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C Fraefel

Publications and source records attributed to C Fraefel.

36 records · Page 2Linked to original sources
PubMed1998

A chimeric fusion protein of cytochrome CYP4B1 and green fluorescent protein for detection of pro-drug activating gene delivery and for gene therapy in malignant glioma.

Quantity and distribution of transgene-expressing tumor cells are central issues in cancer gene therapy. These are critical for the efficiency of tumor killing and for the bystander effect. In an attempt to combine the advantages of a potent bioactivating "suicide" gene with a marker gene for living cells, cDNA encoding cytochrome CYP4B1 was fused to the green fluorescent protein (GFP) cDNA. The resulting chimeric fusion protein, 4B1EGFP, was expressed in rodent and human glioma cell lines in culture. The ability of this recombinant enzyme to destroy tumor cells by converting the prodrug 4-ipomeanol (4-IM) into alkylating metabolites was evaluated in comparison with the cytotoxicity of the native CYP4B1 enzyme. The most sensitive 4B1EGFP-expressing glioma cell clone had a LD50 of 0.75 microgram/ml for 4-IM, as compared to a 4-IM LD50 of 0.5 microgram/ml in glioma cells expressing the native CYP4B1. A strong bystander effect mediated by cell-to-cell contact was present in the 4B1EGFP clones, allowing for more than 50% bystander kill at a ratio of expressing to non-expressing cells of 1:100. A herpes-simplex amplicon (pHSVPrPUC delta Hind) was constructed with the 4B1EGFP fusion protein, and recombinant helper-free HSV particles were packaged in Vero cells. Fisher 344 rats were inoculated with 4 x 10(5) 9L tumor cells to produce epidural tumor. Recombinant HSV particles were injected into the tumor at a dose of 1 x 10(7) pfu. Tumor was resected in living anesthetized animals 24, 48, and 72 hours after virus injection, and cryostat sections were evaluated by fluorescent microscopy. HSV-mediated delivery of the fusion protein to tumor cells was successfully demonstrated. In conclusion, the chimeric fusion protein 4B1EGFP retains essentially all features of the native CYP4B1 enzyme, and, moreover, offers advantages in terms of gene transfer visualization, which may lead to improvement of gene transfer strategies.

Animals↗
PubMed1997

Green fluorescent protein as a reporter for retrovirus and helper virus-free HSV-1 amplicon vector-mediated gene transfer into neural cells in culture and in vivo.

Green fluorescent protein (GFP) is an effective marker for retrovirus and herpes virus vector-mediated gene transfer into various central nervous system-derived cells, both proliferative and non-proliferative, in culture and in vivo. Retrovirus vectors were used to stably transduce several rat and human glioma lines, and a multipotent mouse neural progenitor line in culture. Implantation of selected pools of transduced glioma cells into rodent brain allowed clear visualization of the tumor and the invading tumor edge. Helper virus-free HSV-1 amplicon vectors successfully transferred gfp into non-dividing primary neural cells in culture and in the rat brain. This study describes the versatility of GFP for: (i) labelling of glioma cells in experimental brain tumor models and neural progenitor cells by retrovirus vectors, and (ii) efficient, non-toxic delivery of genes to post mitotic cells of the nervous system using helper-virus free HSV-1 amplicon vectors.

Animals↗
PubMed1997

HSV/AAV hybrid amplicon vectors extend transgene expression in human glioma cells.

Novel hybrid vectors, which incorporate critical elements of both herpes simplex virus type 1 (HSV-1) amplicon vectors and adeno-associated virus (AAV) vectors, are able to sustain transgene expression in dividing glioma cells for over 2 weeks. These vectors combine the high infectibility and large transgene capacity of HSV-1 vectors with the potential for episomal amplification and chromosomal integration of AAV vectors. The hybrid vectors contain the HSV-1 origin of DNA replication, oriS, and the DNA cleavage/packaging signal, pac, which allow amplicon replication and packaging in HSV-1 virions. The lacZ reporter gene under control of the CMV IE1 promoter is flanked by AAV inverted terminal repeat (ITR) sequences, which facilitate replication and genomic integration of this cassette in the host cell nucleus. Constructs were generated with or without the AAV rep gene (rep+ and rep-) to assess its importance in extending transgene expression. Expression of Rep proteins was confirmed by Western blot analysis. An HSV-1 amplicon construct containing the reporter gene, but no AAV sequences, was used as a control. Constructs were packaged into HSV-1 virions with or without helper virus and these vector stocks were used to infect human U87 glioma cells in culture. The hybrid vectors supported transgene retention and expression for over 2 weeks, whereas the control amplicon vector lost the transgene after 10 days. Expression was somewhat longer for the rep+ as compared to the rep- hybrid vectors. Toxicity due to the HSV-1 helper virus was eliminated using helper virus-free amplicon vector stocks. Transgene constructs could also be packaged in AAV virions, using AAV and adenovirus or HSV-1 helper functions. These HSV/AAV hybrid vectors should allow long-term, nontoxic gene delivery of DNA constructs to both dividing and nondividing cells.

Animals↗
PubMed1997

Helper virus-free herpes simplex virus-1 plasmid vectors for gene therapy of Parkinson's disease and other neurological disorders.

Vectors based on herpes simplex virus type 1 (HSV-1) have potential for gene therapy of neurological disorders. HSV-1 plasmid vectors (amplicons) contain only approximately 1% of the 150-kb HSV-1 genome and have been packaged into virus particles by using a helper virus. We have demonstrated that HSV-1 plasmid vectors which express tyrosine hydroxylase can cause long-term biochemical and behavioral recovery in the 6-hydroxydopamine rat model of Parkinson's disease. Furthermore, we and others have used HSV-1 plasmid vectors which express a wide range of genes that affect neuronal physiology. Because of the pathogenicity of the HSV-1 helper virus, however, the use of this vector system has been limited to studies in animal models or primary cultures of neural cells. Thus, to increase the safety of HSV-1 plasmid vectors, we recently developed a helper virus-free packaging system that may facilitate studies on neuronal physiology and potential therapeutic applications.

Animals↗
PubMed1997

Gene transfer into hepatocytes mediated by helper virus-free HSV/AAV hybrid vectors.

BACKGROUND: Vectors based on herpes simplex virus type 1 (HSV-1) can efficiently transduce hepatocytes in the mouse liver, and vector genomes can persist for at least 2 months. However, 24 hr after gene transfer, the number of cells that express the transgene decreases rapidly and no transduced cells are detectable after 7 days. In this study, we examined the capability of a helper virus-free HSV/AAV hybrid amplicon vector to extend transgene expression in hepatocytes in vivo. MATERIALS AND METHODS: HSV-1 amplicon or HSV/AAV hybrid amplicon vectors that express reporter genes from different transcriptional regulatory sequences were packaged into HSV-1 virions using a helper virus-free packaging system. To determine relative transduction efficiencies, vector stocks were titered on four different cell lines, including hamster kidney (BHK21) and human lung (Hs913T) fibroblasts, and mouse (G6Pase-/-) and human (NPLC) hepatocytes. After in vivo injection of vector stocks into mouse liver, tissue sections were examined for reporter gene expression and cellular inflammatory response. Blood samples were collected to measure serum transaminase levels as a biochemical index of liver toxicity. RESULTS: Expression of a reporter gene from liver-specific promoter sequences was consistently more effective in hepatic cells compared with fibroblasts, whereas the opposite was true when using an HSV-1 immediate-early promoter. Expression in hepatocytes in vivo was markedly longer from HSV/AAV hybrid vector compared with traditional HSV-1 amplicon vector: the number of transduced cells (approximately 2% of all hepatocytes) remained stable over 7 days after injection of HSV/AAV hybrid vector, whereas no transduced cells were detected 7 days after gene transfer with standard HSV-1 amplicon vector. The rapid decline in reporter gene expression from standard amplicons was not solely caused by a B or T lymphocyte-mediated immune response, as it also occurred in RAG2-/- mice. Hepatocyte toxicity and cellular inflammatory effects associated with HSV/AAV hybrid vector-mediated gene transfer were minimal, and readministration of vector stock proved equally effective in naive mice and in animals that received a first vector dose 4 weeks earlier. CONCLUSIONS: HSV/AAV hybrid amplicon vectors support gene expression in vivo for considerably longer than do traditional HSV-1 amplicon vectors. Moreover, expression from these vectors does not provoke an overt inflammatory or immune response, allowing efficacious expression following repeated in vivo dosing. These characteristics suggest that such vectors may hold future promise for hepatic gene replacement therapy.

Animals↗
PubMed1996

A method to codetect introduced genes and their products in gene therapy protocols.

To monitor the presence of introduced genes and the distribution of the encoded proteins in host tissues after gene transfer, we combined fluorescence in situ hybridization (FISH) and immunohistochemistry in two separate gene therapy paradigms. In brain tissue sections from animals injected with pHSVlac vector, we localized nuclei containing vector DNA both in cells expressing and not expressing beta-galactosidase (beta-gal). This suggests that the efficiency of gene transfer is affected not only by gene delivery, but also by cellular controls on gene expression. In a second paradigm, following myoblast transplantation, we detected donor nuclei in the muscle of a patient with Duchenne's muscular dystrophy. The donor nuclei were either surrounded by host nuclei or apparently fused in the patient's muscle fiber producing dystrophin. The combined FISH and immunohistochemistry assay offers greater sensitivity and more information than currently used polymerase chain reaction and protein detection methods.

Adult↗
PubMed1996

Identification and characterization of BICP27, an early protein of bovine herpesvirus 1 which may stimulate mRNA 3' processing.

Sequence analysis of the left genomic terminus of bovine herpesvirus 1 (BHV-1) revealed two convergently transcribed genes with 3' ends about 300 bp apart. The gene on the left is the previously described circ gene; that on the right was found to encode a protein of 400 amino acids which was designated BICP27 because of its homology to ICP27 (Vmw63) of herpes simplex virus 1 (HSV-1) and related proteins from other alpha- beta- and gammaherpesviruses. Rabbit antisera raised against a synthetic oligopeptide representing the amino terminus of the predicted polypeptide demonstrated the presence of BICP27 in the nuclei of infected cells by in situ immunoadsorbent assays. In Western immunoblots, BICP27 was detected as a 50 kDa BHV-1 specific protein expressed with early kinetics, in contrast to HSV-1 ICP27 which is an immediate early (IE) protein. A DNA fragment containing BICP27 coding sequences was inserted into a baculovirus genome. The recombinant BICP27 protein, identified by its reactivity with the antipeptide sera, exhibited the same electrophoretic mobility as BICP27 specified by BHV-1. Transient expression assays using target genes differing only in their poly(A) sites showed that BICP27, like its HSV-1 counterpart, may be involved in 3' processing of mRNA.

Amino Acid Sequence↗
PubMed1996

Helper virus-free transfer of herpes simplex virus type 1 plasmid vectors into neural cells.

Herpes simplex virus type 1 (HSV-1) plasmid vectors have promise for genetic intervention in the brain, but several problems caused by the helper virus have compromised their utility. To develop a helper virus-free packaging system for these vectors, the DNA cleavage/packaging signals were deleted from a set of cosmids that represents the HSV-1 genome. Following cotransfection into cells, this modified cosmid set supported replication and packaging of vector DNA. However, in the absence of the DNA cleavage/packaging signals, the HSV-1 genome was not packaged, and consequently vector stocks were free of detectable helper virus. In the absence of helper virus, the vectors efficiently infected rat neural cells in culture or in the brain with minimal cytopathic effects. beta-galactosidase-positive cells were observed for at least 1 month in vivo, and vector DNA persisted for this period. This system may facilitate studies on neuronal physiology and potential therapeutic applications.

Animals↗
PubMed1996

Recombinant bovine herpesvirus-1 (BHV-1) lacking transactivator protein BICPO entails lack of glycoprotein C and severely reduced infectivity.

The immediate-early transactivator protein BICPO is a key regulatory element of bovine herpesvirus 1 (BHV-1) replication based on transient expression assays. To examine BICPO function in the context of the viral genome, we created recombinant BHV-1 expressing beta-galactosidase instead of BICPO. To complement the defect, a neomycin resistant MDBK cell line (M164) expressing BICPO was established, permitting selection of a blue-staining BHV-1 recombinant (A2G2). Southern blot and PCR analysis confirmed that the BICPO gene was interrupted by the beta-galactosidase gene and that wt progeny was absent. Compared with wt BHV-1, A2G2 reached lower titers in M164 cells but replicated with similar kinetics. Once isolated, A2G2 also grew in MDBK cells although the titer was reduced a further 10-fold and the virus remained strongly cell-associated. Thus, BICPO is not absolutely required for replication in cell culture. Gene expression of A2G2 was investigated by Western blots and immunofluorescence. Surprisingly, not only was BICPO absent, but glycoprotein C (gC) was also missing. Other viral genes were expressed normally. Semiquantitative PCR showed that A2G2 produced similar amounts of viral DNA as wt but a much smaller number of infectious particles. Cotransfection of A2G2 DNA and a plasmid containing the BICPO gene yielded revertant virus with fully restored wt properties. We conclude that BICPO is required for gC expression, and that the missing gC partly accounts for the reduced A2G2 infectivity.

Animals↗
PubMed1994

BICP22 of bovine herpesvirus 1 is encoded by a spliced 1.7 kb RNA which exhibits immediate early and late transcription kinetics.

Kinetic analysis of the two divergent immediate early (IE) transcription units of bovine herpesvirus 1 (BHV-1) revealed an unexpected behaviour. The IE1.7 promoter was not turned off at the end of the IE period but acted as a late promoter, unlike the adjacent IE4.2/2.9 promoter which was active only under IE conditions. The genome region specifying the IE1.7 gene was sequenced (0.814 to 0.839 map units). The IE1.7 promoter was found to overlap with duplicated sequence elements bearing close similarity to herpesvirus origins of replication, which may explain the biphasic transcription kinetics. Exons 1 and 2 of the spliced IE1.7 transcript were non-coding. Exon 3 was found to contain a single open reading frame encoding a protein of 300 amino acids that was designated BICP22 because of its homology to ICP22 (Vmw68) of herpes simplex virus type 1 and related proteins from other herpesviruses. The protein probably represents IEP-55, the most abundant BHV-1 phosphoprotein observed under IE conditions.

Amino Acid Sequence↗
PubMed1994

Identification of the bovine herpesvirus 1 circ protein, a myristylated and virion-associated polypeptide which is not essential for virus replication in cell culture.

We have recently reported immediate-early (IE) transcription over covalently joined genome ends of bovine herpesvirus 1 (BHV-1). A spliced 1.5-kb IE RNA (IER1.5) is coterminal with an unspliced 1.1-kb late RNA (LR1.1) which is transcribed from the left end of the genome. Sequence analysis reveals an open reading frame common to IER1.5 and LR1.1 predicted to encode the 247-amino-acid circ polypeptide. This paper reports on the identification of circ as a protein. Using a rabbit antiserum raised against a synthetic oligopeptide representing the carboxy terminus of the predicted circ polypeptide for Western blot (immunoblot) analyses and immunofluorescence assays, we identified a 34-kDa virion-associated protein which accumulated in the cytoplasm of infected cells. To confirm that LR1.1 indeed encoded the 34-kDa polypeptide, we inserted a DNA fragment containing circ coding sequences into the Autographa californica baculovirus genome. A group of recombinant polypeptides with sizes of 32, 34, and 35 kDa were identified by their reactivity with the antipeptide serum. Chicken egg yolk antibodies raised against total proteins of insect cells infected with the recombinant baculovirus identified the 34-kDa circ protein specified by BHV-1. The recombinant circ polypeptides and the circ protein specified by BHV-1 were both myristylated, as determined by radiolabeling with [3H]myristic acid. It was noted that the circ gene could be deleted from the BHV-1 genome without impairing virus replication in cell culture.

Amino Acid Sequence↗
PubMed1994

Identification and zinc dependence of the bovine herpesvirus 1 transactivator protein BICP0.

Bovine herpesvirus 1 (BHV-1) specifies and unspliced early 2.6-kb RNA (ER2.6) which is 3' coterminal with exon 2 of the 2.9-kb immediate-early (IE) RNA. The two transcripts have a common open reading frame (676 codons). The predicted protein, designated BHV-1 infected cell protein 0 (BICP0), contains a zinc finger domain with homology to ICP0 of herpes simplex virus type 1 and protein 61 of varicella-zoster virus, and depending on the promoter, it acts as a strong activator or as a repressor in transient expression assays. In situ immunoadsorbent assays using antisera against synthetic oligopeptides demonstrated that BICP0 accumulates in nuclei of BHV-1-infected cells, as expected for an IE gene product involved in gene regulation. Western blots (immunoblots) revealed a BHV-1-specific 97-kDa protein which was detectable during the IE phase and also at later periods of infection, indicating that the kinetics of BICP0 synthesis is consistent with the switch from IER2.9 to ER2.6. To confirm that ER2.6 encoded the 97-kDa BICP0 protein, a DNA fragment containing BICP0-coding sequences was inserted into the Autographa californica baculovirus genome. A recombinant protein, identified by its reactivity with antipeptide sera, exhibited the same electrophoretic mobility as BICP0 specified by BHV-1. We microinjected Xenopus oocytes with a BICP0 effector plasmid and a promoter-chloramphenicol acetyltransferase plasmid. BICP0-induced stimulation of this promoter was strongly reduced when intracellular zinc was chelated by thionein, indicating that the effect of BICP0 is zinc dependent.

Amino Acid Sequence↗
PubMed1993

Promoter, spliced leader, and coding sequence for BICP4, the largest of the immediate-early proteins of bovine herpesvirus 1.

We report the complete nucleotide sequence of the bovine herpesvirus 1 (BHV-1) immediate-early gene encoding BICP4, the homolog of the ICP4 protein of herpes simplex virus. Combined with previous mapping studies, the sequence analysis revealed that the transcript for BICP4 consisted of a noncoding leader RNA (exon 1; 0.35 kb) separated by an intron (0.46 kb) from the main body (exon 2; 4.1 kb). The open reading frame for BICP4 (1343 amino acid residues) started 27 nt after the splice site and extended across exon 2 for most of its length, BICP4 contained two domains of high homology (regions 2 and 4), which had been recognized earlier to be most conserved in the ICP4 homologs of alpha-herpesviruses and to be functionally important. These domains were flanked by three regions of lower but still discernible homology. Unique features of BICP4 were two large clusters of glutamic acid residues near the end of region 3, and the displacement of a polyserine tract to region 5, which in all other ICP4 homologs residues near the end of region 1. Transient expression assays showed that BICP4 repressed its own promoter and activated other herpes-virus genes. The 8.1-kb sequence summarized here completes analysis of the inverted repeats of the BHV-1 genome; it includes a segment (2.5 kb) upstream of the BICP4 gene apparently devoid of coding sequences but containing numerous scattered transcription signals.

Alphaherpesvirinae↗
PubMed1993

Methods to investigate the expression of lignin peroxidase genes by the white rot fungus Phanerochaete chrysosporium.

Two methods allowing the analysis of expression of specific lignin peroxidase (LPO) genes from white rot fungi are presented. In the first method, degenerate oligonucleotide primers derived from amino acid sequence motifs held in common among all members of the LPO gene family are used to prime the polymerase chain reaction (PCR) amplification of LPO-related nucleotide sequences from cDNA prepared by using RNA from ligninolytic cultures. The PCR products are cloned and analyzed by restriction cleavage and DNA sequencing. This method was applied to the analysis of transcripts from carbon-limited cultures of Phanerochaete chrysosporium BKM-F-1767, revealing two major classes of PCR products. One class showed DNA sequences with a high degree of similarity to the previously described CLG4 cDNA sequence (H. A. De Boer, Y. Zhang, C. Collins, and C. A. Reddy, Gene 60:93-102, 1987), whereas the other harbored DNA sequences with similarities to the L18 cDNA sequence previously described for P. chrysosporium OGC101 (T. G. Ritch, Jr., V. J. Nipper, L. Akileswaran, A. J. Smith, D. G. Pribnow, and M. H. Gold, Gene 107:119-126, 1991). The second method is based on nuclease protection assays involving isoenzyme-specific RNA probes. By using this method, the L18-related gene of P. chrysosporium BKM-F-1767 was found to be expressed under conditions of carbon and of nitrogen limitation, although the transcript levels were found to be higher in carbon-limited cultures. Furthermore, it was found that omission of veratryl alcohol addition to the culture did not affect the levels of the L18-related transcripts in carbon-limited cultures.

Amino Acid Sequence↗
PubMed1993

Immediate-early transcription over covalently joined genome ends of bovine herpesvirus 1: the circ gene.

Herpesvirus genomes are linear molecules in virions. Prior to replication in host cells, they form circular templates by unknown mechanisms. Examining lytic infection with bovine herpesvirus 1, we observed immediate-early transcription over joined genome ends, which suggested that circles are present at the initial stage of infection. Among the transcripts was a spliced immediate-early RNA (1.5 kb) sharing exon 1 with previously described major immediate-early transcripts from the right genome end and exon 2 with a late transcript located near the left genome end. Exon 2 encodes a putative circ-encoded protein with homology to the varicella-zoster virus open reading frame 2 and equine herpesvirus 1 open reading frame 3 products. The novel features reported here for bovine herpesvirus 1 may constitute a more general property of herpesviruses.

Amino Acid Sequence↗
PubMed1992

Immediate-early RNA 2.9 and early RNA 2.6 of bovine herpesvirus 1 are 3' coterminal and encode a putative zinc finger transactivator protein.

Bovine herpesvirus 1 (BHV-1) contains three major immediate-early (IE) genes involved in regulation of the productive cycle of replication. Two spliced IE RNAs, IER4.2 (4.2 kb) and IER2.9 (2.9 kb), are under the control of a single promoter; IER1.7 (1.7 kb) is transcribed from a different promoter in the opposite direction. Examining the kinetics of transcription, we found that the IER4.2/2.9 promoter was turned off at the end of the IE period. An alternative promoter became active, directing synthesis of an unspliced early RNA, ER2.6 (2.6 kb), which was colinear with the second exon of IER2.9 except for its 5' end in the intron about 10 bases upstream of the splice site. Sequence analysis revealed a single open reading frame common to IER2.9 and ER2.6 with a coding potential of 676 amino acids. The putative protein, named p135, contained a cysteine-rich zinc finger domain near the N terminus with homology to ICP0 of herpes simplex virus type 1, to protein 61 of varicella-zoster virus, to early protein 0 of pseudorabies virus, and to other viral and cellular proteins. The remaining parts of p135 exhibited only limited homology, mainly with pseudorabies virus protein 0, but the entire sequence was highly conserved between two strains of BHV-1 (K22 and Jura). The latency-related antisense transcript covered a large portion of ER2.6 excluding the zinc finger coding region. In transient expression assays, p135 activated a variety of promoters, including that for ER2.6, but repressed the IER1.7 promoter. Thus, p135 combines functional characteristics of ICP0, a strong transactivator, and of protein 61, a repressor. BHV-1 seems to have evolved a subtle mechanism to ensure the continued synthesis of p135 while turning off IER4.2, which encodes p180, the herpes simplex virus type 1 ICP4 homolog.

Amino Acid Sequence↗
PubMedDate not supplied

HSV-1 infected cell proteins influence tetracycline-regulated transgene expression.

BACKGROUND: This study investigates elements of herpes simplex virus type 1 (HSV-1) which influence transgene expression in tetracycline-regulated expression systems. METHODS: Different HSV-1 mutants were used to infect Vero cells that had been transfected with plasmids containing the luciferase gene under the control of tet-off or tet-on tetracycline-regulation systems. RESULTS: The baseline level of luciferase expression was elevated after infection with HSV-1 mutants lacking one or more immediate early genes encoding transactivating factors: ICP27, ICP4 and ICP0. With the tet-off system, not only was baseline expression elevated, but there was a complete loss of induction upon removal of tet when this regulatory system was brought into the cell by infection with helper virus-free amplicon vectors. Elevation of luciferase expression was also observed upon infection with the same HSV-1 mutants following transfection with a plasmid containing only a CMV minimal promoter driving luciferase (pUHC13-3). Only one HSV mutant (14Hdelta3), which bears a disruption in the transactivation domain of VP16 and is deleted for both ICP4 genes, did not increase baseline luciferase expression after transfection of pUHC13-3. The disregulating effects were dependent on virus dose and were not influenced by treatment with interferon (IFN)-alpha, which suppresses viral gene expression. Additional assays involving cotransfection of pUHC13-3 with a plasmid encoding of the HSV-1 transactivating factor ICP4 revealed that ICP4 was the most potent inducer of gene expression from the tetO/CMV minimal promoter. CONCLUSION: These results indicate that proteins encoded in the HSV-1 genome, especially the transactivating immediate early gene products (ICP4, ICP27 and ICP0) and the VP16 tegument protein can activate the tetO/ minimal CMV promoter and thereby interfere with the integrity of tetracycline-regulated transgene expression.

Animals↗