Induction of hepatic injury by hepatitis C virus-specific CD8+ murine cytotoxic T lymphocytes in transgenic mice expressing the viral structural genes

In the present study, we generated killer cells specific for hepatitis C virus (HCV) structural protein by re-stimulation of immune spleen cells from H-2(d) haplotype transgenic (Tg) mice, expressing the core, E1, E2, and NS2 genes of HCV regulated by the Cre/loxP switching system. The generated killer cells were conventional CD8(+)L(d) class-I MHC molecule-restricted cytotoxic T lymphocytes (CTLs) and specific for the HCV E1 structural protein. Because the CTLs could also kill hepatocytes from the Tg mice expressing HCV structural proteins in vitro, we attempted to transfer those CTLs intravenously into interferon regulatory factor-1 (IRF-1) negative, CD8-deficient Tg mice representing the HCV structural genes on hepatocytes to examine whether the inoculated CD8(+) CTLs can eliminate hepatocytes expressing the HCV genes in vivo. We observed an elevation of serum ALT level as well as damage of the liver tissue histologically. To our knowledge, this is the first demonstration to show that HCV-specific CD8(+) CTLs specifically attack hepatocytes expressing the HCV structural proteins both in vitro and in vivo.     

Oligomeric structure of the human asialoglycoprotein receptor: nature and stoichiometry of mutual complexes containing H1 and H2 polypeptides assessed by fluorescence photobleaching recovery

The interactions between H1 and H2, the two polypeptides comprising the human asialoglycoprotein receptor (ASGP-R), were investigated by immunofluorescence and lateral mobility measurements combined with antibody-mediated cross-linking and immobilization. Immunofluorescence microscopy revealed two ASGP-R populations on the cell surface, one homogeneously distributed and the other in micropatches. This was observed both in stably transfected NIH 3T3 lines expressing H1 and/or H2, and in the human hepatoma cell line HepG2. In transfected cells expressing both polypeptides (the 1-7-1 line), H1 and H2 were colocalized in the same micro aggregates. Moreover, enhancement of the patching of, e.g., H1 by IgG-mediated crosslinking was accompanied by copatching of H2. To quantify H1-H2 complex formation, the lateral diffusion of H1 and H2 was measured at 12 degrees C (to avoid internalization) by fluorescence photobleaching recovery. H1 (or H2) was immobilized by crosslinking with specific IgG molecules; the other chain was labeled with fluorescent monovalent Fab' fragments, and is lateral mobility was measured. In HepG2 cells, immobilization of either H1 or H2 led to an equal immobilization of the other, indicating that all the mobile H1 and H2 are in stable heterooligomers. In 1-7-1 cells, immobilization of H2 immobilized H1 to the same degree, but immobilization of H1 reduced the mobile fraction of H2 only by 2/3. Thus, in 1-7-1 cells all surface H1 molecules are associated with H2, but 1/3 of the H2 population is independent of H1. From these data and from measurements of the relative surface densities of H1 and H2, conclusions are drawn regarding the oligomeric structure and stoichiometry of the ASGP-R.     

Activation of the CKI-CDK-Rb-E2F pathway in full genome hepatitis C virus-expressing cells

Hepatitis C virus (HCV) causes persistent infection in hepatocytes, and this infection is, in turn, strongly associated with the development of hepatocellular carcinoma. To clarify the mechanisms underlying these effects, we established a Cre/loxP conditional expression system for the precisely self-trimmed HCV genome in human liver cells. Passage of hepatocytes expressing replicable full-length HCV (HCR6-Rz) RNA caused up-regulation of anchorage-independent growth after 44 days. In contrast, hepatocytes expressing HCV structural, nonstructural, or all viral proteins showed no significant changes after passage for 44 days. Only cells expressing HCR6-Rz passaged for 44 days displayed acceleration of CDK activity, hyperphosphorylation of Rb, and E2F activation. These results demonstrate that full genome HCV expression up-regulates the CDK-Rb-E2F pathway much more effectively than HCV proteins during passage.     

E2F/p107 and E2F/p130 complexes are regulated by C/EBPalpha in 3T3-L1 adipocytes.

We have previously found that loss of C/EBPalpha in hepatocytes of newborn livers leads to increased proliferation, to a reduction in p21 protein levels and to an induction of S phase-specific E2F/p107 complexes. In this paper, we investigated C/EBPalpha-dependent regulation of E2F complexes in a well-characterized cell line, 3T3-L1, and in stable transformants that conditionally express C/EBPalpha. C/EBPalpha and C/EBPbeta proteins are induced in 3T3-L1 preadipocytes during differentiation with different kinetics and potentially may regulate E2F/Rb family complexes. In pre-differentiated cells, three E2F complexes are observed: cdk2/E2F/p107, E2F/p130 and E2F4. cdk2/E2F/p107 complexes are induced in nuclear extracts of 3T3-L1 cells during mitotic expansion, but are not detectable in nuclear extracts at later stages of 3T3-L1 differentiation. The reduction in E2F/p107 complexes is associated with elevation of C/EBPalpha, but is independent of C/EBPbeta expression. Bacterially expressed, purified His-C/EBPalpha is able to disrupt E2F/p107 complexes that are observed at earlier stages of 3T3-L1 differentiation. C/EBPbeta, however, does not disrupt E2F/p107 complexes. A short C/EBPalpha peptide with homology to E2F is sufficient to bring about the disruption of E2F/p107 complexes from 3T3-L1 cells in vitro. Induction of C/EBPalpha in stable 3T3-L1 clones revealed that C/EBPalpha causes disruption of p107/E2F complexes in these cells. In contrast, E2F/p130 complexes are induced in cells expressing C/EBPalpha. Our data suggest that induction of p130/E2F complexes by C/EBPalpha occurs via up-regulation of p21, which, in turn, leads to association with and inhibition of, cdk2 kinase activity. The reduction in cdk2 kinase activity correlates with alterations of p130 phosphorylation and with induction of p130/E2F complexes in 3T3-L1 stable clones. Our data suggest two pathways of C/EBPalpha-dependent regulation of E2F/Rb family complexes: disruption of S phase-specific E2F/p107 complexes and induction of E2F/p130 complexes.     

The minor subunit splice variants, H2b and H2c, of the human asialoglycoprotein receptor are present with the major subunit H1 in different hetero-oligomeric receptor complexes

The hepatic asialoglycoprotein receptor (ASGP-R) is an endocytic receptor that mediates the internalization of desialylated glycoproteins and their delivery to lysosomes. The human ASGP-R is a hetero-oligomeric complex composed of H1 and H2 subunits. There are three naturally occurring H2 splice variants, designated H2a, H2b, and H2c, although the expression of the H2c protein had not been reported. Following deglycosylation of purified ASGP-R, we detected the H2b and H2c proteins in HepG2 and HuH-7 hepatoma cells, using an antibody directed against a COOH-terminal peptide common to all H2 isoforms (anti-H2-COOH) and another antibody against a 19-amino acid cytoplasmic insert found only in H2b (anti-H2-Cyto19). H1 and both H2b and H2c were co-purified by affinity chromatography, using asialo-orosomucoid (ASOR)-, anti-H1-, or anti-H2-COOH-Sepharose, whereas only H1 and H2b were immunoprecipitated with anti-H2-Cyto19. These results indicate that H2b and H2c are not present in the same ASGP-R complexes with H1. Similar to the H2b isoform, H2c was also palmitoylated, indicating that the 19-residue cytoplasmic insert does not regulate palmitoylation. Stably transfected SK-Hep-1 cell lines expressing ASGP-R complexes containing H1 and either H2b or H2c had similar binding affinities for ASOR and endocytosed and degraded ASOR at similar rates. The pH dissociation profiles of ASOR.ASGP-R complexes were also identical for complexes containing either H2b or H2c. We conclude that the H2b and H2c isoforms are both functional but are not present with H1 in the same hetero-oligomeric ASGP-R complexes. This structural difference between two functional subpopulations of ASGP-Rs may provide a molecular basis for the existence of two different pathways, designated State 1 and State 2, by which several types of recycling receptors mediate endocytosis.     

High,Broad, Polyfunctional,and Durable T Cell Immune Responses Induced in Mice by a Novel Hepatitis C Virus (HCV) Vaccine Candidate (MVA-HCV) Based on Modified Vaccinia Virus Ankara Expressing the Nearly Full-Length HCV Genome

A major goal in the control of hepatitis C infection is the development of a vaccine. Here, we have developed a novel HCV vaccine candidate based on the highly attenuated poxvirus vector MVA (referred to as MVA-HCV) expressing the nearly full-length (7.9-kbp) HCV sequence, with the aim to target almost all of the T and B cell determinants described for HCV. In infected cells, MVA-HCV produces a polyprotein that is subsequently processed into the structural and nonstructural HCV proteins, triggering the cytoplasmic accumulation of dense membrane aggregates. In both C57BL/6 and transgenic HLA-A2-vaccinated mice, MVA-HCV induced high, broad, polyfunctional, and long-lasting HCV-specific T cell immune responses. The vaccine-induced T cell response was mainly mediated by CD8 T cells; however, although lower in magnitude, the CD4⁺ T cells were highly polyfunctional. In homologous protocol (MVA-HCV/MVA-HCV) the main CD8⁺ T cell target was p7+NS2, whereas in heterologous combination (DNA-HCV/MVA-HCV) the main target was NS3. Antigenic responses were also detected against other HCV proteins (Core, E1-E2, and NS4), but the magnitude of the responses was dependent on the protocol used. The majority of the HCV-induced CD8⁺ T cells were triple or quadruple cytokine producers. The MVA-HCV vaccine induced memory CD8⁺ T cell responses with an effector memory phenotype. Overall, our data showed that MVA-HCV induced broad, highly polyfunctional, and durable T cell responses of a magnitude and quality that might be associated with protective immunity and open the path for future considerations of MVA-HCV as a prophylactic and/or therapeutic vaccine candidate against HCV.     

Prohibitin deficiency causes opposing lipid metabolism between 3T3-L1 adipocytes and Clone 9 hepatocytes

Prohibitin (PHB) is a highly conserved protein in eukaryotic cells that are present in multiple cellular compartments and has potential roles as a tumor suppressor, an anti-proliferative protein, a regulator of cell-cycle progression and in apoptosis. In the present study, we generated PHB-deficient 3T3-L1 adipocytes and Clone 9 (C9) hepatocytes by oligonucleotide siRNA and investigated whether PHB affect lipid metabolism. It was revealed that PHB deficiency caused opposing lipid metabolism between the two cell models. PHB deficiency increased expression of adipogenic, lipogenic, and other lipid metabolic proteins in 3T3-L1 adipocytes, whereas significantly decreased the levels of those proteins in C9 cells. Collectively, PHB deficiency promoted lipid metabolism in 3T3-L1 adipocytes while it aggravated lipid metabolism in C9 hepatocytes.     

Nonpalmitoylated human asialoglycoprotein receptors recycle constitutively but are defective in coated pit-mediated endocytosis,dissociation, and delivery of ligand to lysosomes

The hepatic asialoglycoprotein receptor (ASGP-R) internalizes desialylated glycoproteins via the clathrin-coated pit pathway and mediates their delivery to lysosomes for degradation. The human ASGP-R contains two subunits, H1 and H2. Cytoplasmic residues Cys(36) in H1, as well as Cys(54) and Cys(58) in H2 are palmitoylated (Zeng, F.-Y., and Weigel, P. H. (1996) J. Biol. Chem. 271, 32454). In order to study the function(s) of ASGP-R palmitoylation, we mutated these Cys residues to Ser and generated stably transfected SK-Hep-1 cell lines expressing either wild-type or nonpalmitoylated ASGP-Rs. Compared with wild-type ASGP-Rs, palmitoylation-defective ASGP-Rs showed normal ligand binding, intracellular distribution and trafficking patterns, and pH-induced dissociation profiles in vitro. However, continuous ASOR uptake, and the uptake of prebound cell surface ASOR were slower in cells expressing palmitoylation-defective ASGP-Rs than in cells expressing wild-type ASGP-Rs. Unlike native ASGP-Rs in hepatocytes or hepatoma cells, which mediate endocytosis via the clathrin-coated pit pathway and are almost completely inhibited by hypertonic medium, only approximately 40% of the ASOR uptake in SK-Hep-1 cells expressing wild-type ASGP-Rs was inhibited by hyperosmolarity. This result suggests the existence of an alternate nonclathrin-mediated internalization pathway, such as transcytosis, for the entry of ASGP-R.ASOR complexes into these cells. In contrast, ASOR uptake mediated by cells expressing palmitoylation-defective ASGP-Rs showed only a marginal difference under hypertonic conditions, indicating that most of the nonpalmitoylated ASGP-Rs were not internalized and processed normally through the clathrin-coated pit pathway. Furthermore, cells expressing wild-type ASGP-Rs were able to degrade the internalized ASOR, whereas ASOR dissociation was impaired and degradation was barely detectable in cells expressing nonpalmitoylated ASGP-Rs. We conclude that palmitoylation of the ASGP-R is required for its efficient endocytosis of ligand by the clathrin-dependent endocytic pathway and, in particular, for the proper dissociation and delivery of ligand to lysosomes.     

Interaction of hepatitis C virus-like particles and cells: a model system for studying viral binding and entry

Hepatitis C virus-like particles (HCV-LPs) containing the structural proteins of HCV H77 strain (1a genotype) was used as a model for HCV virion to study virus-cell interaction. HCV-LPs showed a buoyant density of 1.17 to 1.22 g/cm(3) in a sucrose gradient and formed double-shelled particles 35 to 49 nm in diameter. Flow cytometry analysis by an indirect method (detection with anti-E2 antibody) and a direct method (use of dye-labeled HCV-LPs) showed that HCV-LPs binds to several human hepatic (primary hepatocytes, HepG2, HuH7, and NKNT-3) and T-cell (Molt-4) lines. HCV-LPs binding to cells occurred in a dose- and calcium-dependent manner and was not mediated by CD81. Scatchard plot analysis suggests the presence of two binding sites for HCV-LPs with high (K(d) approximately 1 microg/ml) and low (K(d) approximately 50 to 60 microg/ml) affinities of binding. Anti-E1 and -E2 antibodies inhibited HCV-LPs binding to cells. While preincubation of HCV-LPs with very-low-density lipoprotein (VLDL), low-density lipoprotein (LDL), or high-density lipoprotein (HDL) blocked its binding to cells, preincubation of cells with VLDL, LDL, HDL, or anti-LDL-R antibody did not. Confocal microscopy analysis showed that, after binding to cells, dye-labeled HCV-LPs were internalized into the cytoplasm. This process could be inhibited with anti-E1 or anti-E2 antibodies, suggesting that E1 and E2 proteins mediate HCV-LPs binding and, subsequently, their entry into cells. Altogether, our results indicate that HCV-LPs can be used to further characterize the mechanisms involved in the early steps of HCV infection.     

Presence of asialoglycoprotein receptors in the Golgi complex in the absence of protein synthesis

In rat hepatocytes the Golgi complex contains a considerable amount of receptors for asialoglycoproteins (ASGP-R). To establish whether the presence of ASGP-R in the Golgi complex originate from de novo synthesis isolated rat hepatocytes were incubated with 100 micrograms/ml cycloheximide to stop protein synthesis. Provided that protein synthesis was completely inhibited by cycloheximide, uptake and degradation of ligand (asialo-orosomucoid) were unaffected. Also intracellular transport of newly synthesized proteins, as determined by monitoring biosynthesis and intracellular transport of albumin and ASGP-R, was not affected. After culturing the cells for 3.5 h in the presence of cycloheximide, no more albumin could be detected in the Golgi complex with immunofluorescence microscopy. However, immunocytochemical assessment showed that the ASGP-R was still in the Golgi complex. These results suggest that the Golgi complex contains a pool of ASGP-R which is independent of neosynthesis for several hours.     

Effect of differentiation on the adenylate cyclase system of 3T3-C2 and 3T3-L1 cells. Determination of choleragen substrates in differentiating 3T3-L1 and nondifferentiating 3T3-C2 cells

3T3-L1 preadipocytes, when treated with 3-isobutyl-1-methylxanthine, dexamethasone, and insulin, differentiate into cells with the morphological and biochemical properties of adipocytes; the closely related 3T3-C2 cells, under identical conditions, exhibit a low frequency of adipocyte conversion. During differentiation, 3T3-L1 preadipocytes acquire an increased responsiveness to certain agonists (e.g. isoproterenol and adrenocorticotropic hormone) that influence lipolysis and lipogenesis through activation of adenylate cyclase, whereas 3T3-C2 cells do not. It has been suggested that changes in hormone responsiveness of 3T3-L1 cells during differentiation result from increased amounts of the guanyl nucleotide-binding protein of adenylate cyclase, as demonstrated by choleragen-catalyzed [32P]ADP ribosylation of 42 and 49-50-kilodalton particulate peptides. Particulate fractions from nondifferentiating 3T3-C2 cells, like those from 3T3-L1 cells, contained choleragen substrates of 42 and 46-47 (doublet) kilodaltons. Incubation of intact 3T3-L1 or 3T3-C2 cells with choleragen prior to preparation of particulate fractions prevented the subsequent in vitro choleragen-dependent [32P]ADP ribosylation of only these peptides. Increased incorporation of radioactivity into both the 42 and 46-47-kilodalton peptides was observed during differentiation of 3T3-L1 cells. However, a similar increase was also observed in nondifferentiating 3T3-C2 cells subjected to the differentiation protocol. Therefore, increased hormone responsiveness of 3T3-L1 adipocytes cannot be explained solely on the basis of increased labeling, and perhaps increased amounts, of the guanyl nucleotide-binding protein.     

Elimination of hepatitis C virus from hepatocytes by a selective activation of therapeutic molecules

To eliminate hepatitis C virus (HCV) from infected hepatocytes, we generated two therapeutic molecules specifically activated in cells infected with HCV. A dominant active mutant of interferon (IFN) regulatory factor 7 (IRF7) and a negative regulator of HCV replication, VAP-C (Vesicle-associated membrane protein-associated protein subtype C), were fused with the C-terminal region of IPS-1 (IFNβ promoter stimulator-1), which includes an HCV protease cleavage site that was modified to be localized on the ER membrane, and designated cIRF7 and cVAP-C, respectively. In cells expressing the HCV protease, cIRF7 was cleaved and the processed fragment was migrated into the nucleus, where it activated various IFN promoters, including promoters of IFNα6, IFNβ, and IFN stimulated response element. Activation of the IFN promoters and suppression of viral RNA replication were observed in the HCV replicon cells and in cells infected with the JFH1 strain of HCV (HCVcc) by expression of cIRF7. Suppression of viral RNA replication was observed even in the IFN-resistant replicon cells by the expression of cIRF7. Expression of the cVAP-C also resulted in suppression of HCV replication in both the replicon and HCVcc infected cells. These results suggest that delivery of the therapeutic molecules into the liver of hepatitis C patients, followed by selective activation of the molecules in HCV-infected hepatocytes, is a feasible method for eliminating HCV.     

Development of mouse hepatocyte lines permissive for hepatitis C virus (HCV)

The lack of a suitable small animal model for the analysis of hepatitis C virus (HCV) infection has hampered elucidation of the HCV life cycle and the development of both protective and therapeutic strategies against HCV infection. Human and mouse harbor a comparable system for antiviral type I interferon (IFN) induction and amplification, which regulates viral infection and replication. Using hepatocytes from knockout (ko) mice, we determined the critical step of the IFN-inducing/amplification pathways regulating HCV replication in mouse. The results infer that interferon-beta promoter stimulator (IPS-1) or interferon A receptor (IFNAR) were a crucial barrier to HCV replication in mouse hepatocytes. Although both IFNARko and IPS-1ko hepatocytes showed a reduced induction of type I interferons in response to viral infection, only IPS-1-/- cells circumvented cell death from HCV cytopathic effect and significantly improved J6JFH1 replication, suggesting IPS-1 to be a key player regulating HCV replication in mouse hepatocytes. We then established mouse hepatocyte lines lacking IPS-1 or IFNAR through immortalization with SV40T antigen. Expression of human (h)CD81 on these hepatocyte lines rendered both lines HCVcc-permissive. We also found that the chimeric J6JFH1 construct, having the structure region from J6 isolate enhanced HCV replication in mouse hepatocytes rather than the full length original JFH1 construct, a new finding that suggests the possible role of the HCV structural region in HCV replication. This is the first report on the entry and replication of HCV infectious particles in mouse hepatocytes. These mouse hepatocyte lines will facilitate establishing a mouse HCV infection model with multifarious applications.     

Low asialoglycoprotein receptor expression as markers for highly proliferative potential hepatocytes.

Development of a reliable method to isolate highly proliferative potential hepatocytes will provide insight into the molecular mechanisms of liver regeneration, as well as proving crucial for the development of a biohybrid artificial liver. The aim of this study is to isolate highly proliferative, e.g., progenitor-like, hepatocytes. To this end, we fractionated hepatocytes expressing low and high levels of the asialoglycoprotein receptor (ASGP-R) based on the difference in their adhesion to poly[N-p-vinylbenzyl-O-beta-d-galactopyranosyl-(1-->4)-d-gluconamide] (PVLA), and examined the proliferative activity and gene expression of these fractionated hepatocytes. The results showed that approximately 0.5 to 1% of the total number of hepatocytes, which showed low adhesion to PVLA, expressed low levels of the ASGP-R, while the rest of hepatocyte population with high adhesion to PVLA expressed high levels of the ASGP-R. Interestingly hepatocytes with low ASGP-R expression levels had much higher DNA synthesizing activity (i.e., are much more proliferative) than those with high ASGP-R expression levels. Moreover, hepatocytes with low ASGP-R expression levels expressed higher levels of epidermal growth factor receptor (EGF-R), CD29 (beta1 integrin) and CD49f (alpha6 integrin) and lower levels of glutamine synthetase than those with high ASGP-R expression. These findings suggested that hepatocytes with low adhesion to PVLA due to their low ASGP-R expression could be potential candidates for progenitor-like hepatocytes due to their high proliferative capacity; hence, the low expression of the ASGP-R could be a unique marker for progenitor hepatocytes. The isolation of hepatocytes with different functional phenotypes using PVLA may provide a new research tool for a better understanding of the biology of hepatocytes and the mechanisms regulating their proliferation and differentiation in health and disease.     

Identification and characterization of the genes encoding the core histones and histone variants of Neurospora crassa

We have identified and characterized the complete complement of genes encoding the core histones of Neurospora crassa. In addition to the previously identified pair of genes that encode histones H3 and H4 (hH3 and hH4-1), we identified a second histone H4 gene (hH4-2), a divergently transcribed pair of genes that encode H2A and H2B (hH2A and hH2B), a homolog of the F/Z family of H2A variants (hH2Az), a homolog of the H3 variant CSE4 from Saccharomyces cerevisiae (hH3v), and a highly diverged H4 variant (hH4v) not described in other species. The hH4-1 and hH4-2 genes, which are 96% identical in their coding regions and encode identical proteins, were inactivated independently. Strains with inactivating mutations in either gene were phenotypically wild type, in terms of growth rates and fertility, but the double mutants were inviable. As expected, we were unable to isolate null alleles of hH2A, hH2B, or hH3. The genomic arrangement of the histone and histone variant genes was determined. hH2Az and the hH3-hH4-1 gene pair are on LG IIR, with hH2Az centromere-proximal to hH3-hH4-1 and hH3 centromere-proximal to hH4-1. hH3v and hH4-2 are on LG IIIR with hH3v centromere-proximal to hH4-2. hH4v is on LG IVR and the hH2A-hH2B pair is located immediately right of the LG VII centromere, with hH2A centromere-proximal to hH2B. Except for the centromere-distal gene in the pairs, all of the histone genes are transcribed toward the centromere. Phylogenetic analysis of the N. crassa histone genes places them in the Euascomycota lineage. In contrast to the general case in eukaryotes, histone genes in euascomycetes are few in number and contain introns. This may be a reflection of the evolution of the RIP (repeat-induced point mutation) and MIP (methylation induced premeiotically) processes that detect sizable duplications and silence associated genes.     

Functional association of the beta 1 subunit with human cardiac (hH1) and rat skeletal muscle (mu 1) sodium channel alpha subunits expressed in Xenopus oocytes

Native cardiac and skeletal muscle Na channels are complexes of alpha and beta 1 subunits. While structural correlates for activation, inactivation, and permeation have been identified in the alpha subunit and the expression of alpha alone produces functional channels, beta 1- deficient rat skeletal muscle (mu 1) and brain Na channels expressed in Xenopus oocytes do not gate normally. In contrast, the requirement of a beta 1 subunit for normal function of Na channels cloned from rat heart or human heart (hH1) has been disputed. Coinjection of rat brain beta 1 subunit cRNA with hH1 (or mu 1) alpha subunit cRNA into oocytes increased peak Na currents recorded 2 d after injection by 240% (225%) without altering the voltage dependence of activation. In mu 1 channels, steady state inactivation was shifted to more negative potentials (by 6 mV, p < 0.01), but the shift of 2 mV was not significant for hH1 channels. Nevertheless, coexpression with beta 1 subunit speeded the decay of macroscopic current of both isoforms. Ensemble average hH1 currents from cell-attached patches revealed that coexpression of beta 1 increases the rate of inactivation (quantified by time to 75% decay of current; p < 0.01 at -30, -40, and -50 mV). Use- dependent decay of hH1 Na current during repeated pulsing to -20 mV (1 s, 0.5 Hz) after a long rest was reduced to 16 +/- 2% of the first pulse current in oocytes coexpressing alpha and beta 1 subunits compared to 35 +/- 8% use-dependent decay for oocytes expressing the alpha subunit alone. Recovery from inactivation of mu 1 and hH1 Na currents after 1-s pulses to -20 mV is multiexponential with three time constants; coexpression of beta 1 subunit decreased all three recovery time constants. We conclude that the beta 1 subunit importantly influences the function of Na channels produced by coexpression with either the hH1 or mu 1 alpha subunits.     

Trans-encapsidation of hepatitis C virus subgenomic replicon RNA with viral structure proteins

A trans-packaging system for hepatitis C virus (HCV) subgenomic replicon RNAs was developed. HCV subgenomic replicon was efficiently encapsidated by the HCV structural proteins that were stably expressed in trans under the control of a mammalian promoter. Infectious HCV-like particles (HCV-LPs), established a single-round infection, were produced and released into culture medium in titers of up to 10³ focus forming units/ml. Expression of NS2 protein with structural proteins (core, E1, E2, and p7) was shown to be critical for the infectivity of HCV-LPs. Anti-CD81 treatment decreased the number of infected cells, suggesting that HCV-LPs infected cells in a CD81-dependent manner. The packaging cell line should be useful both for the production of single-round infectious HCV-LPs to elucidate the mechanisms of HCV assembly, particle formation and infection to host cells, and for the development of HCV replicon-based vaccines.     

p110beta is up-regulated during differentiation of 3T3-L1 cells and contributes to the highly insulin-responsive glucose transport activity

Activation of p85/p110 type phosphatidylinositol kinase is essential for aspects of insulin-induced glucose metabolism, including translocation of GLUT4 to the cell surface and glycogen synthesis. The enzyme exists as a heterodimer containing a regulatory subunit (e.g. p85alpha) and one of two widely distributed isoforms of the p110 catalytic subunit: p110alpha or p110beta. In the present study, we compared the two isoforms in the regulation of insulin action. During differentiation of 3T3-L1 cells into adipocytes, p110beta was up-regulated approximately 10-fold, whereas expression of p110alpha was unaltered. The effects of the increased p110 expression were further assessed by expressing epitope tagged p110beta and p110alpha in 3T3-L1 cells using adenovirus transduction systems, respectively. In vitro, the basal lipid kinase activity of p110beta was lower than that of p110alpha. When p110alpha and p110beta were overexpressed in 3T3-L1 adipocytes, exposing cells to insulin induced each of the subunits to form complexes with p85alpha and tyrosine-phosphorylated IRS-1 with similar efficiency. However, whereas the kinase activity of p110beta, either endogenous or exogeneous, was markedly enhanced by insulin stimulation, only very small increases of the activity of p110alpha were observed. Interestingly, overexpression of p110beta increased insulin-induced glucose uptake by 3T3-L1 cells without significantly affecting basal glucose transport, whereas overexpression of p110alpha increased both basal and insulin-stimulated glucose uptake. Finally, microinjection of anti-p110beta neutralizing antibody into 3T3-L1 adipocytes abolished insulin-induced translocation of GLUT4 to the cell surface almost completely, whereas anti-p110alpha neutralizing antibody did only slightly. Together, these findings suggest that p110beta plays a crucial role in cellular activities evoked acutely by insulin.