected HepG2 cells which far exceed levels found in HepG2 2.2.15 cells. HBV baculovirus infection of HepG2 cells lends itself readily to experimental manipulation as follows: 1) HBV expression can be initiated any time relative to seeding of HepG2 cells; 2) levels of HBV replication can be regulated over a wide range simply by changing the baculovirus multiplicity of infection; 3) HBV replication is readily detectable by one day post infection with HBV baculovirus and persists at least through day eleven post infection; and (4) the transient nature of the infection can be extended and/or enhanced by superinfecting the cultures. We conclude that infection of HepG2 cells by HBV recombinant baculovirus represents a simple to use and highly flexible system for studying the effects of antivirals and/or cytokines on HBV production and for understanding HBV replication and pathogenesis at the molecular level.

Human biliary glycoproteins function as receptors for interspecies transfer of mouse hepatitis virus.

A variant Mouse Hepatitis virus (MHV), designated MHV-H2, was isolated by serial passage in mixed cultures of permissive DBT cells and nonpermissive Syrian Hamster Kidney (BHK) cells. MHV-H2 replicated efficiently in hamster, mouse, primate kidney (Vero, Cos 1, Cos 7), and human adenocarcinoma (HRT) cell lines but failed to replicate in porcine testicular (ST), feline kidney (CRFK), and canine kidney (MDCK) cells. To understand the molecular basis for coronavirus cross-species transfer into human cell lines, the replication of MHV-H2 was studied in hepatocellular carcinoma (HepG2) cells which expressed high levels of the human homologue of the normal murine receptor, biliary glycoprotein (Bgp). MHV-H2 replicated efficiently in human HepG2 cells, at low levels in breast carcinoma (MCF7) cells, and poorly, if at all, in human colon adenocarcinoma (LS 174T) cell lines which expressed high levels of carcinoembryonic antigen (CEA). These data suggested that MHV-H2 may utilize the human Bgp homologue as a receptor for entry into HepG2 cells. To further study MHV-H2 receptor utilization in human cell lines, blockade experiments were performed with a panel of different monoclonal or polyclonal antiserum directed against the human CEA genes. Pretreatment of HepG2 cells with a polyclonal antiserum directed against all CEA family members, or with a monoclonal antibody, Kat4c (cd66abde), directed against Bgp1, CGM6, CGM1a, NCA and CEA, significantly reduced virus replication and the capacity of MHV-H2 to infect HepG2 cells. Using another panel of monoclonals with more restricted cross reactivities among the human CEA's, Col-4 and Col-14, but not B6.2 B1.13, Col-1, Col-6 and Col-12 blocked MHV-H2 infection in HepG2 cells. These antibodies did not block sindbis virus (SB) replication in HepG2 cells, or block SB, MHV-A59 or MHV-H2 replication in DBT cells. Monoclonal antibodies Col-4, Col-14, and Kat4c (cd66abde) all reacted strongly with human Bgp and CEA, but displayed variable binding patterns with other CEA genes. Following expression of human Bgp in normally nonpermissive porcine testicular (ST) and feline kidney (CRFK) cells, the cells became susceptible to MHV-H2 infection. These data suggested that phylogenetic homologues of virus receptors represent natural conduits for virus xenotropism and cross-species transfer.

Human herpesvirus 6 induces IL-8 gene expression in human hepatoma cell line, Hep G2

The infectivity of human herpesvirus 6 (HHV-6) in a human hepatoma cell line, Hep G2 cells, and the effect of HHV-6 on production of inflammatory cytokines in these cells were examined to analyze pathogenesis of HHV-6 in the liver. We demonstrated that Hep G2 cells were susceptible to infection with HHV-6, and produced infectious virus. Moreover, infection of Hep G2 cells by HHV-6 induced the expression of IL-8 mRNA, but not IL-1 beta. The effect on induction of IL-8 gene expression was observed only in Hep G2 cells infected with infectious virus, whereas both heat-inactivated HHV-6 and UV-irradiated HHV-6 did not change the IL-8 mRNA level in these cells. These data suggest that HHV-6 may induce the cytokine-mediated inflammatory response by infecting liver cells, which could result in liver dysfunction in vivo.

Protease-induced infectivity of hepatitis B virus for a human hepatoblastoma cell line

The human hepatoblastoma cell line HepG2 produces and secretes hepatitis B virus (HBV) after transfection of cloned HBV DNA. Intact virions do not infect these cells, although they attach to the surface of the HepG2 cell through binding sites in the pre-S1 domain. Entry of enveloped virions into the cell often requires proteolytic cleavage of a viral surface protein that is involved in fusion between the cell membrane and the viral envelope. Recently, we observed pre-S-independent, nonspecific binding between hepatitis B surface (HBs) particles and HepG2 cells after treatment of HBs antigen particles with V8 protease, which cleaves next to a putative fusion sequence. Chymotrypsin removed this fusion sequence and did not induce binding. In this study, we postulate that lack of a suitable fusion-activating protease was the reason why the HepG2 cells were not susceptible to HBV. To test this hypothesis, virions were partially purified from the plasma of HBV carriers and treated with either staphylococcal V8 or porcine chymotrypsin protease. Protease-digested virus lost reactivity with pre-S2-specific antibody but remained morphologically intact as determined by electron microscopy. After separation from the proteases, virions were incubated with HepG2 cells at pH 5.5. Cultures inoculated with either intact or chymotrypsin-digested virus did not contain detectable levels of intracellular HBV DNA at any time following infection. However, in cultures inoculated with V8-digested virions, HBV-specific products, including covalently closed circular DNA, viral RNA, and viral pre-S2 antigen, could be detected in a time-dependent manner following infection. Immunofluorescence analysis revealed that 10 to 30% of the infected HepG2 cells produced HBV antigen. Persistent secretion of virus by the infected HepG2 cells lasted at least 14 days and was maintained during several reseeding steps. The results show that V8-digested HBV can productively infect tissue cultures of HepG2 cells. It is suggested that proteolysis-dependent exposure of a fusion domain within the envelope protein of HBV is necessary during natural infection.

Expression of Human Recombinant Granzyme A Zymogen and Its Activation by the Cysteine Proteinase Cathepsin C

Human granzyme A is one of the serine proteinases present in the granules of cytotoxic T lymphocytes and natural killer cells. Granzymes are synthesized as inactive proenzymes with an amino-terminal prodipeptide, which is processed during transport of granzymes to the cytotoxic granules, where they are stored as active proteinases. In this study, we explored the possibility of producing recombinant granzymes. Recombinant human granzyme A zymogen was expressed in several eukaryotic cell lines (HepG2, Jurkat, and COS-1) after infection with a recombinant vaccinia virus containing full-length granzyme A cDNA. Immunoblot analysis of cell lysates showed that all infected cells produced a disulfide-linked homodimer of identical molecular weight as natural granzyme A. Infected HepG2 cells produced the largest amount of this protease (approximately 160 times more than lymphokine activated killer (LAK) cells). The recombinant protein only had high mannose type oligosaccharides as did the natural protein. Although infected HepG2 and COS cells contained high granzyme A antigen levels, lysates from these cells did not show any granzyme A proteolytic activity. However, the inactive proenzyme could be converted into active granzyme A by incubation with the thiol proteinase cathepsin C (dipeptidyl peptidase I). This study is the first to demonstrate expression of an active recombinant human cytotoxic lymphocyte proteinase and conversion of inactive progranzyme A into an active enzyme by cathepsin C. We suggest that a similar approach can be used for the production of other granzymes and related proteinases.

Activated THP-1 cells depress mitochondrial respiration in HEP G2 cells infected with influenza B virus

Influenza B virus has been aetiologically linked to Reye Syndrome (RS), but the mechanism(s) by which this pathogen could disrupt liver metabolism and produce the hepatic mitochondrial injury characteristic of the syndrome are unknown. In this study, two mechanisms by which infection of hepatocytes with influenza B virus could disrupt cellular metabolism were investigated. (1) virus-induced increase in pro-oxidant iron with subsequent iron-induced lipid peroxidation (LP) and (2) increased membrane permeability. Hep G2 cells, a well-differentiated continuous human liver cell line derived from a hepatoblastoma, were infected with allantoic-fluid derived influenza B Lee/40 virus (AFDV) at a multiplicity of infection of 10 for 24 h; productive infection was confirmed by both haemagglutination of chick erythrocytes and by plaque assay. Infection of Hep G2 cells preloaded with 59Fe-transferrin resulted in increased release of 59Fe (153 ± 17% of controls, P <0·03). However, the iron released did not result in increased LP (assessed by thiobarituric acid reactive substances; TBARS). To confirm that this lack of increase in TBARS was not due to insensitivity of the cell line to pro-oxidant iron, cells were exposed to 15 μM iron ascorbate for 60 min. Production of TBARS was increased (122 ± 4%) of controls, P <0·0003). Release of 51Cr from infected cells was also increased (128 ± 12% of controls, P<0·05); thus the infected cells exhibited a generalized increase in membrane permeability. However, infection did not depress mitochondrial respiration (as assessed by the formation of MTT-f3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide-formazan. To determine if the combination of viral infection and soluble products of activated macrophages would affect mitochondrial respiration, infected hepatocytes were expose to the supernatant fluid from THP-1 cells which had previously been incubated with lipopolysaccharide at 100 ng ml -1 for 18 h. This supernate did depress the formation of MTT-f (81 ± 5% of controls, P<0·03). We conclude that influenza B virus does productively infect Hep G2 cells, and does increase hepatocyte membrane permeability. This effect does not impair mitochondrial respiration directly. However, infection does act in concert with soluble products of activated macrophages to depress hepatic mitochondrial respiration. Whether this interaction can be explained by virus-induced permeability changes and/or other effects of infection deserves further investigation.

Effects of Hormones and Cysteine Protease Modulators on Infection of HEPG2 Cells by Plasmodium berghei Sporozoites In vitro Determined by ELISA Immunoassay

An enzyme-linked immunosorbent assay (ELISA) detecting a Plasmodium berghei liver-stage-specific protein Pbl-1 is described. The quantitative detection limits ranged from 0.01 to 0.05 microgram of parasite protein. Qualitatively the assay detected as little as 0.001 microgram Pbl-1 per well. Using the ELISA dexamethasone and insulin together was shown to promote higher parasite infections in HepG2 cells compared to unsupplemented medium. Anti-cowpea-protease cysteine inhibitor significantly increased hepatocyte invasion as compared to controls, whereas a significant decrease was recorded in the presence of the protease inhibitor E64. Partial involvement of cysteine proteases in HepG2 invasion by P. berghei sporozoites is therefore suggested.

Comparative growth of different rotavirus strains in differentiated cells (MA104, HepG2, and CaCo-2)

The production of viral antigen after infection of MA104, HepG2 (derived from human liver), and CaCo-2 (derived from human colon) cells with various cultivatable human and animal rotavirus strains was compared using immunofluorescence tests. All rotavirus strains examined expressed antigen in CaCo-2 cells and MA104 cells, but only some virus strains, namely, SA11-CI3 (simian), RRV (simian), CU-1 (canine), and Tyl (turkey), produced antigen in numbers of infected HepG2 cells comparable to infections in MA104 and CaCo-2 cells. FI-14 (equine), OSU (porcine), NCDV (bovine), and Ch2 (chicken) strains were found to infect moderate numbers of HepG2 cells. Most human rotaviruses (representing viruses in serotypes 1, 2, 3, 4, 8, and 9), a simian rotavirus variant (SA11-4F), lapine (Ala, C-11 and R-2) viruses and porcine (Gottfried) virus infections resulted either in no detectable antigen or antigen synthesis in a low percentage of HepG2 cells. Human rotavirus isolates obtained from the stool specimens of an immunocompromised child with rotavirus antigen in his liver showed two different patterns of replication in HepG2 cells. Examination of the replication of a subset of viruses in the liver and intestinal tissues of orally infected suckling mice showed the CU-1 and Ty1 strains replicated well, while the OSU and human rotavirus strains did not. These results indicate that growth restriction in HepG2 cells is not serotype-specific, and growth of a virus in HepG2 cells does not necessarily correlate with the hepatotropic potential of a virus strain. Factors that may influence these differences of virus infectivity in HepG2 cells are discussed.

Rotavirus genome segment 4 determines viral replication phenotype in cultured liver cells (HepG2)

One-step growth determinations were performed with five strains of rotavirus in HepG2, a cell line derived from human liver. Three virus strains (SA11-C13, SA11-C14, and RRV) replicated in HepG2 cells and attained yields 10- to 100-fold above input titers. Two virus strains (B223 and SA11-4F) failed to replicate above input titer. Analysis of reassortants that segregated the genes of parental virus pairs able and unable to replicate revealed that the HepG2 cell growth phenotype segregated with genome segment 4. Immunofluorescence analysis of infected HepG2 cells showed that the production of detectable antigen correlated with the growth phenotype and also segregated with genome segment 4. Thus, we conclude that (i) some virus strains were capable of replication in cultured liver cells while other strains could not replicate under identical conditions and that (ii) the inability of some virus strains to replicate resulted from a segment 4-associated block in replication before protein synthesis. These results are discussed in terms of what is known of the functions of VP4.

Lack of correlation between infection with reovirus 3 and extrahepatic biliary atresia or neonatal hepatitis

Infection with reovirus 3 (Reo-3) has been suggested as the cause of extrahepatic biliary atresia and idiopathic neonatal hepatitis, but confirmation has been lacking. Therefore we have searched for a specific anti-Reo-3 antibody response in the sera of patients with biliary atresia or neonatal hepatitis and for Reo-3 antigens in their hepatobiliary tissues. Sera from 23 infants with extrahepatic biliary atresia, 12 with neonatal hepatitis, 30 age-matched control patients with other liver diseases, and 55 control patients without liver disease were tested by an enzyme-linked immunosorbent assay for total (IgA, IgG, and IgM) anti-Reo-3 antibodies; sera of infants younger than 6 months of age were tested also for IgM anti-Reo-3 antibodies alone. There was no difference between either total or IgM anti-Reo-3 antibody levels in infants with extrahepatic biliary atresia or neonatal hepatitis and levels in control infants. Reo-3 antigens were not detected in the hepatobiliary tissues of 19 infants (18 with biliary atresia, one with neonatal hepatitis) by an immunoperoxidase method that readily demonstrated Reo-3 in control infected HEp-G2 cells. Our data do not support a relationship between neonatal liver diseases and infection with Reo-3.

Protein synthesis by HepG2 cells infected with influenza B virus.

Reye's syndrome is an acute hepatopathy and encephalopathy affecting children during the convalescent period of a viral infection, frequently influenza B. The role of influenza B in the pathogenesis of Reye's syndrome is unknown. To investigate this relationship, an in vitro system was designed to examine the interaction of influenza B virus with a cell line of human hepatocytes (HepG2) which retains many specific hepatic synthetic characteristics. HepG2 was capable of supporting a productive infection by influenza B, although greater virus inputs were required than in fully permissive Madin-Darby canine kidney cells. Because of the recent association of Reye's syndrome with aspirin use, the kinetics of influenza B growth were studied in the presence of acetylsalicylic acid (25 and 100 micrograms/ml) and were not found to be altered. Protein synthesis by HepG2 cells was decreased by 80% in influenza B-infected cells when compared to uninfected controls. Acetylsalicylic acid, 100 micrograms/ml, did not affect the rate of 35S-methionine incorporation by infected or uninfected HepG2 cells. The rates of synthesis of specific proteins (albumin, transferrin, and apoprotein B) by HepG2 cells were determined by immunoprecipitation of 35S-methionine labeled cell lysates. After 12 h of infection, synthesis of all three plasma proteins was decreased by 40-60%. These studies describe a useful system for delineation of mechanisms by which influenza B virus interacts with host hepatocytes and the host-cell protein synthetic machinery. The studies could be pertinent to the pathogenesis of Reye's syndrome.