Bioluminescence imaging of caspase-3 activity in mouse liver.

Apoptosis is an essential process for the maintenance of liver physiology. The ability to noninvasively image apoptosis in livers would provide unique insights into its role in liver disease processes. In the present work, we established a stable mouse model by hydrodynamics methods to study the activity of caspase-3 and evaluate the effect of the apoptosis inhibitors in mouse livers under true physiological conditions by bioluminescence imaging. The reporter plasmid attB-ANLuc(DEVD)BCLuc that contains fragment of attB and ANLuc(DEVD)BCLuc was codelivered with the mouse-codon optimized φC31 (φC31o) integrase plasmids specifically to mouse liver by hydrodynamic injection procedure. Then, φC31o integrase mediated intramolecular recombination between wild-type attB and attP site in mice, and thus the reporter expression cassette attB-ANLuc(DEVD)BCLuc was integrated permanently into mouse liver chromosome. We used these mice to characterize in vivo activation of caspase-3 upon treatment with LPS/D-GalN. Our data show that liver apoptosis could be reflected by the activity of luciferase. The shRNA targeting caspase-3 protein or apoptosis inhibitors could effectively downregulate luciferase activity in vivo. Also, this model could be used to measure caspase-3 activation during inflammatory and infectious events in vivo as verified by infected with MHV-3. This model could be used for screening anti-apoptosis compounds target mouse livers.

Detection of hepatitis B surface antigen by target-induced aggregation monitored by dynamic light scattering.

In the current work, a one-step, washing-free, homogeneous nanosensor assay has been constructed to sensitively detect hepatitis B surface antigen (HBsAg) based on the light scattering property of gold nanoparticles (GNPs) through a sandwich model. The two nanoprobes in this study were designed by conjugating monoclonal and polyclonal hepatitis B surface antibody (HBsAb) onto the GNPs of different diameters. First, the detection behavior of the combinations of different sizes of GNPs was evaluated and the optimized combination was determined. In analyzing HBsAg in Tris-HCl buffer, such bioassay composed of GNPs of approximately 50 and 100 nm has a limit of detection (LOD) as high as 0.005 IU/ml and a dose-dependent response ranging from 0.005 to 1 IU/ml, which indicates its good diagnostic capability and provides a useful means to analyze protein biomarkers with low virus loads. Observation with transmission electron microscopy (TEM) provides direct evidence that the increase of hydrodynamic diameters resulted from the aggregation induced by immunological reactions. The bioassay also exhibits satisfactory specificity in analyzing HBsAg in serum media. Therefore, with its simple preparation, easy readout, and good stability, this bioassay has the potential to be developed into an automated and widely used biosensor assay.

Noninvasive molecular imaging of interferon beta activation in mouse liver.

BACKGROUND/AIMS: Interferon beta (IFN-ß) is the priming cytokine in the interferons (IFNs) response that plays essential roles in innate immune system. Only very few studies on IFN activation in animals have been reported before, therefore, we embarked to develop a novel method to dynamically examine IFN-ß activation in mouse liver by noninvasive molecular imaging. METHODS: Interferon beta promoter-directed firefly luciferase gene was integrated into chromosomes of hepatocytes by hydrodynamic injection. Mouse hepatitis virus type 3 (MHV-3) and polyinosinic-polycytidylic acid [poly(I:C)] were used to stimulate the activation of IFN-ß. Luciferase activity was used as an indicator of the IFN-ß promoter activity in vitro and in vivo. The expression level of IFN-ß in the sera and firefly luciferase in the liver was assessed by ELISA and bioluminescence imaging respectively. Western blot was used for detecting proteins expression. RESULTS: A rapid and elevated luciferase expression in the mouse liver induced by poly (I:C) and MHV-3 was detected by bioluminescence imaging. The detectable level of IFN-ß in the sera was not induced by MHV-3. Moreover, IFN-ß activation was significantly inhibited by the hepatitis C virus (HCV) NS3/4A protease in mouse liver. These results were consistent with IFN-ß production in the sera. Therefore, a novel visual method to monitor IFN-ß promoter activity was established in the current study. CONCLUSION: This novel sensitive method can be used for not only assessing IFN-ß activation or inhibition in the liver under different conditions, but also screening drug candidates of stimulating or inhibiting of IFN-ß production.

Long-term hepatitis C internal ribosome entry site-dependent gene expression mediated by phage phiC31 integrase in mouse model.

BACKGROUND: The lack of a robust small animal model for hepatitis C virus (HCV) has hindered the development of novel drugs, including internal ribosome entry site (IRES) inhibitors. Phage phiC31 integrase has emerged as a potent tool for achieving long-term gene expression in vivo. This study utilized phiC31 integrase to develop a stable, reproducible and easily accessible HCV IRES mouse model. METHODS: phiC31 integrase plasmid and the reporter vector, HCV-IRES-luciferase expression cassette (containing an attB site), was codelivered to murine livers using high pressure tail vein injection. HCV IRES-dependent translation reflected by luciferase expression was accurately monitored in vivo by bioluminescence imaging. Genomic integration of the transgene was confirmed by partial hepatectomy and nested PCR. An HCV IRES-targeted short hairpin RNA (shRNA) expression plasmid, sh184, was hydrodynamically transfected into mouse liver to study its inhibition efficacy in vivo. RESULTS: phiC31 integrase mediated intramolecular recombination between wild-type attB and attP sites in mice. The expression of luciferase was stable after 30 days post-transfection and remained so for 300 days only in the livers of mice that were coinjected with the integrase-encoding plasmid. Luciferase levels reduced dramatically after hydrodynamic transfection of sh184. CONCLUSIONS: These results indicate that this mouse model provides a powerful tool for accurate and long-term evaluation of potential anti-IRES compounds in vivo.

PhiC31 integrase and liver-specific regulatory elements confer high-level, long-term expression of firefly luciferase in mouse liver.

To improve the efficiency of expression of reporter transgenes delivered by hydrodynamic injection, we generated expression cassettes carrying different liver-specific regulatory elements using the firefly luciferase gene as a reporter. From our studies the human alpha-antitrypsin promoter together with the apolipoprotein E/C-I and albumin enhancer was the combination of choice for prolonged transgene expression, but reporter gene expression in vivo lasted for no more than 7 weeks. Subsequently, phage phiC31 integrase was introduced as a potential tool to improve further the efficiency of expression of the delivered transgene. Long-term transgene expression in vivo was achieved by specific integration of the target gene into mouse livers. This study demonstrates the use of a combination of phage phiC31 integrase and liver-specific regulatory elements for generation of transgenic mice.

The modulation of hepatitis C virus 1a replication by PKR is dependent on NF-kB mediated interferon beta response in Huh7.5.1 cells.

Protein kinase R (PKR), a sensor of double-stranded RNA, plays an important role in the host response to viral infection. Hepatitis C genotype 2a virus (HCV2a) has been shown to induce PKR activation to suppress the translation of antiviral interferon stimulated genes (ISGs), suggesting that PKR inhibitor can be beneficial for treating chronically HCV-infected patients in conjunction with interferon alpha and ribavirin. However, in this study, we found that PKR inhibition using siRNA PKR, shRNA PKR or PKR inhibitor enhanced HCV 1a replication and rendered Huh7.5.1 cells more susceptible to HCV1a infection. Additionally, PKR silencing suppressed NF-kB activation and NF-kB mediated STAT1 phosphorylation in Huh7.5.1 cells and HCV1a persistently infected Huh7.5.1 cells (2HDD4). These effects were accompanied by a reduction of interferon beta response and thereby enhanced HCV1a replication in Huh7.5.1 cells. We conclude that host cells can employ PKR activation to restrict HCV1a replication through regulation of NF-kB expression.

A mouse model for studying the clearance of hepatitis B virus in vivo using a luciferase reporter.

Hepatitis B virus(HBV) infection remains a global problem, despite the effectiveness of the Hepatitis B vaccine in preventing infection. The resolution of Hepatitis B virus infection has been believed to be attributable to virus-specific immunity. In vivo direct evaluation of anti-HBV immunity in the liver is currently not possible. We have developed a new assay system that detects HBV clearance in the liver after the hydrodynamic transfer of a reporter gene and over-length, linear HBV DNA into hepatocytes, followed by bioluminescence imaging of the reporter gene (Fluc). We employed bioluminescence detection of luciferase expression in HBV-infected hepatocytes to measure the Hepatitis B core antigen (HBcAg)-specific immune responses directed against these infected hepatocytes. Only HBcAg-immunized, but not mock-treated, animals decreased the amounts of luciferase expression, HBsAg and viral DNA from the liver at day 28 after hydrodynamic infection with over-length HBV DNA, indicating that control of luciferase expression correlates with viral clearance from infected hepatocytes.

High levels of gene expression in the hepatocytes of adult mice, neonatal mice and tree shrews via retro-orbital sinus hydrodynamic injections of naked plasmid DNA.

Hydrodynamic-based gene delivery has emerged as an efficient and simple method for the intracellular transfection of naked plasmid DNA (pDNA) in vivo. In this system, a hydrodynamic injection via the tail vein is the most effective non-viral method of liver-targeted gene delivery. However, this injection is often technically challenging when used in animals whose tail veins are difficult to visualize or too small to operate on. To overcome this limitation, an alternative in vivo gene delivery method, the rapid injection of large volume of pDNA solution through retro-orbital sinus, was established. Using this technique, we successfully delivered pDNA to the tissue of adult mice, neonatal mice and tree shrews. The efficient expression of exogenous genes was specifically detected in the liver of test animals treated with this gene delivery method. This study demonstrates for the first time that the hydrodynamic gene delivery via the retro-orbital sinus can not only reach the same transgene efficiency as a tradition hydrodynamic-based intravascular injection but also be used in animals that are difficult to inject via the tail vein. This method could open up new areas in gene function studies and gene therapy disease treatment.

A novel, universal and sensitive lateral-flow based method for the detection of multiple bacterial contamination in platelet concentrations.

In the present study, we aimed to develop a nucleic acid lateral-flow method for the rapid and sensitive detection of multiple bacteria that contaminate platelet concentrations (PCs). Polymerase chain reaction (PCR) amplicons were produced by a set of board-range primers that recognize the conserved region of bacteria 16S rDNA, followed by hybridization with both an FITC (fluorescein isothiocyanate)-labelled probe and biotin-labelled probe, and then a nucleic acid lateral-flow dipstick (LFD) assay. The LFD accurately identified 7 species of bacteria, but had no cross-reactivity with human genomic DNA. The limit of detection (LOD) of the LFD assay was as low as 10(1) copies/µL of 16S rDNA for plasmid. In the case of spiked PCs without enrichment, the detection limit of LFD for K. pneumonia was 5 CFU/mL, 6.5 × 10(4) CFU/mL for the S. epidermidis and 35 CFU/mL for P. aeruginosa.

Relative quantification of protein-protein interactions using a dual luciferase reporter pull-down assay system.

The identification and quantitative analysis of protein-protein interactions are essential to the functional characterization of proteins in the post-proteomics era. The methods currently available are generally time-consuming, technically complicated, insensitive and/or semi-quantitative. The lack of simple, sensitive approaches to precisely quantify protein-protein interactions still prevents our understanding of the functions of many proteins. Here, we develop a novel dual luciferase reporter pull-down assay by combining a biotinylated Firefly luciferase pull-down assay with a dual luciferase reporter assay. The biotinylated Firefly luciferase-tagged protein enables rapid and efficient isolation of a putative Renilla luciferase-tagged binding protein from a relatively small amount of sample. Both of these proteins can be quantitatively detected using the dual luciferase reporter assay system. Protein-protein interactions, including Fos-Jun located in the nucleus; MAVS-TRAF3 in cytoplasm; inducible IRF3 dimerization; viral protein-regulated interactions, such as MAVS-MAVS and MAVS-TRAF3; IRF3 dimerization; and protein interaction domain mapping, are studied using this novel assay system. Herein, we demonstrate that this dual luciferase reporter pull-down assay enables the quantification of the relative amounts of interacting proteins that bind to streptavidin-coupled beads for protein purification. This study provides a simple, rapid, sensitive, and efficient approach to identify and quantify relative protein-protein interactions. Importantly, the dual luciferase reporter pull-down method will facilitate the functional determination of proteins.

Bioluminescence imaging of Hepatitis C virus NS3/4A serine protease activity in cells and living animals.

The lack of robust small animal models has been an obstacle to the screening of Hepatitis C virus (HCV) NS3/4A protease inhibitors in vivo. Here, we described a reporter assay system for in vivo noninvasive imaging of NS3/4A serine protease activity using split firefly luciferase complementation strategy. The reporter construct ANluc(NS5A/B)BCluc constitutes the split N- and C-terminal fragments of luciferase, fused to interacting peptides, pepA and pepB, respectively, with an intervening HCV NS3/4A cleavage motif of NS5A/B. We proved that the reporter molecule could be proteolytically cleaved by NS3/4A at the NS5A/B motif in cells and living animals. Association of pepA and pepB brought inactive fragments of luciferase into close proximity, thereby restoring bioluminescence activity. The increase in luciferase activity was proportional to the dose of active NS3/4A protease. The ANluc(NS5A/B)BCluc reporter also could be used to detect the activity of NS3/4A-specific shRNA and IFN-alpha. Therefore, the reporter assay system using split firefly luciferase complementation strategy should prove useful for evaluating NS3/4A protease activity in cells and living animals.

Screening compounds against HCV based on MAVS/IFN-ß pathway in a replicon model.

AIM: To develop a sensitive assay for screening compounds against hepatitis C virus (HCV). METHODS: The proteolytic cleavage of NS3/4A on enhanced yellow fluorescent protein (eYFP)-mitochondrial antiviral signaling protein (MAVS) was examined by reporter enzyme secreted placental alkaline phosphatase (SEAP), which enabled us to perform ongoing monitoring of anti-HCV drugs through repeated chemiluminescence. Subcellular localization of eYFP-MAVS was assessed by fluorescence microscopy. Cellular localization and protein levels were examined by Western blotting. RESULTS: HCV NS3/4A protease cleaved eYFP-MAVS from mitochondria to block the activation of interferon (IFN)-ß promoter, thus resulting in downregulation of SEAP activity. The decrease in SEAP activity was proportional to the dose of active NS3/4A protease. Also this reporter assay was used to detect anti-HCV activity of IFN-α and cyclosporine A. CONCLUSION: Our data show that this reporter system is a sensitive and quantitative reporter of anti-HCV inhibitors. This system will constitute a new tool to allow the efficient screening of HCV inhibitors.

Formation and function of hepatitis C virus replication complexes require residues in the carboxy-terminal domain of NS4B protein.

During replication, hepatitis C virus (HCV) NS4B protein rearranges intracellular membranes to form foci, or the web, the putative site for HCV replication. To understand the role of the C-terminal domain (CTD) in NS4B function, mutations were introduced into NS4B alone or in the context of HCV polyprotein. First, we show that the CTD is required for NS4B-induced web structure, but it is not sufficient to form the web nor is it required for NS4B membrane association. Interestingly, all the mutations introduced into the CTD impeded HCV genome replication, but only two resulted in a disruption of NS4B foci. Further, we found that NS4B interacts with NS3 and NS5A, and that mutations causing NS4B mislocalization have a similar effect on these proteins. Finally, we show that the redistribution of Rab5 to NS4B foci requires an intact CTD, suggesting that Rab5 facilitates NS4B foci formation through interaction with the CTD.

Participation of rab5, an early endosome protein, in hepatitis C virus RNA replication machinery.

Like most positive-strand RNA viruses, hepatitis C virus (HCV) is believed to replicate its genome on the surface of rearranged membranes. We have shown previously that HCV NS4AB, but not the product NS4B, inhibits endoplasmic reticulum (ER)-to-Golgi protein traffic (K. V. Konan, T. H. Giddings, Jr., M. Ikeda, K. Li, S. M. Lemon, and K. Kirkegaard, J. Virol. 77:7843-7855). However, both NS4AB and NS4B can induce "membranous web" formation, first reported by Egger et al. (D. B Egger, R. Gosert, L. Bianchi, H. E. Blum, D. Moradpour, and K. Bienz, J. Virol. 76:5974-5984), which is also observed in HCV-infected cells (Y. Rouille, F. Helle, D. Delgrange, P. Roingeard, C. Voisset, E. Blanchard, S. Belouzard, J. McKeating, A. H. Patel, G. Maertens, T. Wakita, C. Wychowski, and J. Dubuisson, J. Virol. 80:2832-2841) and cells that bear a subgenomic NS5A-green fluorescent protein (GFP) replicon (D. Moradpour, M. J. Evans, R. Gosert, Z. Yuan, H. E. Blum, S. P. Goff, B. D. Lindenbach, and C. M. Rice, J. Virol. 78:7400-7409). To determine the intracellular origin of the web, we examined NS4B colocalization with endogenous cellular markers in the context of the full-length or subgenomic replicon. We found that, in addition to ER markers, early endosome (EE) proteins, including Rab5, were associated with web-inducing protein NS4B. Furthermore, an immunoisolated fraction containing NS4B was found to contain both ER and EE proteins. Using fluorescence microscopy, we showed that wild-type and constitutively active Rab5 proteins were associated with NS4B. Interestingly, expression of dominant-negative Rab5 resulted in significant loss of GFP fluorescence in NS5A-GFP replicon cells. We also found that a small reduction in Rab5 protein expression decreased HCV RNA synthesis significantly. Furthermore, transfection of labeled Rab5 small interfering RNAs into NS5A-GFP replicon cells resulted in a significant decrease in GFP fluorescence. Finally, Rab5 protein was found to coimmunoprecipitate with HCV NS4B. These studies suggest that EE proteins, including Rab5, may play a role in HCV genome replication or web formation.

[Liver tissue-specific stable expression of human CD81 molecule].

AIM: To express stably human CD81 gene on mouse hepatoma cell line Hepa 1-6 using liver specific promoter. METHODS: RNA were isolated from human HepG2 cells which could be infected with hepatitis C virus. RT-PCR was carried out using human CD81 gene specific primers. Amplified fragments were cloned into pGEM-T vector. Albumin promoter and enhancer which were liver tissue specific were ligated to the 5'end of human CD81 gene and SV40 polyA sequence was fused with 3'end of CD81. The fused CD81 gene was inserted into eukaryotic expression vector pcDNA3 to construct a recombinant vector pcDNA3-Alb p-CD81 which was then transfected into Hepa 1-6 cells through lipofectamine mediation. Human CD81 mRNA transcription and its protein expression were detected by RT-PCR and FACS, respectively. RESULTS: Sequence analysis showed that the cloned gene segment was human CD81 gene sequence. After transfection, transcripted human CD81 mRNA was obtained and human CD81 molecules were expressed stably on Hepa 1-6 cells. CONCLUSION: The obtained positive cell clones which stably express HCV receptor human CD81 lay the foundation for further study on interactions between HCV envelope proteins and human CD81, screening of HCV infection blocking drugs and development of HCV infection mouse model.