Faculty Bibliography

The biological consequences of radiation leukaemia virus (RadLV) infection include the stimulation of H-2 antigen expression soon after injection of the virus. Early studies demonstrated that resistance to RadLV-induced leukaemia in certain mouse strains is mediated by genes in the H-2D region of the major histocompatibility complex (MHC). Recent studies have shown that elevated H-2Dd expression on the thymocyte cell surface of resistance mouse strains results from increased mRNA transcription and is correlated with elevated levels of a DNA-binding activity that recognizes a short DNA sequence 5' of the start of transcription for the H-2Dd gene. This binding activity has been termed H-2 binding factor 1 (H-2 BF1) and is found exclusively in the thymus. In an effort to examine the H-2 genes of RadLV-susceptible mice for the presence of the H-2 BF1 binding target, we have cloned class I genes from the highly susceptible B10.S mouse strain and have identified both the Ds and the Ks genes. The entire genomic sequence for the Ds gene has been determined and is reported here. In addition, the 5' regulatory region of the previously cloned Dq gene has been sequenced; mice of the Dq haplotype are also susceptible to RadLV-induced leukaemia. In this report, we show that the H-2 BF1 DNA binding sequence is present 5' of each of these three class I genes

Sindbis virus can infect a broad range of insect and vertebrate cell types due to the widespread distribution of the cellular receptor for the virus. The development of Sindbis virus vectors that target specific cell types could have important implications for the design of gene therapy strategies. To achieve this goal we have designed and constructed Sindbis virus particles displaying the IgG-binding domain of protein A. The protein A-envelope chimeric Sindbis virus vector has minimal infectivities against baby hamster kidney and human cell lines. When used in conjunction with monoclonal antibodies that react with cell-surface antigens, however, the protein A-envelope chimeric virus was able to infect human cell lines with high efficiency. Infection rates were 90% or higher for human lymphoblastoid cells. A variety of cells could be targeted by changing the monoclonal antibody without generating a new recombinant virus

Resistance to radiation leukemia virus-induced leukemia is correlated with an increase in H-2D expression on the thymocyte surface. Recently, it has been shown that elevated H-2Dd expression on the infected thymocyte is a result of elevated mRNA transcription and that the transcriptional increase is correlated with elevated levels of a DNA binding activity, H-2 binding factor 1 (H-2 BF1), which recognizes the 5'-flanking sequences (5'-TGACGCG-3') of the H-2Dd gene. This target for transcription factor binding has been found to be identical in the 5'-regulatory region of 12 rodent class I genes, nine of which have been shown to be functional genes. Furthermore, this cis-element is found 5' of 20 primate class I genes (15 human genes), seven of which are known to be functional. Here, we demonstrate that activation transcription factor 1 (ATF-1) is one component of H-2 BF1. In addition, the levels of ATF-1 mRNA in uninfected and radiation leukemia virus-infected thymocytes parallel those of H-2Dd mRNA, and therefore, it is suggested that ATF-1 up-regulates the transcription of the H-2Dd gene after radiation leukemia virus infection of thymocytes. Transfection experiments also demonstrate that ATF-1 activates a reporter plasmid that contains the H-2 BF1 motif, but not a reporter lacking this motif. This is the first demonstration of the interaction of ATF-1 with 5'-regulatory sequences of major histocompatibility complex class I genes

Tissue-specific delivery of variety of molecules has been a valuable technique for biological and medical research and for the diagnosis and therapy of cancer. We have therefore examined the ability of streptavidin-protein A (ST-PA) fusion protein complexed with monoclonal antibodies (mAbs) to transfer biotinylated proteins into specific type of cells. ST-PA/mAbs complexes could efficiently deliver biotinylated beta-galactosidase into a variety of cancer cell lines through molecules expressed on their surface. In addition, ST-PA/mAb complexed with either biotinylated glucose oxidase or biotinylated ribonuclease A could be transferred to specific cell types and made to display cytotoxic activity against the transduced cell. The flexibility of the system was enhanced by the fact that the cell-targeting specificity could be altered by just changing the mAb used and the 'payload' molecule could be replaced by substituting one biotinylated protein or enzyme with another. This flexibility was achieved without the need to generate a covalent chemical link or engineering new recombinant molecules. Results obtained to date suggest that the ST-PA fusion protein may be used as a nearly 'universal carrier' to transfer a variety of effector molecules into target cells with a high degree of specificity. Essentially, the ST-PA fusion protein effectively serves as a high-efficiency, modular 'molecular bridge' for the transfer into cells of a wide variety of effector molecules

Elucidation of the leukemogenic process induced by fractionated X-irradiation (FX) requires the identification of molecules that mediate the differentiation and regeneration of T cells. To isolate cDNA transcripts associated with FX-induced leukemia in C57BL/6 mice, a cDNA library was constructed from FX-induced thymoma mRNA and differentially screened with cDNA probes. A novel cDNA transcript, FX-induced transcript 1 (FXI-T1), showed strong differential mRNA expression in all C57BL/6 FX-induced thymomas examined when compared with normal thymus tissue. FXI-T1 was not universally expressed in proliferative or other neoplastic cells. Expression of FXI-T1 mRNA in untreated mouse organs was not restricted to the thymus; highest expression was observed in brain and skeletal muscle tissue. The translated FXI-T1 sequence encodes a basic, prolinerich protein that contains a RING-H2-finger motif. The COOH-terminal region of the putative FXI-T1 protein has sequence similarity with the COOH-terminal domain of the Drosophila deltex protein, a component of a signal pathway that functions during cell differentiation. The described observations suggest an association of FXI-T1 with FX-induced leukemogenesis. The study of FXI-T1 should contribute to an understanding of the processes of T-cell differentiation and regeneration in addition to leukemogenesis

The construction of a new retroviral vector, pSKV, is described. This vector carries two unique cloning sites, located between two Moloney leukemia virus-derived LTR, into which genes of interest may be introduced. The gene encoding hygromycin resistance (HyR) was subsequently introduced into one of the two sites, producing a second vector (pSKV/HyR) containing a unique SfiI site for the introduction of cDNA clones under the control of the cytomegalovirus (CMV) promoter (P-CMV). The cDNA (mH13), encoding a protein that has been shown to serve as a murine ecotropic retroviral receptor in transient assays, was cloned into the SfiI site (pSKV/HyR/mH13). Both constructs can be packaged into retroviral particles following transfection into an appropriate packaging cell line. Stable transfectants of the human glioblastoma cell line (U118MG) carrying each of these two constructs were generated by transfection and subsequent Hy selection. Clones expressing both the selectable marker and the mH13 gene, but not those expressing only the selectable marker, are shown to be susceptible to infection with murine ecotropic retroviral particles. These cells (HyR and mH13 positive) were then exposed to CRE/Xtk culture supernatant, a packaging cell line producing ecotropic retroviral particles carrying the HSV-TK (Herpes simplex virus-thymidine kinase) and neoR (neomycin-resistance) genes. Selection was in the presence of G418. In vitro growth of the U118MG/HyR/mH13/TK cells, but not that of the U118MG/HyR/mH13 cells, was inhibited by ganciclovir (GCV), indicating the successful transfer of HSV-TK by infection of human cells with murine retroviruses via the mH13 product

Tissue-specific delivery of a variety of molecules has been a valuable technique for biological and medical research. Therefore, we have constructed a recombinant plasmid containing the coding regions for streptavidin core and mature human transforming growth factor-alpha (TGF-alpha). The recombinant plasmid has been expressed in Escherichia coli to produce a chimeric protein with both streptavidin and TGF-alpha activity. The streptavidin-TGF-alpha chimeric protein (ST-TGF-alpha) could efficiently transfer biotinylated beta-galactosidase into A431 cells via the epidermal growth factor receptor. More than 99% of the cells contained the enzyme transferred. Furthermore, ST-TGF-alpha complexed with biotinylated-glucose oxidase had a significant cytotoxic effect when incubated with A431 cells. These findings suggest that the ST-TGF-alpha chimeric protein could be used to deliver proteins of interest into target cells without the need for chemical linkage or genetic construction. Essentially, ST-TGF-alpha serves as a high-modular 'molecular bridge' for the passage of a wide variety of effector molecules into target cells

We have previously cloned a human gene (H13) homologous to the murine ecotropic retrovirus (E-MuLV) receptor, which, however, does not confer susceptibility to E-MuLV infection. The extracellular domain 3 (ECD3) of H13 contains amino acid residues critical for E-MuLV binding in that the modified H13 gene (mH13), substituted with amino acids from the actual receptor, has the ability to bind E-MuLV. Here we have expressed a fusion protein consisting of mH13/ECD3 and transforming growth factor-alpha in Escherichia coli and demonstrated its binding activity to both ecotropic AKR virus and the epidermal growth factor receptor expressed on the cell surface. Fusion proteins of mH13/ECD3 and ligands to cell surface molecules might be useful for specific cell targeting in E-MuLV-based gene delivery systems

BACKGROUND: Hypericin is a potent virucidal agent with activity against a broad range of enveloped viruses and retroviruses. The effective virucidal activity emanates from a combination of photodynamic and lipophilic properties. Hypericin binds cell membranes (and, by inference, virus membranes) and crosslinks virus capsid proteins. This action results in a loss of infectivity and an inability to retrieve the reverse transcriptase enzymatic activity from the virion. STUDY DESIGN AND METHODS: Since hypericin is devoid of adverse action in most blood components and blood analyses, it is investigated as an additive with potential to inactivate infective viruses in blood components intended for transfusion. RESULTS: Complete inactivation of 10(6) tissue culture-infective doses of human immunodeficiency virus was obtained in whole blood and in diluted packed red cells after illumination with fluorescent light for 1 hour. Loss of viral infectivity to cultured CEM cells has been monitored by use of a detection assay for human immunodeficiency virus p55 in enzyme-linked immunosorbent assay and cytopathic assays. In physiologic media, hypericin interacts with albumin and lipoproteins, retaining the virucidal activity in bound form. The molecule is negatively charged and forms organic and inorganic monobasic salts (ion pairs) in physiologic pH. Various ion pairs differ in virucidal efficacy. CONCLUSION: The apparent transfusibility of hypericin, taken together with the efficacy of the virucidal activity, the broad range of enveloped viruses affected, and the absence of adverse effects on stored red cells, may render hypericin useful for inactivation of infectious viruses in red cells