Inducible CYP4F12 enhances Hepatitis C virus infection via association with viral nonstructural protein 5B.

Hepatitis C virus (HCV) nonstructural protein 5B (NS5B) functions as an RNA-dependent RNA polymerase in the HCV replication complex derived from the endoplasmic reticulum in hepatic cells. In this study, NS5B was used as bait in a yeast two-hybrid assay to screen a human liver cDNA library. We confirmed that CYP4F12, a member of the cytochrome P450 superfamily, interacted with NS5B. Furthermore, overexpression of CYP4F12 facilitated HCV replication. In contrast, knockdown of CYP4F12 by specific shRNA decreased HCV replication and viral protein expression. Moreover, our results demonstrated that HCV infection increased the binding of the transcription factor SREBP1 to the CYP4F12 promoter and activated the promoter activity, which indicated that HCV infection increased the expression of CYP4F12 through the SREBP1 pathway. Our results showed that HCV infection induced expression of CYP4F12 protein, which bound to the HCV replication complex to facilitate viral replication.

Soluble interleukin-6 receptor is elevated during influenza A virus infection and mediates the IL-6 and IL-32 inflammatory cytokine burst.

Influenza A virus (IAV) infection is a major worldwide public health problem. However, the factors involved in mediating the inflammatory response to this infection and their relationships remain poorly understood. Here, we show that IAV infection stimulates the expression of the soluble IL-6 receptor (sIL-6R), a multifunctional protein involved in IL-6 signaling. Interestingly, sIL-6R expression upregulated the levels of its own ligand, IL-6 and those of the pro-inflammatory cytokine IL-32. shRNA-mediated knockdown of sIL-6R suppressed IL-6 and IL-32, indicating that this regulation is dependent on sIL-6R during IAV infection. Furthermore, our results demonstrate that IL-32 participates in a negative feedback loop that inhibits sIL-6R while upregulating IL-6 expression during IAV infection. Therefore, we show that sIL-6R is a critical cellular factor involved in the acute inflammatory response to viral infection.

HCV NS4B induces apoptosis through the mitochondrial death pathway.

The hepatitis C virus (HCV) NS4B protein is known to induce the formation of a membranous web that is thought to be the site of viral RNA replication. However, the exact functions of NS4B remain poorly characterized. In this study, we found that NS4B induced apoptosis in 293T cells and Huh7 cells, as confirmed by Hoechst staining, DNA fragmentation, and annexin V/PI assays. Furthermore, protein immunoblot analysis demonstrated that NS4B triggered the cleavage of caspase 3, caspase 7, and poly(ADP-ribose) polymerase (PARP). Further studies revealed that NS4B induced the activation of caspase 9, the reduction of mitochondrial membrane potential and the release of cytochrome c from the mitochondria. However, NS4B expression did not trigger XBP1 mRNA splicing and increase the expression of binding immunoglobulin protein (BiP, or GRP78) and C/EBP homologous protein (CHOP), which serves as the indicators of ER stress. Taken together, our results suggest that HCV NS4B induces apoptosis through the mitochondrial death pathway.

[High-titer preparation of HIV-1-based defective lentivector and it mediated efficient gene transfer].

Lentivectors have drawn considerable attention recently and become important delivery vehicles for gene transfer manipulation. By Transiently co-transfecting 293T packaging cells with three DNA plasmids system encoding lentivector constituents, a protocol for bulky preparation of human immunodeficiency virus type-1 ( HIV-1)-based defective lentivector with high titer has been established. Transient co-transfection of 293T packaging cells resulted in production of high-titer vector (1.1 x 10 IU/ml), which can be further concentrated over 100-fold through a single step centrifugation. The vector was capable of efficiently transducing a variety of cells from both primate and non-primate sources, including of human T-lymphoblastoid cell line. Long-term culture of vector transduced cells showed a stable expression of foreign gene over 18 months detected by RT-PCR. Assessment of potential generation of replication-competent virus revealed no detection of p24 antigen protein or infectious particles in vector-transduced cells.

Nucleocapsid amino acids 211 to 254, in particular, tetrad glutamines, are essential for the interaction between the nucleocapsid and membrane proteins of SARS-associated coronavirus.

GST pull-down assays were used to characterize the SARS-CoV membrane (M) and nucleocapsid (N) interaction, and it was found that the amino acids 211-254 of N protein were essential for this interaction. When tetrad glutamines (Q) were replaced with glutamic acids (E) at positions of 240-243 of the N protein, the interaction was disrupted.

Expression, purification and characterization of the Hepatitis B virus entire envelope large protein in Pichia pastoris.

The current HBsAg vaccine has performed a vital role in preventing the transmission of HBV during the past 20 years. However, a number of individuals still show no response or a low response to the vaccine. In the present study, the HBV envelope large protein gene was cloned into the eukaryotic expression vector pPIC9k and was subsequently expressed in the yeast Pichia pastoris. The HBV large protein (L protein) was produced and secreted into the medium, where some of the L protein formed particles. The soluble L protein and particles were purified by column chromatography and sucrose density gradient centrifugation. Western blot analysis demonstrated that the particle was composed of both HBV L and S protein. To compare the antigenicity of the L protein and HBsAg, rabbits were immunized with the soluble L protein and the commercially available HBV vaccine and the increasing level of antibodies was determined by ELISA. The results showed that the anti-HBsAg antibody, from rabbits injected with the L protein at a dose of 2 and 10microg, was detected on day 14, whereas rabbits vaccinated with 10 and 2microg HBsAg did not develop antibodies until day 21 and 28, respectively. The antibody level in groups inoculated with the L protein was approximately 50% higher than in the group injected with HBsAg using the same dose. Furthermore, 2microg L protein induced a significant and rapid anti-HBsAg antibody response than 10microg HBsAg. Therefore, we suggest that the L protein is an ideal candidate for a new generation HB vaccine to protect people from HBV infection.

HCV NS2 protein inhibits cell proliferation and induces cell cycle arrest in the S-phase in mammalian cells through down-regulation of cyclin A expression.

Chronic hepatitis C virus (HCV) infection often leads to liver cancer. NS2 protein is a HCV hydrophobic transmembrane protein that associates with several cellular proteins in mammalian cells. In this report, we investigated the functions of NS2 protein by examining its effects on cell growth and cell cycle progression. Stable NS2-expressing HeLa and Vero cell lines were established by transfection of the cells with pcDNA3.1(-)-NS2 followed by selection of the transfected cells in the presence of G418. We found that the proliferation rates of both NS2-expressing cell lines were inhibited by 40-50% compared with the control cells that were transfected with pcDNA3.1(-) control vector. Cell cycle analysis of these NS2-expressing cell lines shows that the proportion of cells in the S-phase increased significantly compared to that of control cells that do not express NS2 protein, suggesting NS2 protein induces cell cycle arrest in the S-phase. Further studies showed that the induction of cell cycle arrest in the S-phase by NS2 protein is associated with the decrease of cyclin A level. In contrast, the expression of NS2 protein does not affect the levels of cyclin-dependent kinase CDK2, CDK4, cyclin D1, or cyclin E. Our results suggest that HCV NS2 protein inhibits cell growth and induces the cell cycle arrest in the S-phase through down-regulation of cyclin A expression, which may be beneficial to HCV viral replication. Our findings not only provide information in the understanding mechanism of HCV infection, but also provide guidance for the future development of potential therapeutics for the prevention and treatment of the viral infection.

Purification and application of bacterially expressed chimeric protein E1E2 of hepatitis C virus.

E1 and E2 glycoproteins are structural components of hepatitis C virus (HCV) virion. They are involved in cellular receptors interaction, neutralising antibodies elicitation, and viral morphogenesis. They are considered as major candidates for anti-HCV vaccine. In this report, we first expressed tandem E1E2 as well as C-terminally truncated E1 fragment and C-terminally truncated E2 fragment, respectively, in Escherichia coli cells and the proteins were purified to homogenesis. All the purified proteins can react specifically with patient sera. Both purified chimeric protein E1E2 and protein E2 can interact with a putative cellular receptor CD81, while purified protein E1 cannot interact with CD81. The sera of rabbit immunized with the E1E2 inhibited the binding of E2 protein to the major extracellular loop of human CD81 and reacted with both proteins E1 and E2, respectively. Anti-E1 and E2 antibodies can be generated simultaneously in the rabbit immunized with the E1E2, and the titers of antibodies were 63 or 56% higher than the titers induced by E1 or E2 alone, respectively. The results suggest that E1 and E2 can enhance their immunogenicity each other in chimeric protein E1E2 and the E. coli-derived chimeric protein E1E2 and corresponding antisera can be used as an useful tools in anti-HCV vaccine research.

In vitro and in vivo protective effects of proteoglycan isolated from mycelia of Ganoderma lucidum on carbon tetrachloride-induced liver injury.

AIM: To investigate the possible mechanism of the protective effects of a bioactive fraction,Ganoderma lucidum proteoglycan (GLPG) isolated from Ganoderma lucidum mycelia, against carbon tetrachloride-induced liver injury. METHODS: A liver injury model was induced by carbon tetrachloride. Cytotoxicity was measured by MTT assay. The activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined with an automatic multifunction-biochemical analyzer and the levels of superoxide dismutase (SOD)and TNF-alpha were determined following the instructions of SOD kit and TNF radioimmunoassay kit. Liver sections were stained with hematoxylin and eosin (H and E) for histological evaluation and examined under light microscope. RESULTS: We found that GLPG can alleviate the L-02 liver cells injury induced by carbon tetrachloride (CCl4) through the measurements of ALT and AST activities and the administration of GLPG to L-02 cells did not display any toxicity. Furthermore, histological analysis of mice liver injury induced by CCl4 with or without GLPG pretreatment indicated that GLPG can significantly suppress the toxicity induced by CCl4 in mice liver. We also found that GLPG reduced TNF-alpha level induced by CCl4 in the plasma of mice, whereas increased SOD activity in the rat serum. CONCLUSION: GLPG has hepatic protective activity against CCl4-induced injury both in vitro and in vivo. The possible anti-hepatotoxic mechanisms may be related to the suppression of TNF-alpha level and the free radical scavenging activity.

[The purification of HBV full-length PreS protein in Pichia pastoris].

The Pichia pastoris strain GS115-PreS could produce a high expression level of full-length PreS protein that secreted to the supernatant after methanol induction in the fermentation. The Western blot analysis showed a single band with expected molecular mass of 48kD and that the major component of the particles was the full-length PreS protein (PreS1 + PreS2 + S) and small envelope protein (S) of 48 and 28 kD, respectively. Electron microscopy image showed PreS particles with 30 nm in diameter. The supernatants of the fermentation were desalted and concentrated. Purified PreS protein was obtained by DEAE-SFF anion exchange column chromatography and the PreS particles were obtained by ultracentrifugation and sucrose density gradient. The ELISA assay results proved that both full-length PreS protein and particles showed high immunogenicity and specificity. P/N ratio further demonstrated that the immunogenicity of the particles is higher than the full-length PreS protein.

Activation of NF-kappaB by the full-length nucleocapsid protein of the SARS coronavirus.

The severe acute respiratory syndrome coronavirus (SARS-CoV) is the major causative agent for the worldwide outbreak of SARS in 2003. The mechanism by which SARS-CoV causes atypical pneumonia remains unclear. The nuclear factor kappa B (NF-kappaB) is a key transcription factor that activates numerous genes involved in cellular immune response and inflammation. Many studies have shown that NF-kappaB plays an important role in the pathogenesis of lung diseases. In this study, we investigated the possible regulatory interaction between the SARS-CoV nucleocapsid (N) protein and NF-kappaB by luciferase activity assay. Our results showed that the SARS-CoV N protein can significantly activate NF-kappaB only in Vero E6 cells, which are susceptible to SARS-CoV infection, but not in Vero or HeLa cells. This suggests that NF-kappaB activation is cell-specific. Furthermore, NF-kappaB activation in Vero E6 cells expressing the N protein is dose-dependent. Further experiments showed that there is more than one function domain in the N protein responsible for NF-kappaB activation. Our data indicated the possible role of the N protein in the pathogenesis of SARS.

Gene expression profiles of HeLa Cells impacted by hepatitis C virus non-structural protein NS4B.

By a cDNA array representing 2308 signal transduction-related genes, we studied the expression profiles of HeLa cells stably transfected by Hepatitis C virus nonstructural protein 4B (HCV-NS4B). The alterations of the expression of four genes were confirmed by real-time quantitative RTPCR; and the aldo-keto reductase family 1, member C1 (AKR1C1) enzyme activity was detected in HCV-NS4B transiently transfected HeLa cells and Huh-7, a human hepatoma cell line. Of the 2,308 genes we examined, 34 were up-regulated and 56 were down-regulated. These 90 genes involved oncogenes, tumor suppressors, cell receptors, complements, adhesions, transcription and translation, cytoskeleton and cellular stress. The expression profiling suggested that multiple regulatory pathways were affected by HCV-NS4B directly or indirectly. And since these genes are related to carcinogenesis, host defense system and cell homeostatic mechanism, we can conclude that HCV-NS4B could play some important roles in the pathogenesis mechanism of HCV.

Hepatitis C virus non-structural 5A protein can enhance full-length core protein-induced nuclear factor-kappaB activation.

AIM: To study the effects of hepatitis C virus (HCV) core and non-structural 5A (NS5A) proteins on nuclear factor-kappaB (NF-kappaB) activity for understanding their biological function on chronic hepatitis caused by HCV infection. METHODS: Luciferase assay was used to measure the activity of NF-kappaB in three different cell lines cotransfected with a series of deletion mutants of core protein alone or together with NS5A protein using pNF-kappaB-Luc as a reporter plasmid. Western blot and indirect immunofluorescence assays were used to confirm the expression of proteins and to detect their subcellular localization, respectively. Furthermore, Western blot was also used to detect the expression levels of NF-kappaB/p65, NF-kappaB/p50, and inhibitor kappaB-a (IkappaB-a). RESULTS: The wild-type core protein (C191) and its mutant segments (C173 and C158) could activate NF-kappaB in Huh7 cells only and activation caused by (C191) could be enhanced by NS5A protein. Moreover, the full-length core protein and its different deletion mutants alone or together with NS5A protein did not enhance the expression level of NF-kappaB. The NF-kappaB activity was augmented due to the dissociation of NF-kappaB-IkappaB complex and the degradation of IkappaB-a. CONCLUSION: NF-kappaB is the key transcription factor that can activate many genes that are involved in the cellular immune response and inflammation. Coexpression of the full-length core protein along with NS5A can enhance the NF-kappaB activation, and this activation may play a significant role in chronic liver diseases including hepatocellular carcinoma associated with HCV infection.

Possible mode of action of antiherpetic activities of a proteoglycan isolated from the mycelia of Ganoderma lucidum in vitro.

A bioactive fraction (GLPG) was extracted and purified from the mycelia of Ganoderma lucidum by EtOH precipitation and DEAE-cellulose column chromatography. GLPG was a proteoglycan and had a carbohydrate:protein ratio of 10.4:1. Its antiviral activities against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) were investigated by the cytopathic effect (CPE) inhibition assay in cell culture. This kind of polysaccharide inhibited the development of the cytopathic effect in dose-dependent manner in HSV-infected cells, moreover did not show any cytotoxic effects on cells even when a concentration was as high as 2000 microg/ml. In order to study the possible mode of action of the antiviral activity of GLPG, cells were treated with GLPG before, during and after infection, and the viral titers in the supernatant of cell culture 48 h post-infection were tested by TCID(50) assay. The antiviral effects in pre-treated and treated during virus infection with GLPG were more remarkable than the treatment of post-infection. Although the precise mechanism has yet to be defined, our work suggested that GLPG inhibits viral replication by interfering with the early events of viral adsorption and entry into target cells. Thus, this proteoglycan seems to be a potential candidate for anti-HSV agents.