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P Hearing

Publications and source records attributed to P Hearing.

At least 19 recordsLinked to original sources
PubMed2003

Regulation of adenovirus packaging.

The application of fundamental concepts about the packaging of the adenovirus genome has contributed significantly to the development of therapeutic viral vectors for gene therapy. The packaging of adenovirus DNA into virus particles requires a cis-acting domain at the left end of the genome. This region contains a series of repeated sequences, termed A repeats due to their AT-rich character, that direct the packaging process. A repeats are believed to represent the binding sites for viral and cellular factors that mediate viral DNA packaging. This review will focus on fundamental aspects of adenovirus DNA packaging as well as how this information has been used and may be used to augment the selectivity of viral DNA packaging in applications pertaining to gene therapy vectors.

Adenoviridae↗
PubMed2001

E2F transcriptional activation requires TRRAP and GCN5 cofactors.

The E2F family of transcription factors regulates the temporal transcription of genes involved in cell cycle progression and DNA synthesis. E2F transactivation is antagonized by retinoblastoma protein (pRb), which recruits chromatin-remodeling proteins such as histone deacetylases and SWI.SNF complexes to the promoter to repress transcription. We hypothesized that E2F proteins must reverse the pRb-imposed chromatin structure to stimulate transcription. If this is true, E2F proteins should recruit proteins capable of histone acetylation. Here we map the E2F-4 transactivation domain and show that E2F-1 and E2F-4 transactivation domains bind the acetyltransferase GCN5 and cofactor TRRAP in vivo. TRRAP and GCN5 co-expression stimulated E2F-mediated transactivation, and c-Myc repressed E2F transactivation dependent on an intact TRRAP/GCN5 binding motif. The transactivation domain of E2F-4 recruited proteins with significant histone acetyltransferase activity in vivo, and this activity required catalytically active GCN5. E2F-4 proteins with subtle mutations in the transactivation domain exhibited a positive correlation among transcriptional activation and GCN5 and TRRAP binding capacity and associated acetyltransferase activity. We conclude that E2F stimulates transcription by recruiting acetyltransferase activity and the essential cofactors GCN5 and TRRAP. These results provide a mechanism for E2F transcription factors to overcome pRb-mediated dominant repression of transcription.

Acetyltransferases↗
PubMed2001

The p107 tumor suppressor induces stable E2F DNA binding to repress target promoters.

E2F transcription factors are key players in the regulation of proliferation, apoptosis, and differentiation in mammalian cells. E2Fs are negatively regulated by members of the retinoblastoma protein family, Rb, p107 and p130. During adenovirus infection, viral proteins are expressed that displace Rb family members from E2Fs and recruit E2F complexes to viral and cellular promoter regions. This recruitment of E2F involves the induction of stable E2F binding to inverted E2F binding sites in the Ad E2a and cellular E2F-1 promoters and induces both viral and cellular gene expression. The cellular p107 tumor suppressor also displays such regulation of E2F DNA binding activity. p107 induces stable E2F-4/DP binding to inverted E2F binding sites in the Ad E2a and cellular E2F-1 promoters. The induction of E2F DNA binding by p107 minimally requires the sequences in p107 that mediate E2F interaction. The related tumor suppressor, p130, also effects this function. p107 levels increase substantially as cells progress through S phase. p107 induction of E2F DNA binding was observed primarily in S phase cells coincident with the increase in p107 protein levels. The results of promoter activity assays directly correlate the induction of E2F DNA binding by p107 with effective transcriptional repression. These results support a model in which p107 and p130 induce the stable binding of E2F complexes to promoters that drive expression of critical regulatory proteins such as E2F-1. Since p107 and p130 bind histone deacetylase complexes (HDACs) which repress promoter activity, p107-E2F and p130-E2F would stably recruit repressor complexes to effect efficient promoter repression.

Binding Sites↗
PubMed2001

Pseudopackaging of adenovirus type 5 genomes into capsids containing the hexon proteins of adenovirus serotypes B, D, or E.

Adenoviruses (Ad) show promise as a vector system for gene delivery in vivo. However, a major challenge in the development of Ad vectors is the circumvention of the host immune responses to Ad infection, including both the host cytotoxic T-cell response and the humoral response resulting in neutralizing antibodies. One method to circumvent the effect of neutralizing antibodies against an Ad vector is to use different Ad serotypes to deliver the transgene of interest. This approach has been demonstrated with Ad genomes of highly related members of subgroup C. However, it is not known whether an Ad5-based vector DNA molecule can be packaged into capsids of evolutionarily more divergent adenoviruses. The aim of these studies was to determine if capsids containing hexon proteins from other Ad subgroups could package the Ad5 genome. A genetic approach utilizing an Ad5 temperature-sensitive (ts) mutant with a mutation in the hexon protein was used. When grown at the nonpermissive temperature, Ad5 ts147 replicates normally, providing a source of Ad5 DNA for virus assembly, but does not produce virus particles due to the hexon protein mutation. Coinfection of Ad5 ts147 with a wild-type virus of other Ad serotypes (Ad3, Ad4, or Ad9), which supply functional hexon proteins, resulted in the pseudopackaging of the Ad5 DNA genome. Furthermore, the pseudopackaged Ad5 DNA virions obtained in the coinfections were infectious. Therefore, switching hexons did not impair the infectivity or uncoating process of the pseudopackaged virion. Since hexon protein is a major antigenic determinant of the Ad capsid, this approach may prove useful to reduce the antigenicity of therapeutic Ad vectors and allow repeated vector administration.

Adenoviridae↗
PubMed2000

The E4-6/7 protein functionally compensates for the loss of E1A expression in adenovirus infection.

The E1A gene products are required and sufficient for activation of adenovirus gene expression in cultured cells. The E4-6/7 gene product induces the binding of the cellular transcription factor E2F to the viral E2a promoter region. The induction of E2F binding to the E2a promoter in vitro is directly correlated with transcriptional activation of the E2a promoter in vivo. The E2 region encodes the viral replication proteins, yet adenoviruses lacking E4-6/7 function demonstrate no defective phenotype in infected cells. Here we show that the E4-6/7 protein can functionally compensate for E1A expression in virus infection. In the absence of the E1A gene products, expression of the E4-6/7 protein is sufficient to displace retinoblastoma protein family members from E2Fs, activate expression of early region 2 via induction of E2F DNA binding to the E2a promoter region, and significantly enhance replication of an E1A-defective adenovirus. These results have implications in the regulation of viral gene expression and for the development of recombinant adenovirus vectors.

Adenovirus E1A Proteins↗
PubMed2000

Adeno-associated virus (AAV) Rep protein enhances the generation of a recombinant mini-adenovirus (Ad) utilizing an Ad/AAV hybrid virus.

Mini-adenoviruses (mAd) deleted of all viral coding regions represent an emerging approach for transgene expression. We have exploited the unique features of the adeno-associated virus (AAV) terminal repeats within the context of an adenovirus-adeno-associated hybrid virus (Ad/AAV) as a strategy for rapid and efficient generation of mAd. Excision and generation of mAd from the parental Ad/AAV hybrid vector was achieved in 293 cells through recombination but without selection for mAd production. Analysis of mAd isolated from 293 cells indicated that mAd DNA exists as monomer and dimer forms within the recombinant viral capsid. Formation of recombinant mAd was significantly increased using an AAV Rep78- or Rep68-expressing cell line through Rep-mediated excision utilizing the AAV terminal repeat sequences present in the Ad/AAV hybrid virus genome. The mAd viruses were infectious and able to transfer functional gene to A549 and HeLa cells. This approach is rapid and efficient, thereby providing a simplified methodology for generating mAd with functional transducing capabilities.

Adenoviridae↗
PubMed2000

Induction of the cellular E2F-1 promoter by the adenovirus E4-6/7 protein.

The adenovirus type 5 (Ad5) E4-6/7 protein interacts directly with different members of the E2F family and mediates the cooperative and stable binding of E2F to a unique pair of binding sites in the Ad5 E2a promoter region. This induction of E2F DNA binding activity strongly correlates with increased E2a transcription when analyzed using virus infection and transient expression assays. Here we show that while different adenovirus isolates express an E4-6/7 protein that is capable of induction of E2F dimerization and stable DNA binding to the Ad5 E2a promoter region, not all of these viruses carry the inverted E2F binding site targets in their E2a promoter regions. The Ad12 and Ad40 E2a promoter regions bind E2F via a single binding site. However, these promoters bind adenovirus-induced (dimerized) E2F very weakly. The Ad3 E2a promoter region binds E2F very poorly, even via a single binding site. A possible explanation of these results is that the Ad E4-6/7 protein evolved to induce cellular gene expression. Consistent with this notion, we show that infection with different adenovirus isolates induces the binding of E2F to an inverted configuration of binding sites present in the cellular E2F-1 promoter. Transient expression of the E4-6/7 protein alone in uninfected cells is sufficient to induce transactivation of the E2F-1 promoter linked to chloramphenicol acetyltransferase or green fluorescent protein reporter genes. Further, expression of the E4-6/7 protein in the context of adenovirus infection induces E2F-1 protein accumulation. Thus, the induction of E2F binding to the E2F-1 promoter by the E4-6/7 protein observed in vitro correlates with transactivation of E2F-1 promoter activity in vivo. These results suggest that adenovirus has evolved two distinct mechanisms to induce the expression of the E2F-1 gene. The E1A proteins displace repressors of E2F activity (the Rb family members) and thus relieve E2F-1 promoter repression; the E4-6/7 protein complements this function by stably recruiting active E2F to the E2F-1 promoter to transactivate expression.

Adenoviridae↗
PubMed1999

Human factor VIII can be packaged and functionally expressed in an adeno-associated virus background: applicability to haemophilia A gene therapy.

Adeno-associated virus (AAV) is a single-stranded DNA parvovirus displaying several attractive features applicable to haemophilia A gene therapy, including nonpathogenicity and potential for long-term transgene expression from either integrated or episomal forms. We have generated and characterized two B-domain-deleted (BDD) fVIII mutants, deleted in residues Phe756 to Ile1679 (fVIIIdelta756-1679) or Thr761 to Asn1639 (fVIIIdelta761-1639). [35S]metabolic labelling experiments and immunoprecipitation demonstrated intact BDD-fVIII of the predicted size in both lysates and supernatants (Mr approximately 155 kD for fVIIIdelta756-1679 and Mr approximately 160 kD for fVIIIdelta761-1639) after transient transfection into COS-1 cells. Functional fVIII quantification appeared maximal using fVIIIdelta761-1639, as evaluated by Coatest and clotting assay (98+/-20mU/ml/1x10(6) cells and 118+/-29 mU/ml/1x10(6) respectively, collection period 48 h). To bypass potential size limitations of rAAV/fVIII vectors, we expressed fVIIIdelta761-1639 using a minimal human 243 bp cellular small nuclear RNA (pHU1-1) promoter, and demonstrated VIII activity approximately 30% of that seen using CMV promoter. This BDD-fVIII (rAAV(pHU1-1) fVIIIdelta761-1639) can be efficiently encapsidated into rAAV (107% of wild type), as demonstrated by replication centre and DNAase sensitivity assays. A concentrated recombinant viral stock resulted in readily detectable factor VIII expression in COS-1 cells using a maximally-achievable MOI approximately 35 (Coatest 15 mU/ml; clotting assay 25+/-20 mU/ml/1x10(6) cells). These data provide the first evidence that rAAV is an adaptable virus for fVIII delivery, and given the recent progress using this virus for factor IX delivery in vivo, provide a new approach towards definitive treatment of haemophilia A.

Cells, Cultured↗
PubMed1998

Cellular components interact with adenovirus type 5 minimal DNA packaging domains.

Adenovirus type 5 DNA packaging is initiated from the left end of the viral genome and depends on the presence of a cis-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 respective cis-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.

Adenoviruses, Human↗
PubMed1997

Bipartite structure and functional independence of adenovirus type 5 packaging elements.

Selectivity and polarity of adenovirus type 5 DNA packaging are believed to be directed by an interaction of putative packaging factors with the cis-acting adenovirus packaging domain located within the genomic left end (nucleotides 194 to 380). In previous studies, this packaging domain was mutationally dissected into at least seven functional elements called A repeats. These elements, albeit redundant in function, exhibit differences in the ability to support viral packaging, with elements I, II, V, and VI as the most critical repeats. Viral packaging was shown to be sensitive to spatial changes between individual A repeats. To study the importance of spatial constraints in more detail, we performed site-directed mutagenesis of the 21-bp linker regions separating A repeats I and II, as well as A repeats V and VI. The results of our mutational analysis reveal previously unrecognized sequences that are critical for DNA encapsidation in vivo. On the basis of these results, we present a more complex consensus motif for the adenovirus packaging elements which is bipartite in structure. DNA encapsidation is compromised by changes in spacing between the two conserved parts of the consensus motif, rather than between different A repeats. Genetic evidence implicating packaging elements as independent units in viral DNA packaging is derived from the selection of revertants from a packaging-deficient adenovirus: multimerization of packaging repeats is sufficient for the evolution of packaging-competent viruses. Finally, we identify minimally sized segments of the adenovirus packaging domain that can confer viability and packaging activity to viruses carrying gross truncations within their left-end sequences. Coinfection experiments using the revertant as well as the minimal-packaging-domain mutant viruses strengthen existing arguments for the involvement of limiting, trans-acting components in viral DNA packaging.

Adenoviridae↗
PubMed1995

Interaction of EF-C/RFX-1 with the inverted repeat of viral enhancer regions is required for transactivation.

The hepatitis B virus (HBV) and polyomavirus (Py) enhancer regions contain multiple cis-acting elements that contribute to enhancer activity. The EF-C binding site was previously shown to be an important functional component of each enhancer region. EF-C is a ubiquitous binding activity that interacts with an inverted repeat sequence in the HBV and Py enhancer regions. Although the EF-C binding site is required for optimal enhancer function, the EF-C site does not possess intrinsic enhancer activity when assayed in the absence of flanking elements. With both the HBV and Py enhancer regions, EF-C stimulates the activity of adjacent enhancer elements in a synergistic manner. EF-C corresponds to RFX-1, a protein that binds to a conserved and functionally important site in major histocompatibility complex (MHC) class II antigen promoter regions. Interestingly, the RFX-1 binding site in MHC class II promoters only contains an EF-C half-site, maintaining one arm of the inverted repeat in an EF-C binding site. We have investigated the binding of purified EF-C and RFX-1 to sites in the Py and HBV enhancer regions that carry mutations that either disrupt one arm of the EF-C inverted repeat, or alter the spacing between the repeats. Our results show that the interaction of EF-C and RFX-1 with an intact inverted repeat is required for functional activity of these viral enhancer regions. Chemical footprinting and modification interference assays show that the interaction of EF-C and RFX-1 with the DRA MHC class II promoter truly represents half-site interaction, and that this binding is unstable. In contrast, the binding of EF-C and RFX-1 to the viral inverted repeats is stable. These results suggest that an additional activity may be required to stabilize EF-C/RFX-1 interaction with the MHC class II promoter, and that viral enhancer regions have evolved high affinity binding sites to sequester dimeric EF-C/RFX-1.

3T3 Cells↗
PubMed1994

A dramatic shift in the transmembrane topology of a viral envelope glycoprotein accompanies hepatitis B viral morphogenesis.

The envelope of hepatitis B virus contains three related glycoproteins (termed L, M and S) produced by alternative translation initiation in a single coding region. The smallest of these, the S protein, is a 24 kDa glycoprotein with multiple transmembrane domains. The M and L proteins contain the entire S domain at their C-termini, but harbor at their N-terminal additional (preS) domains of 55 or 174 amino acids, respectively. Most of these preS residues are displayed on the surface of mature virions and hence would be expected to be translocated into the endoplasmic reticulum (ER) lumen during biosynthesis. Using a coupled, in vitro translation/translocation system we now demonstrate that, contrary to expectation, virtually all preS residues of the L protein are cytoplasmically disposed in the initial translocation product. This includes some preS sequences which in the M protein are indeed translocated into the ER lumen. Since preS sequences are found on the external surface of the virion envelope, our results indicate that during or following budding a dramatic reorganization of either the envelope proteins or the lipid bilayer (or both components) must occur to allow surface display of these sequences. These findings imply that some membrane budding events can have remarkable and previously unsuspected topological consequences.

Base Sequence↗
PubMed1994

Mutually exclusive interaction of the adenovirus E4-6/7 protein and the retinoblastoma gene product with internal domains of E2F-1 and DP-1.

The binding of E2F to the adenovirus (Ad) E2a promoter is stimulated by the Ad E4-6/7 protein. E2F DNA binding activity is composed of a heterodimer of related but distinct proteins of the E2F-1 and DP-1 families. The E4-6/7 protein induces the cooperative and stable binding of E2F to an inverted repeat binding site in the E2a promoter apparently by providing a dimerization interface to two adjacent E2F heterodimers. The product of the retinoblastoma gene product (Rb) represses the transcriptional activity of E2F by direct protein-protein interaction. In this report, we have examined the regions of E2F-1 and DP-1 that are required for the induction of cooperative E2F binding to the E2a promoter by the E4-6/7 protein. Our results demonstrate that an internal segment of E2F-1, that is conserved among members of the E2F family, is required for functional interaction with the E4-6/7 product. Consistent with this observation, other members of the E2F family (E2F-2 and E2F-3) productively interact with E4-6/7. DP-1 also is necessary for stable interaction with E4-6/7 and an internal segment of DP-1 is required that is positioned in a location similar to that of the conserved E2F-1 domain. Interestingly, the binding of E4-6/7 and the binding of Rb to E2F are mutually exclusive, and our results show that the same internal segments of E2F-1 and DP-1 that are required for E4-6/7 binding are also required for stable interaction with Rb. These results suggest that the Ad E4-6/7 protein mimics Rb in part for the protein interaction requirements for E2F binding, although with different functional consequences. While Rb binding represses E2F activity, the E4-6/7 protein stimulates transactivation of the Ad E2a promoter.

Adenoviridae↗
PubMed1994

The adenovirus E4-6/7 protein transactivates the E2 promoter by inducing dimerization of a heteromeric E2F complex.

Binding of the mammalian transcription factor E2F to the adenovirus E2a early promoter is modulated through interaction with the viral E4-6/7 protein. E4-6/7 induces the cooperative and stable binding of E2F in vitro to two correctly spaced and inverted E2F binding sites in the E2a promoter (E2F induction) by physical interaction in the protein-DNA complex. The E2a promoter is transactivated in vivo by the E4-6/7 product. The C-terminal 70 amino acids of E4-6/7 are necessary and sufficient for induction of E2F binding and for transactivation. To assess the mechanism(s) of E2a transactivation and the induction of cooperative E2F binding by the E4-6/7 protein, we have analyzed a series of point mutants in the functional C-terminal domain of E4-6/7. Two distinct segments of E4-6/7 are required for interaction with E2F. Additionally, and E4-6/7 mutant with a phenylalanine-to-proline substitution at amino acid 125 (F-125-P) efficiently interacts with E2F but does not induce E2F binding to the E2a promoter and is defective for transactivation. Induction of E2F stable complex formation at the E2a promoter by the F-125-P mutant protein is restored by divalent E4-6/7-specific monoclonal antibodies, but not a monovalent Fab fragment, or by appending a heterologous dimerization domain to the N terminus of the mutant protein. These and other data support the involvement of E4-6/7 dimerization in the induction of cooperative and stable E2F binding and transactivation of the E2a promoter. We present evidence that at least two cellular components are involved in E2F DNA binding activity and that both are required for E2F induction by the E4-6/7 product. The recently cloned E2F-related activities E2F-1 and DP-1 individually bind to an E2F binding site weakly, but when combined generate an activity that is indistinguishable from endogenous cellular E2F. Recombinant E2F-1, DP-1, and E4-6/7 are sufficient to form the induced E2F complex at the E2a promoter.

Adenovirus E2 Proteins↗
PubMed1993

The NFIII/OCT-1 binding site stimulates adenovirus DNA replication in vivo and is functionally redundant with adjacent sequences.

The inverted terminal repeat (ITR) of adenovirus type 5 (Ad5) is 103 bp in length and contains the origin of DNA replication. Cellular transcription factors NFI/CTF and NFIII/OCT-1 bind to sites within the ITR and participate in the initiation of viral DNA replication in vitro. The ITR also contains multiple copies of two conserved sequence motifs that bind the cellular transcription factors SP1 and ATF. We have analyzed a series of viruses that carry deletions at the left terminus of Ad5. A virus carrying a deletion of the NFIII/OCT-1, SP1, and ATF sites within the ITR (mutant dl309-44/107) was wild type for virus growth. However, the deletion of these elements in addition to sequences immediately flanking the ITR (mutant dl309-44/195) resulted in a virus that grew poorly. The analysis of growth parameters of these and other mutants demonstrate that the NFIII/OCT-1 and adjacent SP1 sites augment the accumulation of viral DNA following infection. The function of these elements was most evident in coinfections with a wild-type virus, suggesting that these sites enhance the ability of a limiting trans-acting factor(s), that stimulates viral DNA replication, to interact with the ITR. The results of these analyses indicate functional redundancy between different transcription elements at the left terminus of the Ad5 genome and demonstrate that the NFIII/OCT-1 site and adjacent SP1 site, previously thought to be nonessential for adenovirus growth, play a role in viral DNA replication in vivo.

Adenoviruses, Human↗
PubMed1993

Functional interaction of nuclear factors EF-C, HNF-4, and RXR alpha with hepatitis B virus enhancer I.

Hepatitis B virus (HBV) enhancer I contains cis-acting elements that are both sufficient and essential for liver-specific enhancer function. The EF-C binding site was previously shown to be a key element in enhancer I. EF-C binding activity is evident in hepatic and nonhepatic cells. Although the EF-C binding site is required for efficient HBV enhancer I function, the EF-C site does not possess intrinsic enhancer activity when assayed in the absence of flanking elements. We have defined a novel region in HBV enhancer I, termed the GB element, that is adjacent to and functions in conjunction with the EF-C binding site. The GB element and EF-C site confer interdependent liver-specific enhancer activity in the absence of flanking HBV enhancer sequences. The nucleotide sequence of the GB element is similar to sequences of the DNA binding sites for members of the steroid receptor superfamily. Among these proteins, we demonstrate that HNF-4, RXR (retinoid X receptor), and COUP-TF bind to the GB element in vitro. HNF-4 transactivates a promoter linked to a multimerized GB/EF-C domain via the GB element in vivo in a manner that is dependent on the integrity of the adjacent EF-C binding site. RXR alpha also transactivates promoter expression via the GB element in vivo in response to retinoic acid but in a largely EF-C-independent manner. Finally, we show that COUP-TF antagonizes the activity of the GB element in human liver cells.

Base Sequence↗