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Spike protein is one of the major structural proteins of severe acute respiratory syndrome-coronavirus. It is essential for the interaction of the virons with host cell receptors and subsequent fusion of the viral envelop with host cell membrane to allow infection. Some spike proteins of coronavirus, such as MHV, HCoV-OC43, AIBV and BcoV, are proteolytically cleaved into two subunits, S1 and S2. In contrast, TGV, FIPV and HCoV-229E are not. Many studies have shown that the cleavage of spike protein seriously affects its function. In order to investigate the maturation and proteolytic processing of the S protein of SARS CoV, we generated S1 and S2 subunit specific antibodies (Abs) as well as N, E and 3CL protein-specific Abs. Our results showed that the antibodies could efficiently and specifically bind to their corresponding proteins from E.coli expressed or lysate of SARS-CoV infected Vero-E6 cells by Western blot analysis. Furthermore, the anti-S1 and S2 Abs were proved to be capable of binding to SARS CoV under electron microscope observation. When S2 Ab was used to perform immune precipitation with lysate of SARS-CoV infected cells, a cleaved S2 fragment was detected with S2-specific mAb by Western blot analysis. The data demonstrated that the cleavage of S protein was observed in the lysate, indicating that proteolytic processing of S protein is present in host cells.

Proteomics was used to identify a protein encoded by ORF 3a in a SARS-associated coronavirus (SARS-CoV). Immuno-blotting revealed that interchain disulfide bonds might be formed between this protein and the spike protein. ELISA indicated that sera from SARS patients have significant positive reactions with synthesized peptides derived from the 3a protein. These results are concordant with that of a spike protein-derived peptide. A tendency exists for co-mutation between the 3a protein and the spike protein of SARS-CoV isolates, suggesting that the function of the 3a protein correlates with the spike protein. Taken together, the 3a protein might be tightly correlated to the spike protein in the SARS-CoV functions. The 3a protein may serve as a new clinical marker or drug target for SARS treatment.

The nucleocapsid (N) protein of severe acute respiratory syndrome-coronavirus (SARS-CoV) is a major virion structural protein. In this study, two epitopes (N1 and N2) of the N protein of SARS-CoV were predicted by bioinformatics analysis. After immunization with two peptides, the peptides-specific antibodies were isolated from the immunized rabbits. The further experiments demonstrated that N1 peptide-induced polyclonal antibodies had a high affinity to bind to E. coli expressed N protein of SARS-CoV. Furthermore, it was confirmed that N1 peptide-specific IgG antibodies were detectable in the sera of severe acute respiratory syndrome (SARS) patients. The results indicated that an epitope of the N protein has been identified and N protein specific Abs were produced by peptide immunization, which will be usefull for the study of SARS-CoV.

The Fc receptor associated pathway might improve the immune responses against hepatitis B virus (HBV) as previously described by us. In addition, the Flt3 ligand (FL) has been reported to potentiate antigen presenting cells in vivo and may act as a potential adjuvant to boost antigen-specific immune responses. In this study, the immune efficacies of a set of fusion proteins of HBsAg and Fc and/or FL were evaluated in HBsAg transgenic mice.The fusion proteins composed of HBsAg and the Fc domain of murine IgG1 (HBsAg-Fc) and/or the Flt3 ligand, and yeast-derived recombinant HBsAg were used as immunogen to immunize HBsAg transgenic mice, respectively. Serum and liver HBsAg levels, serum anti-HBsAg and cytokine profile, and the activities of alanine aminotransferase (ALT)/AST were investigated after immunization.After six injections, the most pronounced decrease in serum and liver HBsAg levels was observed in the HBsAg-Fc immunized group. In addition, serum Th1 cytokines and ALT/AST activities were highest in this group, indicating an effective induction of a favorable cellular immune response. Interestingly, the fusion protein containing HBsAg-Fc and the Flt3 ligand stimulated an alternative Th1-type immune response featured with high level productions of tumor necrosis factor α (TNF-α) and monocyte chemoabstractant protein 1 (MCP-1), causing a more severe cytotoxicity in hepatocytes while showed less effective in reducing serum HBsAg level.HBsAg-Fc is effective in eliciting both the humoral and cellular immune responses against HBsAg in HBsAg transgenic mice, which makes it a potential immunogen for the immunotherapy of chronic hepatitis B.

The genome of hepatitis B virus (HBV) consists of four open reading frames, encoding the envelope proteins (Pre-S/S), the core proteins (Pre-C/C), the polymerase (P) and the transactivating X protein (X). In the sera of HBV-infected patients, hepatitis B surface antigen (HBsAg) particles without the viral genome can outnumber virions by more than 1000-fold. To analyse the interactions between HBsAg and host cells, global gene-expression profiles of a small HBsAg (SHBs)-secreting stable cell line (HepG2-S-G2) and its counterpart control cell line (HepG2-Neo-F4) were compared. Marked upregulation of lymphoid enhancer-binding factor 1 (LEF-1), a transcription factor in the Wnt pathway, was found in SHBs-expressing cells and was confirmed by interference experiments with small interfering RNA. However, compared with the control cells, HepG2-S-G2 did not show higher proliferative competence in culture or increased tumorigenesis in nude mice. A possible mechanism to explain the discrepancy between the upregulation of LEF-1 and the lack of increased tumorigenesis is SHBs expression resulting in altered expression and distribution of LEF-1 protein in cell compartments and upregulation of LEF-1 isoforms that could suppress, rather than enhance, the Wnt pathway.

Human nuclear receptor hb1 f(nr5a2) was cloned and characterized as a novel member of the Ftz-F1 (nr5a) nuclear receptor subfamily,whose its biological function remained largely unidentified. The aim of this study was to establish transgenic mouse model that specifically expressed hB1F in the liver to faciliate the functional study of hB1F. hb1f cDNA was placed downstream of mouse albumin gene enhancer/promoter to construct a liver-specific hb1f expression vector. Transgene fragments were microinjected into fertilized eggs of mice. The manipulated embryos were transferred into the oviducts of pseudopregnant female mice. Four offspring were identified as carrying the transgenes by PCR,from which one was also verified by Southern blotting. RT-PCR and Western blotting results showed that the transgene was expressed in the liver of the transgenic mice. Transgenic founder mice were used to establish transgenic mouse lineages. The F1 mice were identified by PCR analysis. Genetic analysis of the transgenic mice demonstrated that the transgene had been integrated into the chromosome at a single site and could be stably transmitted.

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.

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.

AIM: To analyze the tissue morphologic phenotype and liver gene expression profile of hB1F transgenic mice. METHODS: Transgene expression was analyzed with RTPCR and Western blotting. For one of the transgenic mouse lines, tissue expression pattern of the transgene was also examined with immunochemical methods. Pathological analysis was used to examine the tissue morphologic phenotype of established transgenic mice. The liver gene expression profile of transgenic mice was analyzed with microchip, and some of the differentially expressed genes were verified with RT-PCR. RESULTS: The expressions of hB1F were shown in livers from 6 of 7 transgenic mouse lines. The overexpression of hB1F transgene did not cause pathological changes. Expressions of three genes were up-regulated, while down-regulation was observed for 25 genes. CONCLUSION: The overexpression of hB1F transgene may cause changes of gene expression profiles in the liver of transgenic mice.

AIM: Human hepatitis B virus enhancer 11131 binding factor (hB1F) was cloned and characterized as a novel member of the Ftz-F1 (NR5A) nuclear receptor subfamily. Although progresses have recently been made, its biological function remains largely unidentified. The aim of this study was to establish an hB1F transgenic mouse model to promote the functional study of hB1F. METHODS: Transgene fragments were microinjected into fertilized eggs of mice. The manipulated embryos were transferred into the oviducts of pseudopregnant female mice. The offsprings were identified by PCR and Southern blot analysis. Transgene expression was analyzed with RT-PCR and Western blot analysis. Transgenic founder mice were used to establish transgenic mouse lineages. The F1 and F2 mice were identified by PCR analysis. RESULTS: Seven mice were identified as carrying copies of transgene. RT-PCR and Western blotting results showed that the transgene was expressed in heart, liver, lung, kidney and stomach in one of the transgenic mouse lineages. Genetic analysis of the transgenic mice demonstrated that the transgene was integrated into the chromosome at a single site, and was transmitted stably. CONCLUSION: In this study we established an hB1F transgenic mouse model, which will facilitate the investigation of the biological function of hB1F in vivo.