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V Mautner

Publications and source records attributed to V Mautner.

At least 37 records · Page 2Linked to original sources
PubMed2000

A versatile system for receptor-mediated gene delivery permits increased entry of DNA into target cells, enhanced delivery to the nucleus and elevated rates of transgene expression.

We have developed a method for stabilisation of polyelectrolyte gene delivery vectors by crosslinking their surfaces with biodegradable multivalent copolymers based on N-(2-hydroxypropyl)methacrylamide (HPMA). The resulting nanoparticulate vectors resist attack by serum proteins and can be modified for cell-specific delivery by incorporation of targeting ligands onto the polymer coating. Here we show that vascular endothelial growth factor (VEGF), transferrin and basic fibroblast growth factor (bFGF) can each be linked to polyHPMA-coated poly(L-lysine)/DNA complexes. All ligand-targeted complexes demonstrated increased uptake into receptor-positive cells (measured using plasmids containing 32P-dCTP), that could be antagonised with excess free ligand. Targeted complexes also showed increased transfection, resistant to inhibition by serum, suggesting the possibility of effective application in vivo. Analysis using fluorescence microscopy confirmed enhanced uptake of ligand-targeted complexes (using Texas Red-labelled plasmid DNA), although VEGF- and transferrin-targeted complexes were restricted to cytoplasmic or perinuclear distributions. In contrast, bFGF-targeted complexes showed efficient delivery into the nucleus, with accumulation of more than 100000 plasmids per cell within distinct intranuclear compartments. This method permits versatile targeting of genes to selected cells and may also permit manipulation of intracellular trafficking. It should find several important applications in gene delivery systems both in vitro and in vivo.

Cell Nucleus↗
PubMed2000

Productive replication of human adenoviruses in mouse epidermal cells.

In contrast to most cells of mouse origin, cell lines derived from mouse epidermis are permissive for replication of human adenovirus type 5. The extent of epidermal cell differentiation correlated with the level of E1A expression and virus replication. Mouse epidermal cells may provide useful models for cancer therapy using replication-competent human adenoviruses.

3T3 Cells↗
PubMed1999

Properties of the adenovirus type 40 E1B promoter that contribute to its low transcriptional activity.

The adenovirus type 5 (Ad5) E1B promoter contains two elements essential for maximal activity, a TATA box and a GC box. The enteric adenovirus type 40 (Ad40) E1B promoter has a TATA box sequence identical to that of Ad5 and a GC box that fits the Sp1 binding site consensus. Nevertheless, Ad40 E1B RNA synthesis is severely impaired in HeLa cells, attributable in part at least to the weak transactivating activity of Ad40 E1A. However, the responsiveness of Ad40 early promoters to E1A transactivation has not been directly demonstrated. Using a transient expression assay with a chloramphenicol acetyl transferase (CAT) reporter gene, the Ad40 E1B promoter was very poorly transactivated by E1A of both Ad40 and Ad5 and showed only a limited response to the promiscuous varicella zoster virus transactivator p140. Construction of Ad5 recombinant viruses expressing the CAT gene under the control of the Ad5 or Ad40 E1B promoter allowed detection and measurement of expression from the Ad40 E1B promoter in a well-defined background and showed that overall activity is some 100-fold lower than for the Ad5 E1B promoter. Deletion analysis revealed that sequences upstream of the Sp1 binding site down-modulated Ad40 E1B promoter responsiveness, and two protein binding sites, identified by DNase footprinting and gel retardation assay, may be implicated in this effect. Gel shift analysis also showed that the Ad40 Sp1 binding site had a reduced affinity for Sp1 protein, relative to the Ad5 site, and that the context as well as the core sequence had an influence on Sp1 recognition.

Adenovirus E1B Proteins↗
PubMed1998

Virus directed enzyme prodrug therapy for ovarian and pancreatic cancer using retrovirally delivered E. coli nitroreductase and CB1954.

Expression of the E. coli enzyme nitroreductase (NTR) in tumour cells enables them to activate the prodrug CB1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide), leading to interstrand DNA crosslinking and cell death. Using transfected or retrovirally transduced SKOV3 ovarian carcinoma cell clones, we show a strong correlation between sensitivity to CB1954 and level of NTR enzyme activity. Importantly for clinical application in ovarian cancer, a cisplatin-resistant ovarian tumour cell line remains as susceptible to the NTR-dependent cytotoxicity of CB1954 as parental cells. In mixed populations of NTR-expressing and non-expressing cells, we observe a marked 'bystander killing' effect with this system. The use of NTR-encoding retroviruses from clonal producer cell lines at titres of 5 x 10(5) c.f.u./ml to transduce either established or low passage primary ovarian carcinoma lines only achieves an average 10-fold sensitisation of the cultures at gene transfer efficiencies of 15-25%. Concentration of the retrovirus to 3 x 10(7) c.f.u./ml elevates gene transfer to 80-90% in a single exposure to target cells, resulting in up to 500-fold sensitisation of the entire, unselected SKOV3 population to CB1954. In an initial investigation of NTR/CB1954 for the treatment of tumours in vivo, we observe regression of tumours expressing NTR following administration of CB1954, resulting in significantly increased median survival.

Animals↗
PubMed1997

Polyethylenimine (PEI) is a simple, inexpensive and effective reagent for condensing and linking plasmid DNA to adenovirus for gene delivery.

A simple and inexpensive method of condensing and linking plasmid DNA to carrier adenovirus particles is described. The synthetic polycation polyethylenimine is used to condense plasmid DNA into positively charged 100 nm complexes. These PEI-DNA complexes are then bound to adenovirus particles through charge interactions with negative domains on the viral hexon. The resulting transfection complexes deliver plasmid DNA to cells by the adenovirus infectious route without interference from virus gene expression because psoralen-inactivated virus is employed. The PEI-DNA-adenovirus complexes display DNA delivery comparable to more sophisticated DNA virus complexes employing streptavidin/biotin linkage, but require no special reagents and are much easier to prepare.

Adenoviridae↗
PubMed1997

Immunohistochemical detection of schwannomin and neurofibromin in vestibular schwannomas, ependymomas and meningiomas.

In addition to schwannomas, patients with neurofibromatosis type 2 (NF2) frequently develop meningiomas and occasionally, ependymomas. Using DNA and protein analyses, we have shown NF2 gene mutations and lack of the gene product schwannomin in 29 schwannomas, 10 meningiomas, and in 7 ependymomas. We have raised antibodies (ABs) to peptides from the C-terminal (5990-AB) and N-terminal (5991-AB) domains of schwannomin. The ABs specifically detected a 65 kDa protein in a Schwann cell line and recognized schwannomin in the cytoplasm of Schwann cells (SCH), perineurial cells, and vestibular ganglion neurons. None of the 29 schwannomas were stained by the 5990-AB. Only 4 schwannomas were stained by the 5991-AB, indicating that most truncated schwannomins were unstable or not expressed in schwannomas. Seven of 10 meningiomas, including 3 tumors from NF2 patients, were not stained by either 5990-AB or 5991-AB. Only 2 of 7 ependymomas lacked schwannomin. Complete lack of schwannomin in these tumors supports a tumor suppressor function for schwannomin in some meningiomas and ependymomas. All tumors showed staining with an antibody to a C-terminal peptide of neurofibromin, confirming that full-length neurofibromin is present in these vestibular schwannomas, meningiomas, and ependymomas. The presence of schwannomin in some meningiomas and in the majority of ependymomas indicates that additional genes are likely to play a role in tumorigenesis of these tumors.

Brain Neoplasms↗
PubMed1996

The complete DNA sequence and genomic organization of the avian adenovirus CELO.

The complete DNA sequence of the avian adenovirus chicken embryo lethal orphan (CELO) virus (FAV-1) is reported here. The genome was found to be 43,804 bp in length, approximately 8 kb longer than those of the human subgenus C adenoviruses (Ad2 and Ad5). This length is supported by pulsed-field gel electrophoresis analysis of genomes isolated from several related FAV-1 isolates (Indiana C and OTE). The genes for major viral structural proteins (Illa, penton base, hexon, pVI, and pVIII), as well as the 52,000-molecular-weight (52K) and 100K proteins and the early-region 2 genes and IVa2, are present in the expected locations in the genome. CELO virus encodes two fiber proteins and a different set of the DNA-packaging core proteins, which may be important in condensing the longer CELO virus genome. No pV or pIX genes are present. Most surprisingly, CELO virus possesses no identifiable E1, E3, and E4 regions. There is 5 kb at the left end of the CELO virus genome and 15 kb at the right end with no homology to Ad2. The sequences are rich in open reading frames, and it is likely that these encode functions that replace the missing El, E3, and E4 functions.

Adenoviruses, Human↗
PubMed1994

Cell type specific regulation of expression from the Ad40 E1b promoter in recombinant Ad5/Ad40 viruses.

The defective growth of the enteric adenovirus type 40 (Ad40) in HeLa cells can only be overcome by supplying an E1B 55K function in trans, and it has been demonstrated that expression of Ad40 E1B mRNA is poor in these cells (V. Mautner et al., 1990, Virology 171, 618-622). To study the control of expression from the Ad40 E1B region in greater detail, two Ad5/Ad40 recombinant viruses were constructed containing the Ad40 E1B region in place of the equivalent Ad5 region, under either the control of the Ad40 E1B promoter (sub40P) or that of Ad5 (sub5P). For both recombinants, E1B mRNAs similar to those seen in a wt Ad40 infection were detected, with late splicing (post DNA replication) occurring predominantly via the Ad40 14S splice acceptor. However, the level of expression from the substituted E1B region differs markedly between the two recombinants, and synthesis of E1B mRNA and proteins was impaired in sub40P-infected cells. In 293 and KB16 cells, expression from the Ad40 E1B promoter was reduced 10- to 20-fold compared with the Ad5 promoter. In HeLa cells, the reduction was 80-fold and mirrored the poor expression of E1B mRNA in Ad40-infected HeLa cells. Furthermore, in contrast to the 293 cells, early expression of E1B proteins could not be detected in sub40P- or wt Ad40-infected HeLa cells. The experiments demonstrate the low activity of the Ad40 E1B promoter and that this promoter is regulated in a cell type specific manner.

Adenovirus E1B Proteins↗
PubMed1994

Phylogenetic relationships among adenovirus serotypes.

Phylogenetic comparisons of adenovirus DNA sequences, including the recently completed genomic sequences of Ad40 and Ad12, have been performed in order to investigate the evolutionary relationships among the various serotypes. Phylogenetic trees were constructed from sequence data for the ITR, E1a, E1b, E2a, E3b, major late promoter, hexon, protease, and fiber regions of the genome using programs contained in the PHYLIP (Phylogeny Inference) package. In general the branching pattern of the human serotypes at each locus correlated well with the classification of the human serotypes into six subgenera (A-F). However, a close evolutionary relationship was inferred between Ad4 (the only member of subgenus E) and the subgenus B viruses Ad3, Ad7, and Ad35, and challenges the placement of Ad4 in a subgenus of its own. In addition, the human viruses of subgenera A (Ad12, Ad18, and Ad31) and F (Ad40 and Ad41), as well as the simian adenoviruses SAV16 (SA7) and SAV8 (SV30), all of which are associated with infections of the gastrointestinal tract, were found to cluster together. The results suggest that these viruses have followed a course of evolution distinct from those of the other subgenera which largely infect the respiratory tract. Analysis of genetic variability between the four complete genomic sequences (Ad2, Ad5, Ad12, and Ad40) identified three regions subject to more rapid change, corresponding to the hexon-, fiber- and E3a-coding regions. Genetic variability at the E3a locus is particularly striking and may relate to the pathogenicity of the various serotypes.

Adenoviridae↗
PubMed1993

The DNA sequence of adenovirus type 40.

The 34,214 bp DNA sequence of adenovirus type 40 strain Dugan was determined directly from random fragments of virion DNA cloned into a bacteriophage M13 cloning vector. The gene layout is similar to that of other human adenoviruses, and in addition contains two potential protein-coding regions that are conserved, but have not been recognized previously, in other adenovirus genomes. One is oriented rightward, contained within the intron in the protein-coding region for the L4 33K gene, and would encode a protein sharing N-terminal sequence with 33K. The other is a leftward oriented exon located between the E3 and L5 IV (fibre) regions which would specify the N terminus of a novel protein. The region encoding the C terminus of this protein is not apparent from sequence data.

Adenoviruses, Human↗
PubMed1993

Enteric adenovirus type 40: expression of E1B proteins in vitro and in vivo.

The genes encoding the enteric adenovirus type 40 E1B proteins designated 19K, 55K, and 15K (55K related) have been cloned into the pET3a expression vector and synthesized by in vitro transcription and translation and by in vivo expression after induction in bacteria. The 19K product expressed in bacteria is recognized by anti-peptide sera specific for the C-terminal region of the open reading frame and has the same M(r) as 19K protein immunoprecipitated from virus-infected cells. The 55K protein synthesized in bacteria is insoluble except under extreme denaturing conditions, but after in vitro transcription followed by translation, a polypeptide of the predicted size is obtained. The 15K protein, equivalent to the first 73 and last 29 of the 476-residue 55K protein with an internal deletion of 374 amino acids, is expressed to a high level in bacteria in a soluble form and interacts weakly but specifically with N- and C-terminal anti-peptide sera. The bacterially expressed 15K protein was used to raise antibodies in rabbits. This serum precipitates the 55K protein expressed by in vitro translation, but only the 15K product can be immunoprecipitated from virus-infected cells. The same antiserum, however, detects the 55K protein in infected cells by Western blotting, at a time broadly coinciding with the onset of DNA replication. This is the first identification of Ad40 55K protein in infected cells and confirms that the Ad40 22S mRNA can be utilized in vivo. The question of whether this protein is functional can now be addressed.

Adenovirus E1B Proteins↗
PubMed1991

Enteric adenovirus type 40: complementation of the E4 defect in Ad2 dl808.

The enteric adenovirus type 40 cannot be passaged in HeLa cells, but will grow productively in cells that express the E1B region of adenovirus types 2 or 5. Even in such permissive cells, the lytic cycle is prolonged, there is an abnormal pattern of E1B early gene expression and a failure to switch off host cell functions, suggesting that other gene functions might be impaired in Ad40. For Ad2, E4 ORF 6 and ORF 3 proteins are known to have an essential role in progressing from the early to the late phase of lytic infection and the shutoff of host functions requires an interaction between the E4 ORF 6 34K protein and the E1B 55K protein. To test whether E4 functions of Ad40 are impaired, complementation tests have been made between Ad40 and the E4 deletion mutant Ad2 dl808, which lacks all but ORF 1 of the E4 region. In HeLa and Vero cells, Ad40 complements dl808 to levels equivalent to an Ad2 wild-type infection, as demonstrated by measuring virion packaged DNA, virus titration, and viral protein synthesis. Surprisingly, Ad2 dl808 fails to reciprocally complement Ad40. The results show that Ad40 produces functional E4 ORF 6 and/or ORF 3 activity, and that their expression precedes DNA replication.

Adenovirus Early Proteins↗
PubMed1991

Enteric adenovirus type 40:E1B transcription map and identification of novel E1A-E1B cotranscripts in lytically infected cells.

Adenovirus 40 (Ad40) is defective for growth in tissue culture but is complemented when the Ad2/5 or Ad12 E1B 55K protein is supplied in trans. Ad40 E1B mRNA has not been detected in E1-transformed cells, or at early times in lytically infected cells. In cells constitutively expressing the E1B region of Ad2, Ad40 E1B mRNAs are detected at late times in infection, after the onset of DNA replication. We have determined the Ad40 E1B transcription map from RNA produced at late times in infected KB16 cells, using S1 nuclease, primer extension, PCR-cDNA analysis, and Northern blotting. E1B transcripts corresponding to Ad2 14 S, 22 S, and 9 S mRNAs were identified but no 13 S mRNA equivalent was detected, a pattern similar to that seen in the Ad12 transcription map. The coding potential for E1B 19K, 55K, and 15K proteins and for ppIX is retained in the Ad40 transcripts. In addition we find novel E1A-E1B cotranscript counterparts of the 14 S and 22 S mRNAs. These contain the first 40 codons of the E1A first exon linked to a site 4-5 nt downstream of the E1B cap site, retaining all the coding potential of the E1B mRNAs. No new open reading frames are created by the junction, and the E1A ORF terminates with one codon added after the junction. Each E1A-E1B cotranscript is present in abundance comparable to that of its authentic E1B counterpart. The E1A-E1B junction is unusual in that it does not conform to splice consensus sequences and thus may not be generated by a conventional splicing mechanism.

Adenovirus Early Proteins↗
PubMed1990

Enteric adenovirus type 40: expression of E1B mRNA and proteins in permissive and nonpermissive cells.

The enteric adenovirus type 40 (strain Dugan) grows well in tissue culture only when the E1B 55K protein of Ad5 or Ad12 is supplied in trans, either constitutively expressed in an established cell line or by coinfection with an appropriate helper virus (V. Mautner, N. Mackay, and V. Steinthorsdottir, 1989, Virology 171, 619-622). The synthesis of Ad40 E1B mRNAs and proteins has been examined under permissive and nonpermissive conditions: At late times postinfection in permissive cells, E1B-specific mRNA species of 22 and 13-14 S are made, as well as 15 and 9 S messages for the late IVa2 and ppIX proteins. None of these are detected before the onset of DNA replication and none of them accumulate in the presence of a cytosine arabinoside block to DNA replication. The failure to detect cytoplasmic mRNAs as early times cannot be attributed to a failure of mRNA transport from the nucleus as there is no accumulation of nuclear E1 RNA. In nonpermissive Hela cells only traces of E1B- and ppIX-specific mRNAs are detectable, at very late times postinfection. Antibodies raised to synthetic oligopeptides corresponding to the N- and C-terminal domains of the putative E1B 19K and 55K proteins show a high titer against the cognate peptide by ELISA, but only the E1B 19K C-terminus-specific sera have detected a unique polypeptide in Ad40-infected cells, at late times postinfection. There is no shut-off of host protein synthesis in permissive cells, despite the expression of Ad2 55K protein.

Adenovirus Early Proteins↗
PubMed1989

Complementation of enteric adenovirus type 40 for lytic growth in tissue culture by E1B 55K function of adenovirus types 5 and 12.

The enteric adenovirus type 40 strain Dugan (Ad40) cannot be passaged in HeLa cells, but will grow in 293 cells, which express Ad5 E1 functions. To determine the reason for this limited host range, KB cell lines expressing Ad2 E1A, E1B, or E1A + E1B (L. E. Babiss, C. S. H. Young, P. B. Fisher, and H. S. Ginsberg, 1983, J. Virol. 46, 454-465) have been tested for their ability to support Ad40 replication. Only cell lines which supply E1B functions, but not those expressing E1A alone, are permissive for Ad40, suggesting that Ad40 may require some function supplied by E1B or induced in E1B-containing cells. In coinfection assays Ad40 complements Ad5 dl312 (delta E1A) but not Ad5 dl313 (delta E1B) and is itself complemented by dl312 but not by dl313. Mutants of Ad2 and Ad12 with lesions in E1B 55K or 19K protein have been used to further delineate the requirements for Ad40 growth in HeLa cells. For mutants lacking 55K function there is minimal complementation in either direction, whereas those lacking only the 19K product are able to complement Ad40.

Adenovirus Early Proteins↗