Modulation of transcriptional corepressor activity of prospero-related homeobox protein (Prox1) by SUMO modification.

Prospero-related homeobox protein (Prox1) plays essential roles in the development of many tissues and organs. In the present study, we show that Prox1 is modified by the small ubiquitin-like protein SUMO-1 in cultured cells. Mutation analysis identified at least four potential sumoylation sites within the repression domain of Prox1. Our data indicate that sumoylation of Prox1 reduces its interaction with HDAC3 and as a result downregulates its corepressor activity. These findings suggest that sumoylation may serve as a novel mechanism for the regulation of Prox1's corepressor activity.

Expression of mouse liver receptor homologue 1 in embryonic stem cells is directed by a novel promoter.

Liver receptor homologue 1 (LRH-1) plays important roles in many physiological processes and embryogenesis. However, little is known about the developmental regulation of lrh-1 expression. We identified a novel transcript of mouse lrh-1 (mlrh-1v2) from embryonic stem (ES) cells. mlrh-1v2 is expressed throughout embryogenesis and in several adult tissues, while the known transcript (mlrh-1v1) appears later during embryogenesis. mlrh-1v2 expression is directed by a new promoter which displays a strong activity in ES cells. The generation of the new transcript is conserved in rats. The identification of novel mlrh-1 variant and promoter is critical for elucidating LRH-1 functions in development and adult tissues.

Expression and purification of the complete PreS region of hepatitis B Virus.

AIM: To express the complete PreS region of HBV in E.coli with good solubility and stability, and to establish an effective method for purification of the recombinant PreS protein. METHODS: The complete PreS region (PreS1 and PreS2) was fused into a series of tags including glutathione S-transferase (GST), dihydrofolate reductase (DHFR), maltose binding protein (MBP), 6x histidine, chitin binding domain (CBD), and thioredoxin, respectively. Expression of recombinant PreS fusion proteins was examined by SDS-PAGE analysis and confirmed by Western blot. Two fusion proteins, thio-PreS, and PreS-CBD, with desirable solubility and stability, were subjected to affinity purification and further characterization. RESULTS: Recombinant PreS fusion proteins could be synthesized with good yields in E.coli. However, most of these proteins except for thio-PreS and PreS-CBD were vulnerable to degradation or insoluble as revealed by SDS-PAGE and Western blot. Thio-PreS could be purified by affinity chromatography with nickel-chelating sepharose as the matrix. However, some impurities were also co-purified. A simple freeze-thaw treatment yielded most of the thio-PreS proteins in solution while the impurities were in the precipitate. Purified thio-PreS protein was capable of inhibiting the binding of HBV virion to a specific monoclonal antibody against an epitope within the PreS1 domain. CONCLUSION: Increased solubility and stability of the complete PreS region synthesized in E.coli can be achieved by fusion with the thioredoxin or the CBD tag. A simple yet highly effective method has been established for the purification of the thio-PreS protein. Purified thio-PreS protein likely assumes a native conformation, which makes it an ideal candidate for studying the structure of the PreS region as well as for screening antivirals.

Screening for PreS specific binding ligands with a phage displayed peptides library.

AIM: To construct a random peptide phage display library and search for peptides that specifically bind to the PreS region of hepatitis B virus (HBV). METHODS: A phage display vector, pFuse8, based on the gene 8 product (pVIII) of M13 phage was made and used to construct a random peptide library. E.coli derived thioredoxin-PreS was purified with Thio-bond beads, and exploited as the bait protein for library screening. Five rounds of bio-panning were performed. The PreS-binding specificities of enriched phages were characterized with phage ELISA assay. RESULTS: A phage display vector was successfully constructed as demonstrated to present a pVIII fused HBV PreS1 epitope on the phage surface with a high efficiency. A cysteine confined random peptide library was constructed containing independent clones exceeding 5+/-10(8) clone forming unit (CFU). A pool of phages showing a PreS-binding specificity was obtained after the screening against thio-PreS with an enrichment of approximately 400 times. Five phages with high PreS-binding specificities were selected and characterized. Sequences of the peptides displayed on these phages were determined. CONCLUSION: A phage library has been constructed, with random peptides displaying as pVIII-fusion proteins. Specific PreS-binding peptides have been obtained, which may be useful for developing antivirals against HBV infection.

Purification and application of C-terminally truncated hepatitis C virus E1 proteins expressed in Escherichia coli.

AIM: To explore the possibility of expressing hepatitis C virus (HCV) envelope protein 1 (E1) in Escherichia coli (E. coli) and to test the purified recombinant E1 proteins for clinical and research applications. METHODS: C-terminally truncated E1 fragments were expressed in E. coli as hexa-histidine-tagged fusion proteins. The expression products were purified under denaturing conditions using immobilized-metal affinity chromatography. Purified E1 proteins were used to immunize rabbits. Rabbit anti-sera thus obtained were reacted with both E. coli- and mammalian cell-expressed E1 glycoproteins as detected by Western blot. RESULTS: Full-length E1 protein proved difficult to express in E. coli. C-terminally truncated E1 was successfully expressed in E. coli as hexa-histidine-tagged recombinant fusion protein and was purified under denaturing conditions on Ni(2+)-NTA agarose. Rabbit anti-sera raised against purified recombinant E1 specifically reacted with mammalian cell-expressed E1 glycoproteins in Western blot. Furthermore, E. coli-derived E1 protein was able to detect animal antibodies elicited by E1-based DNA immunization. CONCLUSION: These results demonstrate that the prokaryotically expressed E1 proteins share identical epitopes with eukaryotically expressed E1 glycoprotein. The E. coli-derived E1 proteins and corresponding antisera can become useful tools in anti-HCV vaccine research.

Molecular mechanism for the potentiation of the transcriptional activity of human liver receptor homolog 1 by steroid receptor coactivator-1.

The liver receptor homolog 1 (LRH-1) belongs to the Fushi tarazu factor 1 nuclear receptor subfamily, and its biological functions are just being unveiled. The molecular mechanism for the transcriptional regulation by LRH-1 is not clear yet. In this report, we use mutagenesis and reporter gene assays to carry out a detailed analysis on the hinge region and the proximal ligand binding domain (LBD) of human (h) LRH-1 that possess important regulatory functions. Our results indicate that helix 1 of the LBD is essential for the activity of hLRH-1 and that the steroid receptor coactivator (SRC)-1 interacts directly with the LBD of hLRH-1 and significantly potentiates the transcriptional activity of hLRH-1. Cotransfection assays demonstrate that overexpressed SRC-1 potentiates hLRH-1 mediated activation of the cholesterol 7-alpha-hydroxylase promoter and increases the transcription of the endogenous cholesterol 7-alpha-hydroxylase in Huh7 cells. The interaction between SRC-1 and hLRH-1 assumes a unique pattern that involves primarily a region containing the glutamine-rich domain of SRC-1, and helix 1 and activation function-2 of hLRH-1 LBD. Mutagenesis and molecular modeling studies indicate that, similar to mouse LRH-1, the coactivator-binding cleft of hLRH-1 LBD is not optimized. An interaction between helix 1 of hLRH-1 LBD and a region containing the glutamine-rich domain of SRC-1 can provide an additional stabilizing force and enhances the recruitment of SRC-1. Similar interaction is observed between hLRH-1 and SRC-2/transcriptional intermediary factor 2 or SRC-3/acetyltransferase. Moreover, transcriptional intermediary factor 2 and acetyltransferase also potentiate the transcriptional activity of hLRH-1, suggesting a functional redundancy among SRC family members. These findings collectively demonstrate an important functional role of helix 1 in cofactor recruitment and reveal a novel molecular mechanism of transcriptional regulation and cofactor recruitment mediated by hLRH-1.

Prospero-related homeobox (Prox1) is a corepressor of human liver receptor homolog-1 and suppresses the transcription of the cholesterol 7-alpha-hydroxylase gene.

Cholesterol 7-alpha-hydroxylase (CYP7A1) catalyzes a rate-limiting step in bile acid synthesis in liver, and its gene transcription is under complex regulation by multiple nuclear receptors in response to bile acids, cholesterol derivatives, and hormones. The liver receptor homolog-1 (LRH-1), a member of the fushi tarazu factor 1 subfamily of nuclear receptors, has emerged as an essential regulator for the expression of cyp7a1. In this report, we demonstrate Prox1, a prospero-related homeobox transcription factor, identified through a yeast two-hybrid screening, can directly interact with human LRH-1 (hLRH-1) and suppresses hLRH-1-mediated transcriptional activation of human cyp7a1 gene. Biochemical analysis demonstrates that Prox1 interacts with both the ligand binding domain (LBD) and the DNA binding domain (DBD) of hLRH-1. An LRKLL motif in Prox1 is important for the interaction with the LBD but not the DBD of hLRH-1. In hLRH-1 LBD, helices 2 and 10 are essential for Prox1 recruitment. The suppression by Prox1 on the transcriptional activity of hLRH-1 can be mediated through its interaction with the LBD or the DBD of hLRH-1. Gel shift assays reveal that Prox1 impairs the binding of hLRH-1 to the promoter of human cyp7a1 gene.

LRH-1/hB1F and HNF1 synergistically up-regulate hepatitis B virus gene transcription and DNA replication.

Enhancer II (ENII) is one of the critical cis-elements in the Hepatitis B Virus (HBV) genome for the hepatic viral gene transcription and DNA replication. The liver-specific activity of ENII is regulated by multiple liver-enriched transcription factors, including LRH-1/hB1F, HNF1, HNF3b, HNF4 and C/EBP. Knowledge on the interplay of these important factors is still limited. In this study, we demonstrate a functional synergism between the orphan nuclear receptor LRH-1/hB1F and the homeoprotein HNF1 in up-regulating the liver-specific activity of ENII. This synergism is sufficient for initiating the viral gene transcription and DNA replication in non-hepatic cells. We have defined the activation domains in hB1F and HNF1 that contribute to the synergism. We further show that hB1F and HNF1 can interact directly in vitro and have mapped the domains required for this interaction.

DNA immunization with fusion genes encoding different regions of hepatitis C virus E2 fused to the gene for hepatitis B surface antigen elicits immune responses to both HCV and HBV.

AIM: Both Hepatitis B virus (HBV) and Hepatitis C virus (HCV) are major causative agents of transfusion-associated and community-acquired hepatitis worldwide. Development of a HCV vaccine as well as more effective HBV vaccines is an urgent task. DNA immunization provides a promising approach to elicit protective humoral and cellular immune responses against viral infection. The aim of this study is to achieve immune responses against both HCV and HBV by DNA immunization with fusion constructs comprising various HCV E2 gene fragments fused to HBsAg gene of HBV. METHODS: C57BL/6 mice were immunized with plasmid DNA expressing five fragments of HCV E2 fused to the gene for HBsAg respectively. After one primary and one boosting immunizations, antibodies against HCV E2 and HBsAg were tested and subtyped in ELISA. Splenic cytokine expression of IFN-gamma and IL-10 was analyzed using an RT-PCR assay. Post-immune mouse antisera also were tested for their ability to capture HCV viruses in the serum of a hepatitis C patient in vitro. RESULTS: After immunization, antibodies against both HBsAg and HCV E2 were detected in mouse sera, with IgG2a being the dominant immunoglobulin sub-class. High-level expression of INF-gamma was detected in cultured splenic cells. Mouse antisera against three of the five fusion constructs were able to capture HCV viruses in an in vitro assay. CONCLUSION: The results indicate that these fusion constructs could efficiently elicit humoral and Th1 dominant cellular immune responses against both HBV S and HCV E2 antigens in DNA-immunized mice. They thus could serve as candidates for a bivalent vaccine against HBV and HCV infection. In addition, the capacity of mouse antisera against three of the five fusion constructs to capture HCV viruses in vitro suggested that neutralizing epitopes may be present in other regions of E2 besides the hypervariable region 1.

Expression of hepatitis C virus envelope protein 2 induces apoptosis in cultured mammalian cells.

AIM: To explore the role of hepatitis C virus (HCV) envelope protein 2 (E2) in the induction of apoptosis. METHODS: A carboxyterminal truncated E2 (E2-661) was transiently expressed in several cultured mammalian cell lines or stably expressed in Chinese hamster ovary (CHO) cell line. Cell proliferation was assessed by 3H thymidine uptake. Apoptosis was examined by Hoechst 33258 staining, flow cytometry and DNA fragmentation analysis. RESULTS: Reduced proliferation was readily observed in the E2-661 expressing cells. These cells manifested the typical features of apoptosis, including cell shrinkage, chromatin condensation and hypodiploid genomic DNA content. Similar apoptotic cell death was observed in an E2-661 stably expressing cell line. CONCLUSION: HCV E2 can induce apoptosis in cultured mammalian cells.

A candidate DNA vaccine elicits HCV specific humoral and cellular immune responses.

AIM: To investigate the immunogenicity of candidate DNA vaccine against hepatitis C virus (HCV) delivered by two plasmids expressing HCV envelope protein 1 (E1) and envelope protein 2 (E2) antigens respectively and to study the effect of CpG adjuvant on this candidate vaccine. METHODS: Recombinant plasmids expressing HCV E1 and E2 antigens respectively were used to simultaneously inoculate mice with or without CpG adjuvant. Antisera were then collected and titers of anti-HCV antibodies were analyzed by ELISA. One month after the last injection, animals were sacrificed to prepare single-cell suspension of splenocytes. These cells were subjected to HCV antigen specific proliferation assays and cytokine secretion assays to evaluate the cellular immune responses of the vaccinated animals. RESULTS: Antibody responses to HCV E1 and E2 antigens were detected in vaccinated animals. Animals receiving CpG adjuvant had slightly lower titers of anti-HCV antibodies in the sera, while the splenocytes from these animals showed higher HCV-antigen specific proliferation. Analysis of cytokine secretion from the splenocytes was consistent with the above results. While no antigen-specific IL-4 secretion was detected for all vaccinated animals, HCV antigen-specific INF-gamma secretion was detected for the splenocytes of vaccinated animals. CpG adjuvant enhanced the secretion of INF-gamma but did not change the profile of IL-4 secretion. CONCLUSION: Vaccination of mice with plasmids encoding HCV E1 and E2 antigens induces humoral and cellular immune responses. CpG adjuvant significantly enhances the cellular immune response.

Full-length core sequence dependent complex-type glycosylation of hepatitis C virus E2 glycoprotein.

AIM: To study HCV polyprotein processing is important for the understanding of the natural history of HCV and the design of vaccines against HCV. The purpose of this study is to investigate the affection of context sequences on hepatitis C virus (HCV) E2 processing. METHODS: HCV genes of different lengths were expressed and compared in vaccinia virus/T7 system with homologous patient serum S94 and mouse anti-serum M( E2116) raised against E.coli -derived E2 peptide, respectively. Deglycosylation analysis and GNA ( Galanthus nivalus ) lectin binding assay were performed to study the post-translational processing of the expressed products. RESULTS: E2 glycoproteins with different molecular weights (-75 kDa and -60 kDa) were detected using S94 and M( E2116), respectively. Deglycosylation analysis showed that this difference was mainly due to different glycosylation. Endo H resistance and its failure to bind to GNA lectin demonstrated that the higher molecular weight form (75 kDa) of E2 was complex-type glycosylated, which was readily recognized by homologous patient serum S94. Expression of complex-type glycosylated E2 could not be detected in all of the core-truncated constructs tested, but readily detected in constructs encoding full-length core sequences. CONCLUSION: The upstream conserved full-length core coding sequence was required for the production of E2 glycoproteins carrying complex-type N-glycans which reacted strongly with homologous patient serum and therefore possibly represented more mature forms of E2. As complex-type N-glycans indicated modification by Golgi enzymes, the results suggest that the presence of full-length core might be critical for E1/E2 complex to leave ER. Our data may contribute to a better understanding of the processing of HCV structural proteins as well as HCV morphogenesis.

Corepressor SMRT specifically represses the transcriptional activity of orphan nuclear receptor hB1F/hLRH-1.

The orphan nuclear receptor hB1F (also known as NR5A2, LRH-1, FTF or CPF) plays important roles in regulating the expression of several cellular and viral genes actively involved in a wide range of biological processes such as the bile acid biosynthesis, liver specific gene regulatory network and hepatitis B virus replication. The activity of nuclear receptors is regulated by multiple mechanisms, including coactivation and corepression. In this study, it was found that the silencing mediator for retinoic acid receptor and thyroid hormone receptor (SMRT) specifically represses the transcriptional activity of hB1F, on either GAL4 dependent reporter system or the hB1F-responsive HBV enhancer II/core promoter. The repression imposed by SMRT is observed in different cell lines. Interestingly, hB1F couldn t interact with SMRT directly, as demonstrated by mammalian two-hybrid analysis or GST pull-down assay. Taken together, it can be concluded for the first time that the transcriptional activity of hB1F is regulated specifically by the corepressor SMRT via an indirect mechanism.

Regulation of Hepatitis B Virus Gene Expression by Its Two Enhancers.

A linearized genome containing the entire HBV 3.5 kb mRNA transcriptional units was constructed. Based on it, a series of mutants with enhancer deletions or point mutations were generated. This system has served as a good model to study the regulation of hepatitis B virus gene expression by its two enhancers (ENI and ENII). The results showed that both ENI and ENII could increase the expression of HBsAg, and the two enhancers worked synergistically. They also could increase the expression of HBeAg, but the deletion of 2C and Ep of ENI had no effect on the expression of Hbe/cAg in the cytoplasm. The expression of C gene might be regulated mainly by ENII, but ENI could cooperate with the action of ENII. The data indicated that ENI and ENII might function differentially but cooperatively in regulation the transcription of the two 3.5 kb mRNAs which functioned distinctively.

Roles of Various Regions of the HBV Core Promoter in the Transcription of Precore and Pregenomic RNA.

To determine the potential functions of different regions in the HBV core promoter, several mutants containing B3 and/or C deletion (p3.6IIdeltaB3, p3.5IIdeltaB3, p3.6IIdeltaC, p3.5IIdeltaB3C) have been constructed based on the two HBV transcription units (p3.6II and p3.5II) which contained ENII/Cp and Cp immediate upstream of the linear HBV genome, respectively. The functional effects of deletions were assessed with respect to antigen production, viral mRNA synthesis, and viral DNA replication. When HepG2 cells were transfected with C deletion mutants, HBeAg became undetectable in the supernatant while the expression of HBcAg in the cell lysate was retained. Consistently, precore RNA disappeared but the synthesis of pregenomic RNA and viral DNA replication could still be observed. On the other hand, deletions of B3 fragment reduced the expression of HBeAg and HBcAg significantly, while maintained the synthesis of precore RNA, pregenomic RNA and viral progeny DNA. The results strongly suggest that both fragment C and fragment D serve as minimal promoter elements for the transcription initiation of 3.5 kb RNAs. Notably, the D fragment which contains a novel nuclear factor binding site close to the start site of pregenomic RNA, is critical for the basal transcription of pregenomic RNA, which has not been reported previously. B3 plays an important role in the efficient transcription of precore and pregenomic RNAs, but is not indispensable for their basal transcription.

[Antiviral activity of a hammerhead ribozyme against HBV in HepG2.2.15 cells].

The carboxy terminal arginine rich domain of core protein is highly conserved among HBV subtypes, and plays important roles in the packaging of pregenomic RNA and viral replication. Therefore, the 3' highly conserved region of HBV C gene is an attractive target for antiviral therapy mediated by ribozymes. A hammerhead ribozyme RzC, targeting the above site, was designed; meanwhile, as controls, the disabled form of RzC, dRzC, was also designed. In order to investigate its antiviral effects in cultured cells, in vitro-transcribed ribozymeRzC, dRzC and ribozyme expression vectors pCRzC, pCdRzC were delivered, respectively, into HepG2.2.15 cells ( clonal cells derived from HepG2 cells that contain integrated HBV ayw DNA). The preliminary results demonstrated that in vitro-transcribed ribozyme RzC had weak inhibition on HBV replication, possibly due to its quick degradation by RNases in cells, while the endogenously expressed ribozyme RzC showed significant inhibitory effect on HBV replication. In conclusion, the results suggested the possibility of the hammerhead ribozyme RzC to be used for the gene therapy of HBV infection.

Characterization of a strong repression domain in the hinge region of orphan nuclear receptor hB1F/hLRH-1.

Human hepatitis B virus enhancer II B1 binding factor (hB1F also known as NR5A2, LRH-1, FTF or CPF) is an orphan nuclear receptor and belongs to the fushi tarazu factor I (FTZ-F1) subfamily. It plays important roles in the transcriptional regulation of a number of genes involved in bile acid biosynthesis pathway, hepatitis B virus (HBV) replication and liver specific regulatory network. Like other nuclear receptors, hB1F is composed of modular functional domains. We characterized a domain in its hinge region that imposes a strong repression on the transcriptional activity of hB1F, which is important for the function of hB1F on regulating the activity of HBV enhancer II/core promoter. Mutations of the core residues in this domain abrogate the repression. Bioinformatic analysis reveals that the amino acid sequence of this region is highly conserved only among members of the FTZ-F1 subfamily. The repression is observed in five cell lines tested, while the degree of the repression varies greatly, which does not parallel with the expression level of the DEAD box protein of 130 kD (DP103), a potential interacting protein of a homologous domain in the steroidogenic factor 1 (SF-1). Moreover, the repression is not affected by the silencing mediator for retinoic acid receptor and thyroid hormone receptor (SMRT) and steroid receptor coactivator 1 (SRC-1). Collectively, these data suggest a novel regulatory mechanism for the transcriptional activity of hB1F.

Hammerhead Ribozymes Suppress HBV(adr) in HepG2 Cells.

Three hammerhead ribozymes (RS3, RC2 and RC1) targeting to the HBV genome have been designed. Plasmids were constructed by inserting the genes of naked and tRNA-embedded ribozymes into RNA trimming vector pRG523 and then were transferred to eukaryotic expression vector. By the similar cloning method the shotgun-type plasmids carrying homogeneous RS3 or RtS3 unitconnected in tandem were obtained. After co-transfecting the above plasmids and HBV genome containing plasmid into human hepatoma cell line HepG2 respectively and selection by G418, the HBV-inhibiting activity of different kinds of ribozyme in G418-resistant cells was achieved by measuring the decrease of HBV-RNA, progeny DNA and the antigens expressed. The results showed that all the ribozymes were active with more than 70% inhibition activity against the HBV and that tRNA-embedded ribozymes had higher activity than naked ribozymes. It is worth particular interest that shotgun-type ribozymes with the connected unit in tandem with 8 and 12 units constructed in the plasmid revealed the highest activity, reaching >90% inhibition.

DNA immunization with recombinant HCV E2 expression plasmids.

DNA-based immunization, the delivery of plasmid DNA by direct inoculation, is a newly developed method of vaccination. Besides of many advantages, DNA immunization was demonstrated to generate weaker antibody and CTL responses than did protein and live attenuated vaccines, respectively. To circumvent this shortage, several methods were tested such as using different vector, changing injection mode, and co-expressing cytokines, etc. The studies showed that many factors could greatly affect the immune responses to DNA vaccine. The aim of this study is to compare different vectors for the presentation of the HCV E2 to generate immune responses by using DNA-based immunization. Four expression plasmids, with different promoter types and with or without signal sequences were constructed, which encode C-terminally truncated E2 (384-660) with HBV preS1 21-47 tag fused to its N-termini. Transient expression in HeLa cells showed that only recombinant plasmids with signal sequence could be expressed and properly processed, and the product could be secreted into medium. Protein expression level is slightly higher in plasmids with CMV promoter than with EF1alpha promoter. After immunization of C57BL/6 mice, all the recombinant constructs could elicit both anti-preS1 and anti-E2 antibodies. But only pCMV Sec-S1E2t660 with both CMV promoter and signal sequence could induce high-level and long-lasting antibody, showing that it is a good vaccine candidate. Possible reasons for the different immune responses to these constructs were discussed.

Purification and Characterization of Recombinant Hepatitis B Virus Surface Antigen SS1 Expressed in Pichia pastoris.

The purification of recombinant hepatitis B surface antigen, SS1 protein, expressed in Pichia pastoris, and the investigation of its physiochemical characters and immunogenicity were described here. Employing McAb immunoaffinity chromatography, this protein was purified to purity of 95%. The results of ELISA and Western blotting showed good antigenicity of this purified SS1 protein. CsCl gradient centrifugation and electron microscopy assay proved that this purified protein could be assembled into particles similar to the HBV subviral particles. Strong antibody responses against both the HBs and PreS1 epitopes were induced in BALB/c mice immunized with this purified protein. The simultaneous injection of a CpG adjuvant induced a Th1-like immune response against both the HBs and PreS1 epitopes.