Host cell factor requirement for hepatitis C virus enzyme maturation.

The cellular chaperone, HSP90, is identified here as an essential factor for the activity of NS2/3 protease of hepatitis C virus. The cleavage activity of NS2/3 protease synthesized in reticulocyte lysate is ATP-dependent, as evidenced by ATP depletion experiments and inhibition with nonhydrolyzable ATP analogs. Geldanamycin and radicicol, ATP-competitive inhibitors of the chaperone HSP90, also inhibit the cleavage of in vitro-synthesized NS2/3. Furthermore, these HSP90 inhibitors prevent NS2/3 cleavage when the protease is expressed in mammalian cells. The physical association of NS2/3 with HSP90 is demonstrated by immunoprecipitation. Thus, by way of a chaperone/folding activity, an HSP90-containing complex is required for maturation of the polyprotein that encodes the enzymes essential for hepatitis C virus replication.

A ubiquitin-based tagging system for controlled modulation of protein stability.

Many biotechnology applications depend on the expression of exogenous proteins in a predictable and controllable manner. A key determinant of the intracellular concentration of a given protein is its stability or "half-life." We have developed a versatile and reliable system for producing short half-life forms of proteins expressed in mammalian cells. The system consists of a series of destabilization domains composed of varying numbers of a mutant form of ubiquitin (UbG76V) that cannot be cleaved by ubiquitin hydrolases. We show that increasing the number of UbG76V moieties within the destabilization domain results in a graded decrease in protein half-life and steady-state levels when fused to heterologous reporter proteins as well as cellular proteins. Cells expressing a destabilized beta-lactamase reporter act as a robust, high-throughput screening (HTS)-compatible assay for proteasome activity within cells.

Development and application of a GFP-FRET intracellular caspase assay for drug screening.

Apoptosis is a crucial biological process, and activation of caspase endoproteases is essential for proper regulation and execution of apoptosis. Because caspases also appear to be central players in several pathological states, there is a practical need within the biopharmaceutical research community for facile, noninvasive cellular assays for the discovery of compounds that modulate caspase activity. Tandem molecules of green fluorescent protein (GFP) stably expressed within cells can serve as a genetically encoded sensor of protease activity. Using this technology, we have developed a stable cellular system for the screening of agents that modulate activation of the caspase cascade. This assay technology allows for the real-time monitoring of apoptosis in situ, using conventional fluorescent plate reader detection. By applying this assay system to an actual compound screen, small-molecule inducers of cell apoptosis were reliably identified. Follow-up pharmacology confirmed that the rank-order potency of primary hits using the intracellular GFP assay corresponded to that found using a conventional, cell lysis-based assay method.

A fluorescent reporter assay for the detection of ligands acting through Gi protein-coupled receptors.

Accompanying the advances in basic biology of G protein-coupled receptors (GPCRs) is the practical need among biopharmaceutical companies for sensitive assays to assess GPCR function, particularly formats that are compatible with high-throughput drug screening. Here we describe a novel cell-based assay format for the high-throughput detection of ligands for Gi protein-coupled receptors. Two Gi-GPCRs, mu-opioid receptor (mu-OPR) and 5-hydroxytryptamine receptor la (5HT1aR) are employed as model receptor targets. The key feature of this assay system is the isolation of stable, clonal Chinese hamster ovary (CHO) cell lines that carry three separate expression plasmids: (1) a chimeric Gq/i5 protein (which re-directs a negative Gi-type signal to a positive Gq-type response), (2) a given Gi-GPCR, and (3) a beta-lactamase (beta1a) reporter gene responsive to Gi-GPCR signaling. Cell-based assays built using this format show appropriate rank order of potency among a reference set of receptor agonist and antagonist compounds. Such assays are also robust, reliable, and can be used for industrial-scale applications such as high-throughput screening for drug leads.

Using GFP in FRET-based applications.

The use of green fluorescent protein (GFP) is a powerful technology that has recently enabled investigators to study dynamic molecular events within living cells. One method for detecting molecular interactions involves fluorescence resonance energy transfer (FRET) between two GFPs or between GFP and a second fluorophore. This review summarizes the use of GFP for FRET and illustrates the theme with specific examples on how GFP has been employed as an intracellular molecular sensor.

Discovery of a novel, potent, and Src family-selective tyrosine kinase inhibitor. Study of Lck- and FynT-dependent T cell activation.

Here, we have studied the activity of a novel protein-tyrosine kinase inhibitor that is selective for the Src family of tyrosine kinases. We have focused our study on the effects of this compound on T cell receptor-induced T cell activation, a process dependent on the activity of the Src kinases Lck and FynT. This compound is a nanomolar inhibitor of Lck and FynT, inhibits anti-CD3-induced protein-tyrosine kinase activity in T cells, demonstrates selectivity for Lck and FynT over ZAP-70, and preferentially inhibits T cell receptor-dependent anti-CD3-induced T cell proliferation over non-T cell receptor-dependent phorbol 12-myristate 13-acetate/interleukin-2 (IL-2)-induced T cell proliferation. Interestingly, this compound selectively inhibits the induction of the IL-2 gene, but not the granulocyte-macrophage colony-stimulating factor or IL-2 receptor genes. This compound offers a useful new tool for examining the role of the Lck and FynT tyrosine kinases versus ZAP-70 in T cell activation as well as the role of other Src family kinases in receptor function.

Role of tyrosine kinases in lymphocyte activation: targets for drug intervention.

Recent developments in our understanding of lymphocyte receptor-associated signalling events have offered many new potential targets for modifying antigen and cytokine receptor signalling events in immune-related diseases such as allergy, autoimmunity and transplant rejection. As discussed below, these targets are largely tissue-restricted and are functionally confined to a limited set of receptors. Therefore, it is anticipated that selective inhibitors of these signalling events would offer safe and effective therapies for immunologically-based diseases. First, we review T and B cell antigen receptor signalling as targets for inhibiting lymphocyte responses. Second, targets in lymphocyte cytokine receptor signalling pathways are discussed. Finally, we review strategies for inhibition of receptor signalling.

Dual EBNA1 promoter usage by Epstein-Barr virus in human B-cell lines expressing unique intermediate cellular phenotypes.

The use of different viral promoters for the expression of the EBNA1 gene product appears to be a critical step in the regulation of Epstein-Barr virus latent gene expression and may reflect the extent of differentiation of B-cell hosts. Low-passage Burkitt lymphoma cell lines resemble immature B cells in that they express CD10 (CALLA) and do not express B-cell activation antigens. In these cells, transcription from a promoter located in the BamHI F fragment of the viral genome results in the exclusive expression of EBNA1, referred to as the latency I pattern of viral gene expression. In contrast, high-passage Burkitt lymphoma cells and lymphoblastoid cell lines resemble activated B cells in that they do not express CD10 but do express activation antigens such as CD23. In these cells, the use of two promoters located in the BamHI W and C fragments of the viral genome leads to the expression of all six EBNA gene products (latency III). We have found that four human B-cell lines, DB, LBW2, LBW14, and Josh 7, stably express a pattern of B-cell differentiation antigens intermediate between those found in latency I and latency III cell lines and characterized by the coexpression of CD10 and CD23. The pattern of EBNA1 promoter usage in these cell lines was examined to determine whether their intermediate cellular phenotype was reflected in their patterns of viral gene expression. DB, LBW2, and LBW14 utilize both the BamHI F promoter region and BamHI W promoter region to transcribe the EBNA1 gene. This stable pattern of mixed promoter usage for the expression of the EBNA gene products in B cells has not previously been described. In addition, these three B-cell lines expressed lower levels of the viral latent gene product EBNA2 than those typically observed in latency III cells. The lower levels of activation of viral and cellular promoters known to be regulated by EBNA2 also correlated with the reduced levels of EBNA2 expression in these cells. These included the viral LMP1 and LMP2A promoters and the cellular CD23B promoter. The fourth B-cell line, Josh 7, expressed EBNA1 mRNAs derived from both the BamHI W promoter and BamHI C promoter, similar to latency III cells. The intermediate cellular phenotype in Josh 7 cells appeared to be due, in part, to a deficiency in the expression of viral LMP1.(ABSTRACT TRUNCATED AT 400 WORDS)

Generation of phosphatidic acid and diacylglycerols following ligation of surface immunoglobulin in human B lymphocytes: potential role in PKC activation.

We have examined signal transduction via membrane IgM (mIgM) in resting and cycling human B cells. Crosslinking mIgM on all of the cell types studied transduced a signal through the phosphatidylinositol pathway, producing inositol 1,4,5-trisphosphate and release of intracellular free calcium. These second messengers were formed regardless of quantitative or qualitative differences in the surface expression of mIgM: cells that had low levels of surface IgM (T-51) or had no light chain associated with surface heavy chain (DB) signaled phosphatidylinositol pathway activation after mIgM crosslinking. Production of specific lipid products in nonquiescent B cells differed from that in normal resting cells. Ligation of surface immunoglobulin on resting B cells resulted in sustained increases of both diacylglycerol and phosphatidic acid, two lipids that can influence PKC activation. Whereas PKC was strongly activated in normal tonsillar B cells, several cell lines had reduced PKC activation following crosslinking of mIgM. The reduction in protein kinase C activation correlated with the absence or reduced levels of phosphatidic acid or diacylglycerol following stimulation: protein kinase C translocated and was activated only in cells that had elevated levels of both diacylglycerides and phosphatidic acid. Anti-IgM-induced phosphorylation of a protein kinase C substrate protein CD20, also increased in those cells having PKC activation and not in cells in which kinase activity was reduced. CD20 phosphorylation also increased following the direct addition of exogenous phosphatidic acid to resting B cells. Together, these observations show that the generation of lipid products following mIgM crosslinking in resting cells can vary from that in cycling cells and may relate to the different levels of PKC activation. In a companion study we report that ligation of surface IgM activates both an acyltransferase and phospholipase D to form phosphatidic acid.

Conserved patterns of somatic mutation and secondary VH gene rearrangement create aberrant Ig-encoding genes in Epstein-Barr virus-transformed and normal human B lymphocytes.

Expression of N-terminal-truncated Ig heavy chains without normal light chain expression has been shown to occur in human B cell tumor lines, and to be due to diverse types of structural alteration within the expressed Ig heavy and light chain genes. Due to the tumor cell origin of these lines, generation of aberrant Ig-encoding genes may only occur after malignant transformation, reflecting the release of the tumor B cell from the need to express functional Ig for continued clonal proliferation. The genetic basis for expression of VH-truncated mu chains without light chains in several Epstein-Barr virus (EBV)-transformed human B cell lines was investigated with the aim that this information would lead to detection of similarly aberrant Ig genes in normal human B lymphocytes. Analysis of the productive mu genes in three truncated mu-only human B cell lines showed a consistent structural change where a secondary VH-VHDJH gene rearrangement had occurred. The site of VH-VH joining was suggestive of a V(D)J recombinase-mediated event. A consistent pattern of mutation was also observed in the normal-sized, but non-functional kappa light chain transcripts, making them incapable of coding for a functional kappa chain. Using genomic DNA from peripheral blood lymphocytes of a donor whose B cells were originally used to make one of the truncated mu-only EBV B cell lines, a similarly mutated V kappa gene was detected and a similar composite VH-VH gene was cloned. The lack of such aberrant VH and V kappa genes in non-lymphoid cells of this individual showed that the structural abnormalities in the expressed Ig genes arose somatically during development of that B cell clone. The presence of these altered Ig heavy and light chain genes in the genomic DNA of untransformed lymphocytes also shows that generation of such variant B cell clones can be a pre-neoplastic event and occurs by genetic mechanisms active during normal B cell development.

Co-expression of full-length and truncated Ig mu-chains in human B lymphocytes results from alternative splicing of a single primary RNA transcript.

A survey of Ig synthesis among mu-heavy chain-producing human B cell lines indicated that roughly one-third co-express full length (mu+) and a particular type of truncated mu-chain (designated mu'). The relative molecular size of the intracellular form of this truncated mu-chain and its normal pattern of N-glycosylation suggested that mu'-chains were missing a single Ig domain at the protein level. Cell-free translation of polyA+ RNA from mu'-producing human B cell lines generated appropriate mu'-translation products, and Northern blot analysis demonstrated the presence of correspondingly truncated mu'-transcripts in these lines. These results pointed to a pretranslational basis for mu+/mu' co-expression. Sequencing of mu+- and mu'-cDNA clones from two human B cell lines showed that mu+- and mu'-transcripts derive from the same primary transcript, with mu'-mRNA formed by a direct leader-to-C mu 1 exon splice such that the heavy chain variable region exon is excluded via a cassette-type alternative splicing mechanism. Southern blot analysis of the rearranged Ig heavy chain genes in one B cell line confirmed that the co-expressed mu+- and mu'-mRNA derive from the same rearranged Ig heavy chain gene. mu'-cDNA clones were readily isolated from normal human bone marrow lymphocytes, whereas peripheral B cells do not appear to express mu'-transcripts. The frequent occurrence of mu'-mRNA in B cell lines, and its high relative expression in untransformed bone marrow lymphocytes attest to a mode of post-transcriptional control of Ig gene expression that may have implications for human B cell development.

VH and VL region structure of antibodies that recognize the (NANP)3 dodecapeptide sequence in the circumsporozoite protein of Plasmodium falciparum.

The sporozoite form of Plasmodium falciparum displays on its surface the circumsporozoite (CS) protein. The central domain of this protein possesses a reiterated tetrapeptide sequence Asn-Ala-Asn-Pro (NANP), and greater than 90% of the sporozoite-specific antibodies obtained from individuals living in malaria endemic areas recognize epitopes within this repeat sequence. Considering the highly repetitive structure of this naturally occurring antigen and its immunodominance, we were interested in analyzing the structural diversity of antibodies that bind to the (NANP)3 sequence. Molecular characterization of immunoglobulin heavy and light chain mRNA was performed for five hybridomas that produce antibodies with binding specificity for the dodecapeptide (NANP)3. These hybridomas were produced in BALB/c mice by inoculation with whole P. falciparum sporozoites. Sequence analysis and Northern blotting showed that for heavy chain, three hybridomas used VH elements that belong to the VHIX family and two to the VHJ558 family. Four different V kappa subgroups were represented among the light chains. Different D and J kappa segments are also utilized, while four heavy chain gene rearrangements involved the JH4 segment. These results indicated that multiple VH-VL gene combinations can code for reactivity to the (NANP)3 sequence, demonstrating that the murine antibody response to this immunodominant region is structurally heterogeneous.

Clonal diversity in the B cell repertoire of patients with X-linked agammaglobulinemia.

Ig protein and mRNA expression was examined in a collection of 18 monoclonal EBV-transformed B cell lines derived from five patients with X-linked agammaglobulinemia (XLA). A diversity of H and L chain isotypes were synthesized by these lines: the majority (12 lines) expressed mu kappa chains, while mu lambda (two lines), gamma kappa (one), gamma lambda (one), delta lambda (one), and alpha kappa (one) isotype expression was also observed. For all the mu kappa-producing XLA B cell lines, the mu and kappa mRNA transcripts were of native size, and sequence analysis across the regions of VHDJH and V kappa J kappa gene joining showed that Ig gene rearrangements occurred in a typical manner. A variety of VHDJH and V kappa J kappa gene rearrangements were observed, not only within the set of mu kappa+ XLA B cells as a whole, but also among the cell lines derived from single patients. Southern blot analysis for genomic Ig H chain gene rearrangements was done to fully assess the extent of clonal heterogeneity among multiple mu kappa+ XLA B cell lines derived from two patients; all the B cell lines possessed distinct gene rearrangement patterns demonstrating their clonal unrelatedness. Our findings indicate that the B cell repertoire in individual XLA patients is clonally diverse and that it is unlikely that the defect in B cell differentiation in XLA is the result of inefficient or ineffective rearrangement of Ig H or L chain genes. Rather, this study provides support for the idea that the XLA defect relates to a more generalized cellular function, such as regulating the proliferation and/or clonal expansion of cells of the B lymphoid lineage.

Characterization of immunoglobulin mRNA expression in Burkitt lymphoma cell lines.

Immunoglobulin heavy- and light-chain mRNA of 11 Burkitt lymphoma (BL) cell lines (9 African and 2 American) were analyzed for various structural characteristics. In agreement with previous results at the protein level, all the BL cell lines express heavy-chain mRNA transcripts of the mu class. Surprisingly, a high mu s/mu m mRNA ratio was found in 2 IgM-producing BL cell lines (Raji and CCL85), that do not secrete immunoglobulin. Variable region gene use was also assessed in the cell lines: while 4 out of 7 endemic BL cell lines use VH genes that belong to the VH3 gene family, no clear bias in the expression of particular VH or VL gene families among this sampling of BL lines was found. Northern blot analysis of immunoglobulin transcripts in endemic BL cell lines did show that 2 such lines (AG876 and HTB62) expressed truncated heavy-chain transcripts; RNA sequence analysis of the VH region demonstrated different abnormal 5'-localized RNA splicing events for the 2 shortened mu transcripts. The light-chain mRNA in these 2 cell lines also showed structural abnormalities and, in the case of HTB62, 3 different kappa light-chain transcripts are produced (of elongated, native and truncated sizes). In vitro translation of mRNA from HTB62 showed mu and kappa chain proteins corresponding with the relative size for each message.

Interleukin 1 and cyclic AMP induce kappa immunoglobulin light-chain expression via activation of an NF-kappa B-like DNA-binding protein.

Interleukin 1 (IL-1) induces the synthesis of kappa immunoglobulin light chains and the expression of surface immunoglobulin in the murine pre-B-cell line 70Z/3 (J. G. Giri, P. W. Kincade, and S. B. Mizel, J. Immunol. 132:223-228, 1984). In the present study, we found that these effects of IL-1 are mimicked by cyclic AMP (cAMP) analogs and cAMP-elevating drugs. The induction of kappa immunoglobulin light-chain gene expression by IL-1 was associated with an increase in intracellular cAMP levels. Incubation of 70Z/3 cells with IL-1 or cAMP resulted in the activation of the kappa immunoglobulin enhancer, as detected by the induction of chloramphenicol acetyltransferase (CAT) in cells transfected with a kappa enhancer-CAT expression plasmid. In contrast, CAT plasmids lacking a kappa immunoglobulin enhancer were inactive in the presence of IL-1 or cAMP. Furthermore, IL-1 and cAMP analogs and inducers were found to induce the activation of a NF-kappa B-like DNA-binding protein that exhibited specificity for the kappa immunoglobulin enhancer. These results suggest that cAMP may play an important role as a second messenger for IL-1 in the induction of kappa immunoglobulin light-chain synthesis in pre-B cells via the activation of a DNA-binding protein that is similar or identical to NF-kappa B.

The coupling between enhancer activity and hypomethylation of kappa immunoglobulin genes is developmentally regulated.

Previous studies have indicated that immunoglobulin enhancers are essential for establishing transcriptional competence but not for maintaining the activity of constitutively transcribed genes. To understand the basis for this developmental shift away from dependence on enhancer function, we have investigated the relationship between transcriptional activity and methylation status of the immunoglobulin kappa light-chain genes (kappa genes) in mouse cell lines representing different stages of B-cell maturation. Using pre-B-cell lines in which the level of a critical kappa enhancer-binding factor, NF-kappa B, was controlled by the administration or withdrawal of lipopolysaccharide and plasmacytoma lines that either contain or lack this factor, we studied the properties of endogenous kappa genes and of transfected kappa genes which were stably integrated into the genomes of these cells. In the pre-B cells, the exogenous (originally unmethylated) kappa genes, as well as endogenous kappa genes, were fully methylated and persistently dependent on enhancer function, even after more than 30 generations in a transcriptionally active state. In plasmacytoma cells, the endogenous kappa genes were invariably hypomethylated, whereas exogenous kappa genes were hypomethylated only in cells that contain NF-kappa B and are thus permissive for kappa enhancer function. These results indicate that the linkage of hypomethylation to enhancer-dependent activation of kappa transcription occurs after the pre-B-cell stage of development. The change in methylation status, together with associated changes in chromatin structure, may suffice to eliminate or lessen the importance of the enhancer for the maintenance of the transcriptionally active state.

Molecular basis of the cell-surface expression of immunoglobulin mu chain without light chain in human B lymphocytes.

Four distinct human B-lymphoid cell lines possess the ability to circumvent the mechanism regulating intracellular transport of immunoglobulin protein. These cells do not produce light chains, yet they express mu heavy chains on the cell surface at comparable levels to B-cell lines that produce native forms of both proteins. The mu-chain mRNA produced in all four cell lines was found to contain an identical deletion of most of the heavy-chain variable (VH) region (75% of the 3' portion), with no apparent alteration in constant (C) region structure. The truncated mu (mu*)-chain mRNA in these cells was created through the use of a cryptic splice donor site found within the human VH gene(s) utilized by these B-cell lines. The truncated mu chains exhibited a decreased ability to associate with the intracellular transport regulatory protein, heavy-chain binding protein (BiP). This result indicates that VH region structure, in addition to C mu 1 region structure, influences the formation of the BiP recognition site on the heavy chain. Furthermore, it suggests that the mechanism allowing for cell-surface expression of the mu* chains in the absence of light-chain pairing is the inability of BiP to bind to the mu* chains and hence prevent their intracellular transport. The high frequency with which the mu-only surface immunoglobulin positive phenotype is present in our collection of human B-cell lines and the isolation of one of the cell lines from a healthy individual also suggest that B cells of this type may represent a significant subpopulation among the normal human B-cell repertoire.

A biological consequence of variation in the site of D-JH gene rearrangement.

One mechanism which generates diversity in immunoglobulin variable (V) regions is flexibility in the site of recombination among the constituent genetic elements. Within a specific antibody family (that is, a particular VH-VL combination), variability in V-D-J rearrangement not only leads to sequence diversity at the boundary of the juxtaposed genes, but also enables the total length of the third complementarity-determining region (CDR-3) of the heavy chain to be conserved. We demonstrate here that the junctional diversity inherent in rearranged immunoglobulin genes can have consequences for the biology of the immune system. Sequence analysis of the expressed immunoglobulin genes of idiotypically variant as opposed to conventional B lymphocytes of a dominant antibody family showed that the variant B cells undergo a novel D-JH joining event such that an extra amino acid is inserted into the heavy chain CDR-3. The unique D-region conformation possessed by the variant B cells accounts for previous observations which showed that variant and conventional B cells could be differentially regulated in vivo by an autologous set of idiotope-specific B lymphocytes. Our findings indicate that D-region structure can determine the expression of regulatory idiotopes and suggest that the conservation of heavy-chain CDR-3 length within an antibody family may reflect regulatory as well as functional constraints.

Identification and characterization of an apparent germline set of auto-anti-idiotypic regulatory B lymphocytes.

By fusing BALB/c splenic lymphocytes from mice immunized with phosphorylcholine (PC) to an immunoglobulin nonproducing plasmacytoma cell line, a B cell hybridoma was isolated (MM-60) that has been shown by multiple criteria to produce a bona fide auto-anti-(anti-T15 idiotype) antibody. In vivo administration of MM-60 antibody suppressed T15+ anti-PC antibody production in an idiotope-specific manner by activation of an intervening set of anti-T15 B cells. These T15-specific B cells i) appeared to express germline-encoded variable region gene products, ii) developed in parallel to, but independent of, T15+ B cells, and iii) suppressed the anti-PC response in a T cell-independent fashion. Variants of T15+ anti-PC B cells possessing aberrant immunoglobulin heavy chain D region structure escaped from the suppression imposed by this anti-T15 B cell set, suggesting that a function of the heavy chain D region may be to contribute to the formation of molecular target sites for idiotype-directed regulatory cells and/or antibodies. The indigenous nature of these particular populations of anti-idiotypic and anti-(anti-idiotypic) B cells and the ability of their immunoglobulin products to regulate antigen-specific B cells in vivo provides strong supportive evidence for the significant role idiotype-directed network interactions play in regulating specific antibody production during a normal immune response.