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 Cellular Components Interact with Adenovirus Type 5 Minimal DNA Packaging Domains

1998 ◽  
Vol 72 (8) ◽  
pp. 6339-6347 ◽  
Author(s):  
Susanne I. Schmid ◽  
Patrick Hearing
Keyword(s):  
Transcription Factor ◽  
Viral Genome ◽  
Adenovirus Type ◽  
Functional Independence ◽  
Binding Activity ◽  
Dna Packaging ◽  
Viral Origin ◽  
Adenovirus Type 5

ABSTRACT Adenovirus type 5 DNA packaging is initiated from the left end of the viral genome and depends on the presence of acis-acting packaging domain located between nucleotides 194 and 380. Multiple redundant packaging elements (termed A repeats I through VII [AI through AVII]) are contained within this domain and display differential abilities to support DNA packaging in vivo. The functionally most important repeats, AI, AII, AV, and AVI, follow a bipartite consensus motif exhibiting AT-rich and CG-rich core sequences. Results from previous mutational analyses defined a fragment containing AV, AVI, and AVII as a minimal packaging domain in vivo, which supports a functional independence of the respectivecis-acting sequences. Here we describe multimeric versions of individual packaging elements as minimal packaging domains that can confer viability and packaging activity to viruses carrying gross truncations within their left end. These mutant viruses directly rate the functional role that different packaging elements play relative to each other. The A repeats are likely to be binding sites for limiting,trans-acting packaging factors of cellular and/or viral origin. We report here the characterization of two cellular binding activities interacting with all of the minimal packaging domains in vitro, an unknown binding activity termed P-complex, and the transcription factor chicken ovalbumin upstream promoter transcription factor. The binding of both activities is dependent on the integrity of the AT-rich, but not the CG-rich, consensus half site. In the case of P-complex, binding affinity for different minimal packaging domains in vitro correlates well with their abilities to support DNA packaging in vivo. Interestingly, P-complex interacts not only with packaging elements but also with the left terminus of the viral genome, the core origin of replication. Our data implicate cellular factors as components of the viral packaging machinery. The dual binding specificity of P-complex for packaging and replication sequences may further suggest a direct involvement of left-end replication sequences in viral DNA encapsidation.

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.

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.

scholarly journals Binding of CCAAT Displacement Protein CDP to Adenovirus Packaging Sequences

2003 ◽  
Vol 77 (11) ◽  
pp. 6255-6264 ◽  
Author(s):  
Ece Erturk ◽  
Philomena Ostapchuk ◽  
Susanne I. Wells ◽  
Jihong Yang ◽  
Keqin Gregg ◽  
...  
Keyword(s):  
Protein S ◽  
Cellular Protein ◽  
Dna Packaging ◽  
Sequence Motif ◽  
Uncharacterized Protein ◽  
Virus Particles ◽  
Ccaat Displacement Protein ◽  
Viral Dna Packaging

ABSTRACT Adenovirus (Ad) type 5 DNA packaging is initiated in a polar fashion from the left end of the genome. The packaging process is dependent upon the cis-acting packaging domain located between nucleotides 194 and 380. Seven A/T-rich repeats have been identified within this domain that direct packaging. 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. Two cellular activities that bind to minimal packaging domains in vitro have been previously identified. These binding activities are P complex, an uncharacterized protein(s), and chicken ovalbumin upstream promoter transcription factor (COUP-TF). In this work, we report that a third cellular protein, octamer-1 protein (Oct-1), binds to minimal packaging domains. In vitro binding analyses and in vivo packaging assays were used to examine the relevance of these DNA binding activities to Ad DNA packaging. The results of these experiments reveal that COUP-TF and Oct-1 binding does not play a functional role in Ad packaging, whereas P-complex binding directly correlates with packaging function. We demonstrate that P complex contains the cellular protein CCAAT displacement protein (CDP) and that full-length CDP is found in purified virus particles. In addition to cellular factors, previous evidence indicates that viral factors play a role in the initiation of viral DNA packaging. We propose that CDP, in conjunction with one or more viral proteins, binds to the packaging sequences of Ad to initiate the encapsidation process.

scholarly journals Reducing the Native Tropism of Adenovirus Vectors Requires Removal of both CAR and Integrin Interactions

2001 ◽  
Vol 75 (23) ◽  
pp. 11284-11291 ◽  
Author(s):  
David A. Einfeld ◽  
Rosanna Schroeder ◽  
Peter W. Roelvink ◽  
Alena Lizonova ◽  
C. Richter King ◽  
...  
Keyword(s):  
Adenovirus Type ◽  
Wild Type ◽  
Base Modification ◽  
Model Following ◽  
Vector Distribution ◽  
High Level ◽  
Adenovirus Type 5

ABSTRACT The development of tissue-selective virus-based vectors requires a better understanding of the role of receptors in gene transfer in vivo, both to rid the vectors of their native tropism and to introduce new specificity. CAR and αv integrins have been identified as the primary cell surface components that interact with adenovirus type 5 (Ad5)-based vectors during in vitro transduction. We have constructed a set of four vectors, which individually retain the wild-type cell interactions, lack CAR binding, lack αv integrin binding, or lack both CAR and αv integrin binding. These vectors have been used to examine the roles of CAR and αv integrin in determining the tropism of Ad vectors in a mouse model following intrajugular or intramuscular injection. CAR was found to play a significant role in liver transduction. The absence of CAR binding alone, however, had little effect on the low level of expression from Ad in other tissues. Binding of αv integrins appeared to have more influence than did binding of CAR in promoting the expression in these tissues and was also found to be important in liver transduction by Ad vectors. An effect of the penton base modification was a reduction in the number of vector genomes that could be detected in several tissues. In the liver, where CAR binding is important, combining defects in CAR and αv integrin binding was essential to effectively reduce the high level of expression from Ad vectors. While there may be differences in Ad vector tropism among species, our results indicate that both CAR and αv integrins can impact vector distribution in vivo. Disruption of both CAR and αv integrin interactions may be critical for effectively reducing native tropism and enhancing the efficacy of specific targeting ligands in redirecting Ad vectors to target tissues.

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

2021 ◽  
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 preexisting 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.

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 Analysis of DNA binding by the adenovirus type 5 E1A oncoprotein

2002 ◽  
Vol 83 (3) ◽  
pp. 517-524 ◽  
Author(s):  
Nikita Avvakumov ◽  
Majdina Sahbegovic ◽  
Zhiying Zhang ◽  
Michael Shuen ◽  
Joe S. Mymryk
Keyword(s):  
Amino Acids ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Adenovirus Type ◽  
Dna Binding Domain ◽  
Activation Domain ◽  
Transcriptional Activation Domain ◽  
Adenovirus Type 5

Adenovirus type 5 E1A proteins interact with cellular regulators of transcription to reprogram gene expression in the infected or transformed cell. Although E1A also interacts with DNA directly in vitro, it is not clear how this relates to its function in vivo. The N-terminal conserved regions 1, 2 and 3 and the C-terminal portions of E1A were prepared as purified recombinant proteins and analyses showed that only the C-terminal region bound DNA in vitro. Deletion of E1A amino acids 201–220 inhibited binding and a minimal fragment encompassing amino acids 201–218 of E1A was sufficient for binding single- and double-stranded DNA. This portion of E1A also bound the cation-exchange resins cellulose phosphate and carboxymethyl Sepharose. As this region contains six basic amino acids, in vitro binding of E1A to DNA probably results from an ionic interaction with the phosphodiester backbone of DNA. Studies in Saccharomyces cerevisiae have shown that expression of a strong transcriptional activation domain fused to a DNA-binding domain can inhibit growth. Although fusion of the C-terminal region of E1A to a strong transcriptional activation domain inhibited growth when expressed in yeast, this was not mediated by the DNA-binding domain identified in vitro. These data suggest that E1A does not bind DNA in vivo.

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