Improved ecdysone receptor-based inducible gene regulation system.

To develop an ecdysone receptor (EcR)-based inducible gene regulation system, several constructs were prepared by fusing DEF domains of Choristoneura fumiferana EcR (CfEcR), C. fumiferana ultraspiracle (CfUSP), Mus musculus retinoid X receptor (MmRXR) to either GAL4 DNA binding domain (DBD) or VP16 activation domain. These constructs were tested in mammalian cells to evaluate their ability to transactivate luciferase gene placed under the control of GAL4 response elements and synthetic TATAA promoter. A two-hybrid format switch, where GAL4 DBD was fused to CfEcR (DEF) and VP16 AD was fused to MmRXR (EF) was found to be the best combination. It had the lowest background levels of reporter gene activity in the absence of a ligand and the highest level of reporter gene activity in the presence of a ligand. Both induction and turn-off responses were fast. A 16-fold induction was observed within 3 h of ligand addition and increased to 8942-fold by 48 h after the addition of ligand. Withdrawal of the ligand resulted in 50% and 80% reduction in reporter gene activity by 12 h and 24 h, respectively.     

Reporter substrates for assessing the activity of the hepatitis C virus NS3-4A serine protease in living cells

We describe a versatile system for monitoring the activity of the NS3-4A serine protease of the hepatitis C virus (HCV) in mammalian cells. The system relies on coexpression of the protease and of an artificial substrate containing a reporter domain and an intracellular targeting sequence separated by a NS3-4A-specific cleavage site. We constructed two different substrates suitable for different applications. The first substrate secretory alkaline phosphatase-1 (SEAP-1) harbors the NS3-4A cleavage site inserted between the SEAP and a membrane anchor featuring an endoplasmic reticulum retention sequence. The arrangement of this substrate is such that SEAP is secreted in the extracellular medium depending on the NS3 protease activity. We show that SEAP-1 can be used to evaluate the activity of NS3-4A inhibitors in living cells. In the second substrate (CD8-1), SEAP is replaced by the extracellular domain of the lymphocyte surface antigen CD8 alpha. The arrangement of this substrate is such that the CD8 alpha domain is transported to the cell surface upon NS3-4Ap cleavage and remains associated with the plasma membrane as an integral membrane protein. We show that CD8-1 can be used for selecting cells capable of supporting HCV replication.     

A collaborative screening program for the discovery of inhibitors of HCV NS2/3 cis-cleaving protease activity

This report describes the development of a cell-based assay for high-throughput screening and detection of small-molecule inhibitors for hepatitis C virus (HCV) NS2/3 protease. The HCV NS2/3 protease is essential for the normal infectious cycle of HCV. Generation of a cell-based assay for this cis-acting viral protease involved reporter constructs in which the NS2/3 protease sequence was inserted between the ,B-lactamase (BLA) reporter and a ubiquitin-based destabilization domain. In stable cell lines, NS2/3 cis cleavage of the NS2/3-BLA fusion protein resulted in differential stability of the cleaved versus uncleaved BLA reporter, providing a robust readout for protease activity. BLA reporter activity was shown to be a function of NS2/3-specific protease activity, by using genetic mutants of the NS2/3 sequence. In addition, the cell-based assay was validated and screened in a 384-well format on a fully automated robotic platform where small-molecule inhibitors of NS2/3 protease activity were identified.     

Functional characterization of ecdysone receptor gene switches in mammalian cells

Regulated expression of transgene is essential in basic research as well as for many therapeutic applications. The main purpose of the present study is to understand the functioning of the ecdysone receptor (EcR)-based gene switch in mammalian cells and to develop improved versions of EcR gene switches. We utilized EcR mutants to develop new EcR gene switches that showed higher ligand sensitivity and higher magnitude of induction of reporter gene expression in the presence of ligand. We also developed monopartite versions of EcR gene switches with reduced size of the components that are accommodated into viral vectors. Ligand binding assays revealed that EcR alone could not bind to the nonsteroidal ligand, RH-2485. The EcR's heterodimeric partner, ultraspiracle, is required for efficient binding of EcR to the ligand. The essential role of retinoid X receptor (RXR) or its insect homolog, ultraspiracle, in EcR function is shown by RXR knockdown experiments using RNAi. Chromatin immunoprecipitation assays demonstrated that VP16 (activation domain, AD):GAL4(DNA binding domain, DBD):EcR(ligand binding domain, LBD) or GAL4(DBD):EcR(LBD) fusion proteins can bind to GAL4 response elements in the absence of ligand. The VP16(AD) fusion protein of a chimera between human and locust RXR could heterodimerize with GAL4(DBD):EcR(LBD) in the absence of ligand but the VP16(AD) fusion protein of Homo sapiens RXR requires ligand for its heterodimerization with GAL4(DBD):EcR(LBD).     

Development of a cell-based assay for monitoring specific hepatitis C virus NS3/4A protease activity in mammalian cells

The hepatitis C virus (HCV) contains a positive-sense RNA genome that encodes a unique polyprotein precursor, which must be processed by proteases to enable viral maturation. Virally encoded NS3/4A protease has thus become an attractive target for the development of antiviral drugs. To establish an assay system for monitoring NS3/4A protease activity in mammalian cells, this study describes a substrate vector, pEG(Delta4AB)SEAP, in which enhanced green fluorescent protein (EGFP) was fused to secreted alkaline phosphatase (SEAP) through the NS3/4A protease decapeptide recognition sequence, Delta4AB, which spans the NS4A and NS4B junction region. Secretion of SEAP into the culture medium was demonstrated to depend on the cleavage of Delta4AB by HCV NS3/4A protease. We demonstrated that the accumulation of SEAP activity in the culture medium depends on time up to 60h with the coexpression of active NS3/4A protease. The amount of SEAP in the culture medium was around 10 times greater than that of cells with coexpression of inactive NS3/4A mutant protease. This strategy has made it possible to monitor NS3/4A activity inside mammalian cells. Moreover, by using cells containing the HCV subgenomic replicon, the EG(Delta4AB)SEAP reporter can be used to detect the anti-HCV activity of interferon-alpha (IFN-alpha). Consequently, this EG(Delta4AB)SEAP reporter can be used to screen for NS3/4A protease inhibitors in the cellular environment and for anti-HCV drugs in replicon cells.     

Ribosomal 18S RNA prevails over glyceraldehyde-3-phosphate dehydrogenase and beta-actin genes as internal standard for quantitative comparison of mRNA levels in invasive and noninvasive human melanoma cell subpopulations

A cell-based assay system for monitoring NF-kappaB activity was developed to determine the influence of activated NF-kappaB in human HaCaT cells. The pNF-kappaB-SEAP-NPT plasmid that permits expression of the secretory alkaline phosphatase (SEAP) reporter gene in response to the NF-kappaB activity and contains the neomycin phosphotransferase (NPT) gene for geneticin resistance in host cells was constructed and transfected into the human keratinocyte cell line HaCaT. Human HaCaT transfectant cells were demonstrated to secrete the SEAP enzyme into the culture medium in a time-dependent manner until 72 h. NF-kappaB activities were measured by the SEAP reporter gene assay using a fluorescence detection method. HaCaT cell transfectants treated with antioxidants [e.g., N-acetyl-l-cysteine and vitamin C] showed reduction of NF-kappaB activity in a time- and concentration-dependent manner, whereas phorbol 12-myristate 13-acetate known as a stimulator of NF-kappaB expression increased NF-kappaB activity in a time- and concentration-dependent manner. This assay system could be used to determine the quantitative measurement of NF-kappaB activity in the human skin and allow the screening of anti-inflammatory agents for dermatological purpose from various synthetic chemicals and natural products.     

Assembly of the Herpes Simplex Virus Capsid: Preformed Triplexes Bind to the Nascent Capsid

The herpes simplex virus type 1 (HSV-1) capsid is a T=16 icosahedral shell that forms in the nuclei of infected cells. Capsid assembly also occurs in vitro in reaction mixtures created from insect cell extracts containing recombinant baculovirus-expressed HSV-1 capsid proteins. During capsid formation, the major capsid protein, VP5, and the scaffolding protein, pre-VP22a, condense to form structures that are extended into procapsids by addition of the triplex proteins, VP19C and VP23. We investigated whether triplex proteins bind to the major capsid-scaffold protein complexes as separate polypeptides or as preformed triplexes. Assembly products from reactions lacking one triplex protein were immunoprecipitated and examined for the presence of the other. The results showed that neither triplex protein bound unless both were present, suggesting that interaction between VP19C and VP23 is required before either protein can participate in the assembly process. Sucrose density gradient analysis was employed to determine the sedimentation coefficients of VP19C, VP23, and VP19C-VP23 complexes. The results showed that the two proteins formed a complex with a sedimentation coefficient of 7.2S, a value that is consistent with formation of a VP19C-VP23₂ heterotrimer. Furthermore, VP23 was observed to have a sedimentation coefficient of 4.9S, suggesting that this protein exists as a dimer in solution. Deletion analysis of VP19C revealed two domains that may be required for attachment of the triplex to major capsid-scaffold protein complexes; none of the deletions disrupted interaction of VP19C with VP23. We propose that preformed triplexes (VP19C-VP23₂ heterotrimers) interact with major capsid-scaffold protein complexes during assembly of the HSV-1 capsid.     

Inhibitory effect of galanin on dopamine-induced enhanced vasopressin secretion in rat neurohypophyseal tissue cultures

The effect of galanin (GAL) on vasopressin (VP) secretion was studied in 13-14-day cultures of isolated rat neurohypophyseal (NH) tissue. The VP content of the supernatant was determined by radioimmunoassay (RIA) after a 1- or 2-h incubation. A significantly decreased content of VP was detected following the administration of 10(-6)-10(-9) M doses of GAL. Dopamine (DA) and the DA-active drugs apomorphine (APM) and Pro-Lys-Gly (PLG) (10(-6) M in each medium) increased the VP level of NH tissue cultures. This VP concentration elevation could be blocked by the administration of GAL together with DA, APM or PLG. The DA-blocking effect of GAL was prevented by previous treatment with the GAL receptor antagonist galantid (M15). The results indicate that VP release is directly influenced by the GAL-ergic system. The GAL-ergic control of VP secretion from NH tissue in rats can occur independently of the hypothalamus, at the level of the posterior pituitary.     

Development of enhancer trap lines for functional analysis of the rice genome

Enhancer trapping has provided a powerful strategy for identifying novel genes and regulatory elements. In this study, we adopted an enhancer trap system, consisting of the GAL4/VP16-UAS elements with GUS as the reporter, to generate a trapping population of rice. Currently, 31 443 independent transformants were obtained from two cultivars using Agrobacterium-mediated T-DNA insertion. PCR tests and DNA blot hybridization showed that about 94% of the transformants contained T-DNA insertions. The transformants carried, on average, two copies of the T-DNA, and 42% of the transformants had single-copy insertions. Histochemical assays of approximately 1000 T0 plants revealed various patterns of the reporter gene expression, including expression in only one tissue, and simultaneously in two or more tissues. The expression pattern of the reporter gene in T1 families corresponded well with the T0 plants and segregated in a 3 : 1 Mendelian ratio in majority of the T1 families tested. The frequency of reporter gene expression in the enhancer trap lines was much higher than that in gene trap lines reported previously. Analysis of flanking sequences of T-DNA insertion sites from about 200 transformants showed that almost all the sequences had homology with the sequences in the rice genome databases. Morphologically conspicuous mutations were observed in about 7.5% of the 2679 T1 families that were field-tested, and segregation in more than one-third of the families fit the 3 : 1 ratio. It was concluded that GAL4/VP16-UAS elements provided a useful system for enhancer trap in rice.     

Stable luciferase reporter cell lines for signal transduction pathway readout using GAL4 fusion transactivators

While GAL4 fusion activators have been widely used for dissecting signal transduction pathways in transient assays, there has been surprisingly little reported on utilizing cell lines with stably integrated fusion activators. To avoid problems with the efficiency and reproducibility inherent to transient transfection, we describe here the generation and characterization of HeLa reporter cell lines, which contain a stably integrated luciferase gene responsive to stably integrated and constitutively expressed GAL4-CREB or GAL4-Elk1 fusion activators. These cell lines exhibited extremely low basal luciferase expression but robust response to various extracellular stimuli or the expression of signaling molecules that resulted in elevated MAP kinase or PKA activities. This integrated two-component reporter system allows one to focus specifically on particular signaling pathway endpoints and the altered transactivation activity of either Elk1 or CREB. With the procedures described here, many novel cell-based assays can be developed by generating new reporter cell lines with medically important but difficult-to-transfect cell types, and by using different reporter genes or different fusion transactivator genes.