scholarly journals A novel general approach to eucaryotic mutagenesis functionally identifies conserved regions within the adenovirus 13S E1A polypeptide.

1986 ◽  
Vol 6 (5) ◽  
pp. 1487-1496 ◽  
Author(s):  
D Kimelman
Keyword(s):  
Shuttle Vector ◽  
Point Mutations ◽  
Mutant Gene ◽  
Adenovirus Type ◽  
Cellular Morphology ◽  
Morphological Alteration ◽  
Morphological Alterations ◽  
E1a Gene ◽  
Adenovirus Type 5

A new approach to the isolation of mutations in mammalian genes was developed which permits both the selection of infrequently occurring mutants that alter the cellular morphology of recipient cells and the rapid reisolation of the mutant gene. The adenovirus type 5 13S early region 1a (E1a) gene was mutagenized in vitro with sodium bisulfite and then efficiently transferred into cells with a retrovirus shuttle vector. Three classes of mutants of the 13S E1a gene product were isolated, each of which induced a distinct morphological alteration. The mutant E1a gene was reisolated from each cell line, and the precise nucleotide changes were determined. The E1a-induced morphological alterations were further examined by the construction of single and double point mutations within different regions of the polypeptides by utilizing the amino acid substitutions obtained from the original mutants. The results suggest that each of the three regions of highly conserved amino acids within the E1a 13S polypeptide has a distinct role in the alteration of cellular morphology and the activation of gene expression.

A novel general approach to eucaryotic mutagenesis functionally identifies conserved regions within the adenovirus 13S E1A polypeptide

1986 ◽  
Vol 6 (5) ◽  
pp. 1487-1496
Author(s):  
D Kimelman
Keyword(s):  
Shuttle Vector ◽  
Point Mutations ◽  
Mutant Gene ◽  
Adenovirus Type ◽  
Cellular Morphology ◽  
Morphological Alteration ◽  
Morphological Alterations ◽  
E1a Gene ◽  
Adenovirus Type 5

A new approach to the isolation of mutations in mammalian genes was developed which permits both the selection of infrequently occurring mutants that alter the cellular morphology of recipient cells and the rapid reisolation of the mutant gene. The adenovirus type 5 13S early region 1a (E1a) gene was mutagenized in vitro with sodium bisulfite and then efficiently transferred into cells with a retrovirus shuttle vector. Three classes of mutants of the 13S E1a gene product were isolated, each of which induced a distinct morphological alteration. The mutant E1a gene was reisolated from each cell line, and the precise nucleotide changes were determined. The E1a-induced morphological alterations were further examined by the construction of single and double point mutations within different regions of the polypeptides by utilizing the amino acid substitutions obtained from the original mutants. The results suggest that each of the three regions of highly conserved amino acids within the E1a 13S polypeptide has a distinct role in the alteration of cellular morphology and the activation of gene expression.

scholarly journals Adenovirus type 5 E1A gene products act as transformation suppressors of the neu oncogene.

1991 ◽  
Vol 11 (3) ◽  
pp. 1745-1750 ◽  
Author(s):  
D H Yu ◽  
K Scorsone ◽  
M C Hung
Keyword(s):  
Nude Mice ◽  
Adenovirus Type ◽  
Transformed Cells ◽  
Gene Products ◽  
Early Region ◽  
Transforming Activity ◽  
Neu Oncogene ◽  
E1a Gene ◽  
Adenovirus Type 5

The adenovirus type 5 early region 1A (E1A) gene was introduced into neu-transformed B104-1-1 cells. Cells that expressed E1A possessed reduced transforming activity in vitro and reduced tumorigenicity in nude mice. These results demonstrate that the E1A gene products can act negatively to suppress the transformed phenotype in neu-transformed cells.

Adenovirus type 5 E1A gene products act as transformation suppressors of the neu oncogene

1991 ◽  
Vol 11 (3) ◽  
pp. 1745-1750
Author(s):  
D H Yu ◽  
K Scorsone ◽  
M C Hung
Keyword(s):  
Nude Mice ◽  
Adenovirus Type ◽  
Transformed Cells ◽  
Gene Products ◽  
Early Region ◽  
Transforming Activity ◽  
Neu Oncogene ◽  
E1a Gene ◽  
Adenovirus Type 5

The adenovirus type 5 early region 1A (E1A) gene was introduced into neu-transformed B104-1-1 cells. Cells that expressed E1A possessed reduced transforming activity in vitro and reduced tumorigenicity in nude mice. These results demonstrate that the E1A gene products can act negatively to suppress the transformed phenotype in neu-transformed cells.

scholarly journals Functional Interaction of the Adenovirus IVa2 Protein with Adenovirus Type 5 Packaging Sequences

2005 ◽  
Vol 79 (5) ◽  
pp. 2831-2838 ◽  
Author(s):  
Philomena Ostapchuk ◽  
Jihong Yang ◽  
Ece Auffarth ◽  
Patrick Hearing
Keyword(s):  
Specific Interaction ◽  
Point Mutations ◽  
Adenovirus Type ◽  
Viral Proteins ◽  
Dna Packaging ◽  
Sequence Motif ◽  
Site Specific ◽  
Adenovirus Type 5

ABSTRACT Adenovirus type 5 (Ad5) DNA packaging is initiated in a polar fashion from the left end of the genome. The packaging process is dependent on the cis-acting packaging domain located between nucleotides 230 and 380. Seven AT-rich repeats that direct packaging have been identified within this domain. A1, A2, A5, and A6 are the most important repeats functionally and share a bipartite sequence motif. Several lines of evidence suggest that there is a limiting trans-acting factor(s) that plays a role in packaging. Both cellular and viral proteins that interact with adenovirus packaging elements in vitro have been identified. In this study, we characterized a group of recombinant viruses that carry site-specific point mutations within a minimal packaging domain. The mutants were analyzed for growth properties in vivo and for the ability to bind cellular and viral proteins in vitro. Our results are consistent with a requirement of the viral IVa2 protein for DNA packaging via a direct interaction with packaging sequences. Our results also indicate that higher-order IVa2-containing complexes that form on adjacent packaging repeats in vitro are the complexes required for the packaging activity of these sites in vivo. Chromatin immunoprecipitation was used to study proteins that bind directly to the packaging sequences. These results demonstrate site-specific interaction of the viral IVa2 and L1 52/55K proteins with the Ad5 packaging domain in vivo. These results confirm and extend those previously reported and provide a framework on which to model the adenovirus assembly process.

scholarly journals Concentration dependence of transcriptional transactivation in inducible E1A-containing human cells.

1988 ◽  
Vol 8 (11) ◽  
pp. 4799-4807 ◽  
Author(s):  
L J Brunet ◽  
A J Berk
Keyword(s):  
Adenovirus Type ◽  
In Vitro Transcription ◽  
Mrna Levels ◽  
Adenovirus E1a ◽  
Transcription System ◽  
Nuclear Extracts ◽  
Tumor Virus ◽  
E1a Gene ◽  
High Level

The adenovirus E1A proteins are essential for the normal temporal activation of transcription from every other adenoviral early promoter. High-level E1A expression in the absence of viral infection would facilitate biochemical studies of E1A-mediated transactivation. Toward this end, we introduced the adenovirus type 2 E1A gene under the control of the murine mammary tumor virus promoter into HeLa cells. Uninduced cells expressed little or no detectable E1A mRNA. Upon induction, mRNA levels accumulated to about 50% of the level observed in 293 cells. The level of E1A expression in these cells could be controlled by varying the concentration of the inducing glucocorticoid. Under these conditions of varying E1A concentrations, it was observed that activation of the E2, E3, and E4 promoters of H5dl312 initiated at the same E1A concentration and that transcription from each promoter increased as the E1A concentration increased. These results indicate that E1A-mediated transactivation is proportional to the concentration of E1A protein. E1A-dependent transcriptional stimulation of the E4 promoter was reproduced in an in vitro transcription system, demonstrating that expression of only the E1A proteins was sufficient to increase the transcriptional activity of nuclear extracts.

scholarly journals In Vitro and In Vivo Evaluation of Human Adenovirus Type 49 as a Vector for Therapeutic Applications

Viruses ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1483
Author(s):  
Emily A. Bates ◽  
John R. Counsell ◽  
Sophie Alizert ◽  
Alexander T. Baker ◽  
Natalie Suff ◽  
...  
Keyword(s):  
Viral Vector ◽  
Ex Vivo ◽  
Human Adenovirus ◽  
Adenovirus Type ◽  
Cell Types ◽  
In Vivo Evaluation ◽  
In Vivo Biodistribution ◽  
Adenovirus Type 5

The human adenovirus phylogenetic tree is split across seven species (A–G). Species D adenoviruses offer potential advantages for gene therapy applications, with low rates of pre-existing immunity detected across screened populations. However, many aspects of the basic virology of species D—such as their cellular tropism, receptor usage, and in vivo biodistribution profile—remain unknown. Here, we have characterized human adenovirus type 49 (HAdV-D49)—a relatively understudied species D member. We report that HAdV-D49 does not appear to use a single pathway to gain cell entry, but appears able to interact with various surface molecules for entry. As such, HAdV-D49 can transduce a broad range of cell types in vitro, with variable engagement of blood coagulation FX. Interestingly, when comparing in vivo biodistribution to adenovirus type 5, HAdV-D49 vectors show reduced liver targeting, whilst maintaining transduction of lung and spleen. Overall, this presents HAdV-D49 as a robust viral vector platform for ex vivo manipulation of human cells, and for in vivo applications where the therapeutic goal is to target the lung or gain access to immune cells in the spleen, whilst avoiding liver interactions, such as intravascular vaccine applications.

Concentration dependence of transcriptional transactivation in inducible E1A-containing human cells

1988 ◽  
Vol 8 (11) ◽  
pp. 4799-4807
Author(s):  
L J Brunet ◽  
A J Berk
Keyword(s):  
Adenovirus Type ◽  
In Vitro Transcription ◽  
Mrna Levels ◽  
Adenovirus E1a ◽  
Transcription System ◽  
Nuclear Extracts ◽  
Tumor Virus ◽  
E1a Gene ◽  
High Level

The adenovirus E1A proteins are essential for the normal temporal activation of transcription from every other adenoviral early promoter. High-level E1A expression in the absence of viral infection would facilitate biochemical studies of E1A-mediated transactivation. Toward this end, we introduced the adenovirus type 2 E1A gene under the control of the murine mammary tumor virus promoter into HeLa cells. Uninduced cells expressed little or no detectable E1A mRNA. Upon induction, mRNA levels accumulated to about 50% of the level observed in 293 cells. The level of E1A expression in these cells could be controlled by varying the concentration of the inducing glucocorticoid. Under these conditions of varying E1A concentrations, it was observed that activation of the E2, E3, and E4 promoters of H5dl312 initiated at the same E1A concentration and that transcription from each promoter increased as the E1A concentration increased. These results indicate that E1A-mediated transactivation is proportional to the concentration of E1A protein. E1A-dependent transcriptional stimulation of the E4 promoter was reproduced in an in vitro transcription system, demonstrating that expression of only the E1A proteins was sufficient to increase the transcriptional activity of nuclear extracts.

scholarly journals Tumorigenicity of fibroblast lines expressing the adenovirus E1a, cellular p53, or normal c-myc genes.

1986 ◽  
Vol 6 (1) ◽  
pp. 7-14 ◽  
Author(s):  
A Kelekar ◽  
M D Cole
Keyword(s):  
Simian Virus 40 ◽  
Adenovirus Type ◽  
Simian Virus ◽  
Primary Cells ◽  
Adenovirus E1a ◽  
Immortalized Cells ◽  
Transforming Activity ◽  
E1a Gene

Cellular and viral oncogenes have been linked to the transformation of established cell lines in vitro, to the induction of tumors in vivo, and to the partial transformation or immortalization of primary cells. Based on the ability to cooperate with mutated ras oncogenes in the transformation of primary cells, the adenovirus E1a and cellular p53 genes have been assigned an immortalizing activity. It is demonstrated in this paper that the adenovirus type 5 E1a gene and simian virus 40 promoter-linked p53 cDNA are able to transform previously immortalized cells to a tumorigenic phenotype without a significant change in cell morphology. It is also shown that, when linked to a constitutive promoter, the normal mouse and human c-myc genes have the same transforming activity. Cells transformed by each of these oncogenes have an increased capacity to grow in the absence of growth factors and a limited anchorage-independent growth capability.

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