Molecular Interactions During Adenovirus DNA Replication

The SNF2-related CBP activator protein, SrCap (pronounced "sir cap"), shares homology with the SNF2/ SWI2 protein family. SrCap was cloned through its ability to bind CBP. SrCap can function as a CBP coactivator and can activate transcription in a reporter assay when expressed as a Gal-SrCap fusion protein.A monoclonal antibody raised against the carboxyl terminus of SrCap coimmunoprecipitates CBP/p300, supporting the model that SrCap is a CBP binding protein and that these proteins can be found together in a cellular protein complex. In addition, several cellular proteins are coimmunoprecipitated by the SrCapspecific antibody. Since adenovirus E1A proteins interact with CBP/p300 proteins, we examined what proteins could be copurified in a SrCap-specific coimmunoprecipitation assay from lysates of adenovirus-infected cells. While E1A proteins were not detected in this complex, to our surprise, we observed the presence of an infected-cell-specific band of 72 kDa, which we suspected might be the adenovirus DNA binding protein, DBP. The adenovirus DBP is a multifunctional protein involved in several aspects of the adenovirus life cycle, including an ability to modulate transcription. The identity of DBP was confirmed by DBP-specific Western blot analysis and by reimmunoprecipitating DBP from denatured SrCap-specific protein complexes. Using in vitro-translated DBP and SrCap proteins, we demonstrated that these proteins interact. To determine whether this interaction could affect SrCap-mediated transcription, we tested whether increasing amounts of DBP could modulate the Gal-SrCap transcription activity. We observed that DBP inhibited Gal-SrCap transcription activity in a dose-dependent manner. These data suggest a novel mechanism of adenovirus host cell control by which DBP binds to and inactivates SrCap, a member of the SNF2 chromatin-remodeling protein family.The adenovirus DNA binding protein, DBP, is well studied for its role in adenovirus replication (3, 7, 12). DBP's replication function can be reconstituted in vitro and involves functions and interactions of at least three viral proteins, including the adenovirus DNA polymerase, precursor terminal protein, and DBP. In vitro replication function can be enhanced by the addition of two cellular transcription factors, nuclear factor I (NFI) and Oct1 (28). However, DBP is also implicated in several other essential functions important in the adenovirus life cycle. These include virion assembly (21), host range determination (1, 9), mRNA stability (5), and transformation (23).In addition, DBP has roles in transcriptional regulation. DBP can enhance its own expression, and mutant studies demonstrate that only the highly phosphorylated forms of DBP grant this activation (20). DBP can regulate transcription directed by virus promoters (2). In these studies, DBP was shown to enhance transcription from the adenovirus E1A, E2A, and major late promoters and the adeno-associated virus P5 promoter but was also found to slightly inhibit the adenovirus E4 promoter. The mechan...

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confidence: 99%

Adenovirus DNA Binding Protein Interacts with the SNF2-Related CBP Activator Protein (SrCap) and Inhibits SrCap-Mediated Transcription

Xu¹,

Chackalaparampil²,

Monroy

et al. 2001

“…The 671-amino-acid E2 preTP serves as the protein primer for viral DNA synthesis when it becomes covalently attached to the 5= ends of newly replicated viral DNA molecules (36,52). Subsequently, this precursor is processed by the viral L3 protease to the mature TP (77,98).…”

Section: Resultsmentioning

confidence: 99%

“…from which viral particles are assembled (reviewed in references 5 and 72). The immature particles initially assembled also contain the precursor of the terminal protein (TP), preTP, which becomes covalently attached to the 5= ends of newly synthesized viral genomes when it serves as the protein primer for initiation of DNA synthesis (36,52). Processing of precursor proteins is essential to form mature, infectious virions: a temperature-sensitive mutation (Ad2ts1) in the L3 coding sequence for the viral cysteine protease that prevents encapsidation of this viral enzyme (67,97) results in the accumulation at nonpermissive temperatures of noninfectious particles containing uncleaved precursor proteins (reviewed in reference 72).…”

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confidence: 99%

Reduced Infectivity of Adenovirus Type 5 Particles and Degradation of Entering Viral Genomes Associated with Incomplete Processing of the Preterminal Protein

Kato

Chahal

Flint

2012

To investigate further the contribution of the adenovirus type 5 (Ad5) E1B 55-kDa protein to genome replication, viral DNA accumulation was examined in primary human fibroblasts and epithelial cells infected with Ad5 or the E1B 55-kDa-null mutant Hr6. Unexpectedly, all cell types were observed to contain a significantly higher concentration of entering Hr6 than of Ad5 DNA, as did an infectious unit of Hr6. However, the great majority of the Hr6 genomes were degraded soon after entry. As this unusual phenotype cannot be ascribed to the Hr6 E1B frameshift mutation (J. S. Chahal and S. J. Flint, J. Virol. 86:3064 -3072, 2012), the sequences of the Ad5 and Hr6 genomes were compared by using high-throughput sequencing. Seven previously unrecognized mutations were identified in the Hr6 genome, two of which result in substitutions in virion proteins, G315V in the preterminal protein (preTP) and A406V in fiber protein IV. Previous observations and the visualization by immunofluorescence of greater numbers of viral genomes entering the cytosol of Hr6-infected cells than of Ad5-infected cells indicated that the fiber mutation could not be responsible for the low-infectivity phenotype of Hr6. However, comparison of the forms of terminal protein present in purified virus particles indicated that the production of mature terminal protein from a processing intermediate is impaired in Hr6 particles. We therefore propose that complete processing of preTP within virus particles is necessary for the ability of viral genomes to become localized at appropriate sites and persist in infected cells. Successful initiation of the human adenovirus infectious cycle depends on a complex set of interactions among viral and cellular components that allow attachment, entry, and partial dismantling of virus particles prior to the transport of viral genomes to and into the infected cell nucleus. The nonenveloped, icosahedrally symmetric virus particles carry distinctive fibers that project from the penton base present at each of the 12 vertices (5, 72). The distal knob of the fiber contains the binding site for attachment to the primary cell surface receptor, the coxsackievirus and adenovirus receptor, Car, in the case of species C adenoviruses such as serotype 5 (Ad5) (4,70,89). Interactions of RGD sequences present in loops that project from the surface of each subunit of the pentameric penton base with ␣v integrins on the cell surface (14, 84) then promote the entry of virus particles by clathrin-mediated endocytosis (18,50,75,80,101; reviewed in reference 1). Subsequent escape from early endosomes into the cytoplasm is coordinated with, and dependent on, initial uncoating reactions that remove capsid proteins.

“…DBP has been reported to be associated with replication, transcription modulation, and host range of Ad (18). A number of Ad mutants have previously been generated within the region of DBP that also encodes the third exon of UXP (7,43,49).…”

Section: Discussionmentioning

confidence: 99%

Identification of a New Human Adenovirus Protein Encoded by a Novel Late l -Strand Transcription Unit

Tollefson

Bai

Doronin

et al. 2007

A short open reading frame named the "U exon," located on the adenovirus (Ad) l-strand (for leftward transcription) between the early E3 region and the fiber gene, is conserved in mastadenoviruses. We have observed that Ad5 mutants with large deletions in E3 that infringe on the U exon display a mild growth defect, as well as an aberrant Ad E2 DNA-binding protein ( An open reading frame (ORF) named the "U exon," located on the l strand (for leftward transcription) between the early E3 region and the fiber gene, was first identified after the sequencing of Ad40 (9). Davison et al. (9) reported that the U exon that is conserved in mastadenoviruses would encode just over 50 amino acids (aa) for most mastadenoviruses and might encode the N terminus of a larger protein. U exon sequences are conserved within each of the four Ad genera but not between genera (10). Davison et al. (10) suggested that the U exon is an ancient feature in adenoviruses (Ads; all Ad genera genomes encode capsid proteins and the E2 proteins within the central region of the Ad genome; the U exon is encoded within this region).In our work with a number of Ad E3 deletion mutants, we noted that mutants with deletions extending into the U exon have a mild growth defect and exhibit an altered Ad DNA-binding protein (DBP) (also known as E2 72K protein) intranuclear localization pattern when analyzed by indirect immunofluorescence. Reconstruction of the U exon DNA sequence reversed the phenotype of these mutants. Polyclonal and monoclonal antibodies were generated against peptides predicted from the putative U exon protein (UXP) ORF. We describe here the identification and characterization of UXP. MATERIALS AND METHODS Cell lines and adenoviruses. A549 cells (American Type Culture Collection[ATCC], Manassas, VA), HEK293 cells (Microbix, Toronto, Ontario, Canada), 21f1 cells (HeLa cells stably expressing DBP) (6), and A549E1 cells (A549 cells stably transfected with E1 sequences [bp 552 to 4090] under control of a mouse mammary tumor virus promoter; obtained from Genetic Therapy, Inc., Rockville, MD) were grown in Dulbecco modified Eagle medium (DMEM) with 10% fetal bovine serum (FBS). HEL299 cells (human diploid embryonic lung cells; ATCC) were grown in DMEM with 10% FBS supplemented with nonessential amino acids and sodium pyruvate (1 mM). KB suspension culture cells were grown in Joklik's suspension culture medium containing 5% horse serum. Wildtype viruses used were Ad5 (ATCC VR-5), Ad1 (ATCC 1078-VR), Ad2, and Ad6 (ATCC 1083-VR). The Ad5-derived mutants used were dl327 (16), VRX-006, VRX-007 (13), VRX-021 (see below), dl7000 (42), and dl7001 (17, 42). The Ad E3 mutants dl722 (⌬12.5K) and dl762 (⌬14.7K) (5) were also used. Figure 1A is a schematic of the E3 genes present in these mutants, and Fig. 1B shows the U exon sequence present in these mutants.Construction of VRX-006 and VRX-021. The construction of mutants VRX-006 and VRX-007 has been described (13). In VRX-006 all E3 sequences between the L4 poly(A) site and a site (bp 30883) downstream ...