Biomedical subjects

J H Ou

Publications and source records attributed to J H Ou.

At least 37 records · Page 2Linked to original sources

PubMed1994

Alteration of the subcellular localization of hepatitis B virus core protein by large but not small surface proteins.

By performing immunofluorescence staining assays in confluent COS-7 cells, hepatitis B virus core, small surface and large surface proteins were found to localize largely in the nucleus, diffusely in the cytoplasm, and accumulatively in a perinuclear area, respectively. When two proteins were co-expressed, the core protein was found co-localized with the large but not small surface protein in the cytoplasm. By performing subcellular fractionation experiments, the large surface protein was found to reduce the amount of the core protein in the nuclear fraction to an undetectable level. These results indicate that the large but not small surface protein can alter the subcellular localization of the core protein.

Blotting, Western↗

PubMed1994

Comparative studies of the core gene products of two different hepatitis C virus isolates: two alternative forms determined by a single amino acid substitution.

We have compared the expression of the core gene of two different hepatitis C virus (HCV) isolates, HCV-1 and HCV-RH, using the in vitro translation assay. In the absence of the downstream E1 envelope protein sequence, a 16-kDa protein (P16) was the dominant protein product synthesized from the HCV-1 core gene sequence. On the other hand, a 21-kDa protein (P21) was the dominant protein product synthesized from the HCV-RH sequence. Domain-swapping and site-directed mutagenesis experiments indicated that codon 9 of the core protein coding sequence played a crucial role on the synthesis of the core protein: a lysine codon at this position led to the synthesis of P16 and an arginine codon at this position led to the synthesis of P21. For HCV-1 and HCV-RH, this codon encodes lysine and arginine, respectively. Further analyses indicated that P16 was likely co-amino-terminal with P21. In the presence of the downstream E1 envelope protein sequence and microsomal membranes, P16 as well as P21 were synthesized from the HCV-1 core gene sequence whereas P21 remained the only detectable protein product synthesized from the HCV-RH core gene sequence. These results indicate that the pathway leading to the synthesis of the HCV core protein may be more complicated than originally envisioned.

Amino Acid Sequence↗

PubMed1994

Early detection of anti-HCc antibody in acute hepatitis C virus (HCV) by western blot (immunoblot) using a recombinant HCV core protein fragment.

Crude extract from Escherichia coli which expressed a recombinant protein containing amino acids 2 to 127 of the hepatitis C virus (HCV) core protein was used to detect the antibody against HCV core protein (anti-HCc). After electrophoretic separation of proteins from the extract, Western blot (immunoblot) analysis was performed with the serum samples. This method was compared with a commercially available second-generation enzyme immunoassay (EIA) which employed synthetic peptides corresponding to highly antigenic segments of both structural and nonstructural portions of HCV. Also, reverse transcription PCR for HCV RNA was used for comparison. Seventy-two serum samples from three groups of patients were tested. Groups I and II represented healthy subjects and subjects with acute hepatitis A or B, respectively. Group III included patients with newly acquired acute hepatitis C. By Western blot analysis, 31 of 31 (100%) samples from group I were negative for anti-HCc antibody, whereas 4 of 22 (18%) samples from group II were positive for anti-HCc. One of these four samples was also positive for anti-HCV antibody by the second-generation EIA (1 of 22 [4.5%]). Among 19 patients diagnosed with newly acquired acute hepatitis C, 4 (21%) were positive for anti-HCV by the second-generation EIA, whereas 12 of 19 (63%) were positive for anti-HCc by Western blot analysis. Of EIA-positive subjects, 4 of 4 (100%) were also positive for anti-HCc by Western blot analysis, whereas among EIA-negative subjects, 8 of 15 (53%) were positive. For HCV RNA detected by reverse transcription PCR, 15 of 19 (80%) of this group of samples were positive. Strikingly, the peak bilirubin level for patients with EIA-negative and Western blot-positive results is significantly higher than that for patients with consistent EIA and Western blot results (22.7 versus 7.2 mg/dl). A series of serum samples from a patient with concurrent hepatitis B and C viral infection was also studied by both tests. Although anti-HCc persisted throughout the course of infection, anti-HCV by EIA converted from negative to positive 20 days after admission and then converted back to negative 30 days later.

Acute Disease↗

PubMed1993

Cell cycle regulation of nuclear localization of hepatitis B virus core protein.

The hepatitis B virus (HBV) core protein has been found in the nucleus, the cytoplasm, or both of HBV-infected hepatocytes. However, the mechanism that regulates the subcellular localization of the HBV core protein is still unclear. In this report, we demonstrate that nuclear localization of the HBV core protein is cell cycle-regulated in two different cell lines. The amount of the core protein in the nucleus was increased during the G1 phase, reduced to an undetectable level during the S phase, and increased again when the cells were confluent and ceased to grow. Thus, the nuclear localization of the core protein during HBV infection can be at least partially attributed to liver injury and regeneration, which cause the hepatocytes to enter cell cycles. Based on the observation that the cytoplasmic core protein was phosphorylated and the nuclear core protein was not, we speculate that nuclear localization of the HBV core protein is negatively regulated by phosphorylation during the cell cycle.

3T3 Cells↗

PubMed1993

Hepatocyte-specific expression of the hepatitis B virus core promoter depends on both positive and negative regulation.

The core promoter of hepatitis B virus shows hepatocyte specificity, which is largely dependent on an upstream regulatory sequence that overlaps with viral enhancer II. Footprint analyses by numerous groups have shown binding by cellular proteins over a large stretch of DNA in this region, but the identity of these proteins and their role in core promoter function remain largely unknown. We present data showing that the transcription factor HNF-4 is one such factor, as it activates the core promoter approximately 20-fold via a binding site within the upstream regulatory sequence. Since HNF-4 is enriched in hepatocytes, its involvement at least partially explains the hepatocyte specificity of this promoter. In addition, however, we have found a region upstream of the HNF-4 site that suppresses activation by HNF-4 in HeLa cells but not in hepatoma cells. Therefore, the cell type specificity of the core promoter appears to result from a combination of activation by one or more factors specifically enriched in hepatocytes and repression by some other factor(s) present in nonhepatocytes, and it may provide a convenient model system for studying this type of tissue-specific transcriptional regulation in mammalian cells.

Base Sequence↗

PubMed1992

Brefeldin A as a regulator of grp78 gene expression in mammalian cells.

We report here that brefeldin A (BFA), which specifically blocks protein transport from an endoplasmic reticulum to the Golgi apparatus and causes resorption of Golgi membrane to the endoplasmic reticulum, specifically induced the endoplasmic reticulum-resident protein GRP78. Treatment of a human hepatoma cell line Alex-PC with BFA at a concentration of 5 micrograms/ml increased the grp78 transcript level by 12-fold. Analyses of the transcriptional rate of grp78 and the transfection with grp78 promoter suggested that this cell line utilized a posttranscriptional mechanism to increase the expression of grp78 in response to BFA. The induction process was partially dependent on de novo protein synthesis. Interestingly, in a hamster lung fibroblast cell line, K12, the induction of grp78 by BFA could be mediated by a transcriptional control mechanism. We further demonstrated that in K12 cells the region of the grp78 promoter responsive to BFA was within a 40-base pair region between -169 and -130, containing the conserved grp core and a 10-base pair region between -99 to -90 that contained a proximal CCAAT element. A model of how BFA regulates grp78 expression at both the transcriptional and posttranscriptional level is presented.

Actins↗

PubMed1992

Characterization of nuclear targeting signal of hepatitis delta antigen: nuclear transport as a protein complex.

Hepatitis delta antigen (HDAg) is the only protein encoded by hepatitis delta virus (HDV). HDAg has been demonstrated in the nuclei of HDV-infected hepatocytes, and its nuclear transport may be important for the replication of HDV RNA. In this report, we investigated the mechanism of nuclear transport of HDAg. By expressing fusion proteins consisting of the different portions of HDAg and alpha-globin, we have identified a nuclear localization signal (NLS) within the N-terminal one-third of HDAg. It consists of two stretches of basic amino acid domains separated by a short run of nonbasic amino acids. Both of the basic domains are necessary for the efficient nuclear transport of HDAg. The nonbasic spacer amino acids could be removed without affecting the nuclear targeting of HDAg significantly. Thus, the HDAg NLS belongs to a newly identified class of NLS which consists of two discontiguous stretches of basic amino acids. This NLS is separated from a stretch of steroid receptor NLS-like sequence, which is also present but not functioning as an NLS, in HDAg. Furthermore, we have shown that subfragments of HDAg which do not contain the NLS can be passively transported into the nucleus by a trans-acting full-length HDAg, provided that these subfragments contain the region with a leucine zipper sequence. Thus, our results indicate that HDAg forms aggregates in the cytoplasm and that the HDAg oligomerization is probably mediated by the leucine zipper sequence. Therefore, HDAg is likely transported into the nucleus as a protein complex.

Amino Acid Sequence↗

PubMed1991

Characterization of the hepatitis B virus EnhI enhancer and X promoter complex.

The hepatitis B virus EnhI enhancer element overlaps the promoter of the X gene. By performing methylation interference experiments, four protein factor binding sites clustered in a 120-bp region were found to control the EnhI enhancer and X promoter activities. Deletion mapping experiments indicated that the two upstream protein factor binding sites constituted a basal enhancer module. This module, likely bound by a liver-specific factor and a ubiquitous factor, could activate the herpes simplex virus thymidine kinase gene promoter by 5- or 10-fold, depending on the orientation, in Huh7 cells, a liver-derived cell line, but not in other cell types tested. The two downstream protein factor binding sites interact with the upstream basal enhancer module in an orientation- and distance-dependent manner to increase the enhancer activity by another 10-fold. In addition, at least one of the two downstream protein factor binding sites is also essential for the X promoter activity.

Base Sequence↗

PubMed1991

Phosphorylation of hepatitis B virus precore and core proteins.

Hepatitis B virus precore and core proteins are related. The precore protein contains the entire sequence of the core protein plus an amino-terminal extension of 29 amino acids. The amino-terminal extension of the precore protein contains a signal sequence for the secretion of the precore protein. This signal sequence is removed after the translocation of the precore protein across the endoplasmic reticulum membrane to produce the precore protein derivative named P22. We demonstrate that both P22 and the core protein can be phosphorylated in cells. Microsomal fractionation and trypsin digestion experiments demonstrate that a fraction of phosphorylated P22 is located in the endoplasmic reticulum lumen. Phosphorylation of P22 likely occurs in the carboxy terminus, since the P22 derivative P16, which lacks the carboxy terminus of P22, is not phosphorylated. Linking the carboxy terminus of the precore-core protein to heterologous secretory and cytosolic proteins led to the phosphorylation of the resulting chimeric proteins. These results indicate that phosphorylation of P22 and the core protein is likely mediated by cellular kinases.

Animals↗

PubMed1991

Proteolytic conversion of hepatitis B virus e antigen precursor to end product occurs in a postendoplasmic reticulum compartment.

At least two proteolytic events are involved in the biogenesis of hepatitis B virus e antigen. The first proteolytic event removes the signal peptide and results in the translocation of the precursor protein, P22, into the lumen of the endoplasmic reticulum (ER). The second proteolytic event removes the carboxy-terminal arginine-rich sequence of P22 and converts it to the 16-kDa hepatitis B virus e antigen end product. In contrast to the first proteolytic event, the second proteolytic event is suppressed by brefeldin A, a chemical that inhibits the transport of protein from the ER to the Golgi apparatus. In subcellular fractionation experiments, P22 was detected in both the ER and the Golgi fractions, but P16 was detected only in the Golgi fraction. On the basis of these results, we conclude that the conversion of P22 to P16 occurs ina post-ER compartment, mostly likely the Golgi apparatus.

Biological Transport↗

PubMed1990

Comparative studies of hepatitis B virus precore and core particles.

Hepatitis B virus core antigen gene expresses two cocarboxy-terminal proteins, termed precore and core proteins. Both precore and core proteins can form nucleocapsid-like particles. In order to understand the mechanism that leads to the formation of the nucleocapsid, we have expressed precore and core protein sequences in COS cells, a monkey kidney cell line, and compared the properties of these two particles. Our results show that core protein can form particles with various densities and they are present mostly in the cytosol. Precore protein, on the other hand, forms particles with one predominant density, and a majority of these particles are present in the lumen of the endoplasmic reticulum (ER). Furthermore, our results show that, when coexpressed in the same cells, core protein and the ER-associated surface antigens (envelope protein) show colocalization, indicating interaction between these two viral structural proteins.

Animals↗

PubMed1990

The arginine-rich domain of hepatitis B virus precore and core proteins contains a signal for nuclear transport.

Precore and core proteins are two related co-carboxy-terminal proteins of hepatitis B virus. Precore protein contains the entire sequence of core protein plus an amino-terminal extension of 29 amino acid residues. Both proteins can display a common antigenic determinant known as core antigen (HBcAg). Clinically, HBcAg is detected in the nucleus, cytoplasm, or both of hepatitis B virus-infected hepatocytes. In order to understand the mechanism that regulates nuclear transport of HBcAg, various portions of precore and core proteins were linked to a reporter protein, human alpha-globin, and expressed in mammalian cells. Our results indicate that the precore protein-specific sequence, although important for nuclear transport, does not contain a nuclear localization signal. Instead, a signal for nuclear transport is located near the carboxy termini of precore and core proteins in the arginine-rich domain. This signal is made up of a set of two direct PRRRRSQS repeats and is highly conserved among mammalian hepadnaviruses.

Amino Acid Sequence↗

PubMed1990

Activation of class I major histocompatibility complex gene expression by hepatitis B virus.

Normal hepatocytes express very few class I major histocompatibility complex (MHC I) molecules, but MHC I expression is elevated in hepatitis B virus (HBV) infection. We report here that hepatoblastoma cells with replicating HBV genomes express three- to fourfold-higher levels of MHC I protein and mRNA than do parent cells without HBV DNA. Transient transfection assays demonstrated that the HBV X protein trans activated transcription from an MHC I promoter and allowed identification of cis elements important for trans activation.

2',5'-Oligoadenylate Synthetase↗

PubMed1990

Preferred translation of human hepatitis B virus polymerase from core protein- but not from precore protein-specific transcript.

In the human hepatitis B virus (HBV) genome, the 5' end of the polymerase coding sequence overlaps with the 3' end of the core protein coding sequence. Recent results obtained from genetic studies have suggested that translation of HBV polymerase initiates from the first ATG codon of the polymerase reading frame and is not a result of frameshift translation from the core protein reading frame, as in the case of retroviruses. By using in vitro-synthesized SP6 RNA transcripts, we now demonstrate that HBV core protein-specific mRNA can direct the synthesis of polymerase from the internal polymerase ATG codon in rabbit reticulocyte lysates and Xenopus oocytes. A related message with an additional 60 nucleotides at the 5' end (pre-core protein mRNA) was not as efficient as the core protein mRNA for translation of polymerase. Furthermore, translation of polymerase from the core protein mRNA was not inhibited by the cap analog m7GpppG. This result, together with the results described above, indicates that translation of HBV polymerase occurs in a novel, cap-independent manner.

Animals↗

PubMed1990

[Medium-chain triglycerides metabolism and its application in total parenteral nutrition].

Twenty patients undergoing uneventful gastrectomy were randomly assigned to receive TPN containing either 10% Intralipid or 10% Lipofundin (MCT/LCT) beginning postoperative day (POD) 1 through POD 6. Patients were given non-protein energy 27 kcal/kg/d and nitrogen 0.14 g/kg/d through peripheral vein along with fat emulsion. Body weight, hemoglobin, blood lymphocyte count, SGPT, serum bilirubin, AKP, BUN, Cr, serum albumin, transferrin, prealbumin, fibronectin, creatine phosphokinase, serum and plasma cholesterol, triglycerides, plasma free fatty acids, blood sugar, serum insulin, urinary creatinine and nitrogen balance were monitored. It was found that MCT-based fat emulsion was tolerated without any side effects. Kinetics study showed that levels of medium chain free fatty acids increased during MCT-based fat emulsion infusion, but returned to normal within 2 hours on cessation of infusion, indicating good clearance of MCT. Plasma concentration of triglycerides in the LCT group was higher than that in the MCT group at 2 hours after infusion. On POD 6, better nitrogen balance was observed in the MCT group, 0.44 +/- 0.21 g/d (-1.4 +/- 0.51 g/d in the LCT group). Urinary excretion of creatinine in the MCT group was also less than in the LCT group (0.76 +/- 0.03 g/d vs. 1.034 +/- 0.11 g/d). LCT was also observed to induce an elevation of serum bilirubin (1.12 +/- 0.11 mg/dl). These results indicate that MCT-based fat emulsion appears to be a safe energy source.

Adult↗

PubMed1989

Transport of hepatitis B virus precore protein into the nucleus after cleavage of its signal peptide.

The precore and core proteins of hepatitis B virus have identical deduced amino acid sequences other than a 29-residue amino-terminal extension (precore region) on the precore protein. The first 19 of these residues serve as a signal sequence to direct the precore protein to the endoplasmic reticulum, where they are cleaved off with formation of precore protein derivative P22 for secretion. In this report, we show that P22 can alternatively be transported into the nucleus following signal peptide cleavage. Experiments with deletion mutants indicated that this nuclear transport proceeds via the cytosol and is dependent on the amino-terminal portion of P22. Thus, the hepatitis B virus precore protein is a secreted, cytosolic, and nuclear protein.

Amino Acid Sequence↗

PubMed1988

Trans-activation of the human immunodeficiency virus long terminal repeat by the hepatitis B virus X protein.

Human immunodeficiency virus 1 has been implicated as the main etiologic agent of the acquired immunodeficiency syndrome. However, other infectious agents may accelerate the progression of this disease. In particular, hepatitis B virus has been suggested as one such cofactor. Therefore, we have investigated the effects of hepatitis B virus gene products on expression of the human immunodeficiency virus I in transient transfection studies of Jurkat lymphoblastic T cells, using as reporter the chloramphenicol acetyltransferase gene coupled to the long terminal repeat of human immunodeficiency virus I. As measured by the amount of chloramphenicol acetyltransferase activity, gene expression directed by the human immunodeficiency virus I long terminal repeat increased approximately 10-fold in response to the hepatitis B virus X protein. This trans-activation by the X protein is multiplicative with the effect of phorbol esters and can be accounted for by an increase in the steady-state level of chloramphenicol acetyltransferase mRNA. Analysis of deletion and clustered point mutants in the long terminal repeat indicated that the X protein exerts its effect through multiple cis-acting sites. These results provide a possible molecular basis for the association of hepatitis B virus and the acquired immunodeficiency syndrome and confirm that the X protein is a transcriptional transactivator.

Acquired Immunodeficiency Syndrome↗

PubMed1988

A signal peptide encoded within the precore region of hepatitis B virus directs the secretion of a heterogeneous population of e antigens in Xenopus oocytes.

Using synthetic hepatitis B virus (HBV) mRNAs, we have shown that expression of HBV core-antigen gene sequences in Xenopus oocytes leads to the stable accumulation of 21-kDa cytoplasmic core protein (P21). In contrast, expression of precore plus core sequences leads mainly to the secretion of a heterogeneous population of proteins ranging in size from 15 to 22 kDa that collectively display viral e antigen (HBeAg) activity. We demonstrate that the precore region contains a cleavable 19 amino acid signal peptide that targets the precore proteins to the secretory pathway. The initial product of translocation (P22) is further processed during migration through the secretory pathway, apparently by a series of cleavage events at the arginine-rich carboxyl terminus, to yield multiple proteins of 15-18 kDa (P15-P18) that are secreted along with some P22. Our results indicate that serum HBeAg is generated by a signal peptide-mediated secretion event dependent on precore sequences.

Animals↗