Publications

2017
Hung, George, and SJ Flint. “Normal human cell proteins that interact with the adenovirus type 5 E1B 55kDa protein.”. Virology 504 (2017): , 504, 12-24. Web.Abstract
Several of the functions of the human adenovirus type 5 E1B 55kDa protein are fulfilled via the virus-specific E3 ubiquitin ligase it forms with the viral E4 Orf6 protein and several cellular proteins. Important substrates of this enzyme have not been identified, and other functions, including repression of transcription of interferon-sensitive genes, do not require the ligase. We therefore used immunoaffinity purification and liquid chromatography-mass spectrometry of lysates of normal human cells infected in parallel with HAdV-C5 and E1B 55kDa protein-null mutant viruses to identify specifically E1B 55kDa-associated proteins. The resulting set of >90 E1B-associated proteins contained the great majority identified previously, and was enriched for those associated with the ubiquitin-proteasome system, RNA metabolism and the cell cycle. We also report very severe inhibition of viral genome replication when cells were exposed to both specific or non-specific siRNAs and interferon prior to infection.
2015
DeHart, Caroline J, David H Perlman, and SJ Flint. “Impact of the adenoviral E4 Orf3 protein on the activity and posttranslational modification of p53.”. J Virol 89.6 (2015): , 89, 6, 3209-20. Web.Abstract
UNLABELLED: Our previous studies have established that the p53 populations that accumulate in normal human cells exposed to etoposide or infected by an E1B 55-kDa protein-null mutant of human adenovirus type 5 carry a large number of posttranslational modifications at numerous residues (C. J. DeHart, J. S. Chahal, S. J. Flint, and D. H. Perlman, Mol Cell Proteomics 13:1-17, 2014, http://dx.doi.org/10.1074/mcp.M113.030254). In the absence of this E1B protein, the p53 transcriptional program is not induced, and it has been reported that the viral E4 Orf3 protein inactivates p53 (C. Soria, F. E. Estermann, K. C. Espantman, and C. C. O'Shea, Nature 466:1076-1081, 2010, http://dx.doi.org/10.1038/nature09307). As the latter protein disrupts nuclear Pml bodies, sites at which p53 is modified, we used mass spectrometry to catalogue the posttranscriptional modifications of the p53 population that accumulates when neither the E1B 55-kDa nor the E4 Orf3 protein is made in infected cells. Eighty-five residues carrying 163 modifications were identified. The overall patterns of posttranslational modification of this population and p53 present in cells infected by an E1B 55-kDa-null mutant were similar. The efficiencies with which the two forms of p53 bound to a consensus DNA recognition sequence could not be distinguished and were lower than that of transcriptionally active p53. The absence of the E4 Orf3 protein increased expression of several p53-responsive genes when the E1B protein was also absent from infected cells. However, expression of these genes did not attain the levels observed when p53 was activated in response to etoposide treatment and remained lower than those measured in mock-infected cells. IMPORTANCE: The tumor suppressor p53, a master regulator of cellular responses to stress, is inactivated and destroyed in cells infected by species C human adenoviruses, such as type 5. It is targeted for proteasomal degradation by the action of a virus-specific E3 ubiquitin ligase that contains the viral E1B 55-kDa and E4 Orf6 proteins, while the E4 Orf3 protein has been reported to block its ability to stimulate expression of p53-dependent genes. The comparisons reported here of the posttranslational modifications and activities of p53 populations that accumulate in infected normal human cells in the absence of both mechanisms of inactivation or of only the E3 ligase revealed little impact of the E4 Orf3 protein. These observations indicate that E4 Orf3-dependent disruption of Pml bodies does not have a major effect on the pattern of p53 posttranslational modifications in adenovirus-infected cells. Furthermore, they suggest that one or more additional viral proteins contribute to blocking p53 activation and the consequences that are deleterious for viral reproduction, such as apoptosis or cell cycle arrest.
Ortega-Esteban, Alvaro, et al.Mechanics of Viral Chromatin Reveals the Pressurization of Human Adenovirus.”. ACS Nano 911 (2015): , 9, 11, 10826-33. Web.Abstract
Tight confinement of naked genomes within some viruses results in high internal pressure that facilitates their translocation into the host. Adenovirus, however, encodes histone-like proteins that associate with its genome resulting in a confined DNA-protein condensate (core). Cleavage of these proteins during maturation decreases core condensation and primes the virion for proper uncoating via unidentified mechanisms. Here we open individual, mature and immature adenovirus cages to directly probe the mechanics of their chromatin-like cores. We find that immature cores are more rigid than the mature ones, unveiling a mechanical signature of their condensation level. Conversely, intact mature particles demonstrate more rigidity than immature or empty ones. DNA-condensing polyamines revert the mechanics of mature capsid and cores to near-immature values. The combination of these experiments reveals the pressurization of adenovirus particles induced by maturation. We estimate a pressure of ∼30 atm by continuous elasticity, which is corroborated by modeling the adenovirus mini-chromosome as a confined compact polymer. We propose this pressurization as a mechanism that facilitates initiating the stepwise disassembly of the mature particle, enabling its escape from the endosome and final genome release at the nuclear pore.
2014
DeHart, Caroline J, et al.Extensive post-translational modification of active and inactivated forms of endogenous p53.”. Mol Cell Proteomics 13.1 (2014): , 13, 1, 1-17. Web.Abstract
The p53 tumor suppressor protein accumulates to very high concentrations in normal human fibroblasts infected by adenovirus type 5 mutants that cannot direct assembly of the viral E1B 55-kDa protein-containing E3 ubiquitin ligase that targets p53 for degradation. Despite high concentrations of nuclear p53, the p53 transcriptional program is not induced in these infected cells. We exploited this system to examine select post-translational modifications (PTMs) present on a transcriptionally inert population of endogenous human p53, as well as on p53 activated in response to etoposide treatment of normal human fibroblasts. These forms of p53 were purified from whole cell lysates by means of immunoaffinity chromatography and SDS-PAGE, and peptides derived from them were subjected to nano-ultra-high-performance LC-MS and MS/MS analyses on a high-resolution accurate-mass MS platform (data available via ProteomeXchange, PXD000464). We identified an unexpectedly large number of PTMs, comprising phosphorylation of Ser and Thr residues, methylation of Arg residues, and acetylation, ubiquitinylation, and methylation of Lys residues-for example, some 150 previously undescribed modifications of p53 isolated from infected cells. These modifications were distributed across all functional domains of both forms of the endogenous human p53 protein, as well as those of an orthologous population of p53 isolated from COS-1 cells. Despite the differences in activity, including greater in vitro sequence-specific DNA binding activity exhibited by p53 isolated from etoposide-treated cells, few differences were observed in the location, nature, or relative frequencies of PTMs on the two populations of human p53. Indeed, the wealth of PTMs that we have identified is consistent with a far greater degree of complex, combinatorial regulation of p53 by PTM than previously anticipated.
2013
Chahal, Jasdave S, and SJ Flint. “The p53 protein does not facilitate adenovirus type 5 replication in normal human cells.”. J Virol 87.10 (2013): , 87, 10, 6044-6. Web.Abstract
Although several adenovirus type 5 (Ad5) proteins prevent deleterious consequences of activation of p53, it has been reported that viral replication proceeds more efficiently when human tumor cells produce wild-type compared to mutant p53. We have now exploited RNA interference and lentiviral vectors to achieve essentially complete knockdown of p53 in normal human cells: no effects on the kinetics or efficiency of viral gene expression or production of infectious particles were observed.
Chahal, Jasdave S, et al.The repression domain of the E1B 55-kilodalton protein participates in countering interferon-induced inhibition of adenovirus replication.”. J Virol 87.8 (2013): , 87, 8, 4432-44. Web.Abstract
To begin to investigate the mechanism by which the human adenovirus type 5 E1B 55-kDa protein protects against the antiviral effects of type 1 interferon (IFN) (J. S. Chahal, J. Qi, and S. J. Flint, PLoS Pathog. 8:e1002853, 2012 [doi:10.1371/journal.ppat.1002853]), we examined the effects of precise amino acid substitution in this protein on resistance of viral replication to the cytokine. Only substitution of residues 443 to 448 of E1B for alanine (E1B Sub19) specifically impaired production of progeny virus and resulted in a large defect in viral DNA synthesis in IFN-treated normal human fibroblasts. Untreated or IFN-treated cells infected by this mutant virus (AdEasyE1Sub19) contained much higher steady-state concentrations of IFN-inducible GBP1 and IFIT2 mRNAs than did wild-type-infected cells and of the corresponding newly transcribed pre-mRNAs, isolated exploiting 5'-ethynyluridine labeling and click chemistry. These results indicated that the mutations created by substitution of residues 443 to 448 for alanine (Sub19) impair repression of transcription of IFN-inducible genes, by the E1B, 55-kDa protein, consistent with their location in a segment required for repression of p53-dependent transcription. However, when synthesized alone, the E1B 55-kDa protein inhibited expression of the p53-regulated genes BAX and MDM2 but had no impact whatsoever on induction of IFIT2 and GBP1 expression by IFN. These observations correlate repression of transcription of IFN-inducible genes by the E1B 55-kDa protein with protection against inhibition of viral genome replication and indicate that the E1B 55-kDa protein is not sufficient to establish such transcriptional repression.
2012
Chahal, Jasdave S, Ji Qi, and SJ Flint. “The human adenovirus type 5 E1B 55 kDa protein obstructs inhibition of viral replication by type I interferon in normal human cells.”. PLoS Pathog 88 (2012): , 8, 8, e1002853. Web.Abstract
Vectors derived from human adenovirus type 5, which typically lack the E1A and E1B genes, induce robust innate immune responses that limit their therapeutic efficacy. We reported previously that the E1B 55 kDa protein inhibits expression of a set of cellular genes that is highly enriched for those associated with anti-viral defense and immune responses, and includes many interferon-sensitive genes. The sensitivity of replication of E1B 55 kDa null-mutants to exogenous interferon (IFN) was therefore examined in normal human fibroblasts and respiratory epithelial cells. Yields of the mutants were reduced at least 500-fold, compared to only 5-fold, for wild-type (WT) virus replication. To investigate the mechanistic basis of such inhibition, the accumulation of viral early proteins and genomes was compared by immunoblotting and qPCR, respectively, in WT- and mutant-infected cells in the absence or presence of exogenous IFN. Both the concentration of viral genomes detected during the late phase and the numbers of viral replication centers formed were strongly reduced in IFN-treated cells in the absence of the E1B protein, despite production of similar quantities of viral replication proteins. These defects could not be attributed to degradation of entering viral genomes, induction of apoptosis, or failure to reorganize components of PML nuclear bodies. Nor was assembly of the E1B- and E4 Orf6 protein- E3 ubiquitin ligase required to prevent inhibition of viral replication by IFN. However, by using RT-PCR, the E1B 55 kDa protein was demonstrated to be a potent repressor of expression of IFN-inducible genes in IFN-treated cells. We propose that a primary function of the previously described transcriptional repression activity of the E1B 55 kDa protein is to block expression of IFN- inducible genes, and hence to facilitate formation of viral replication centers and genome replication.
Kato, Sayuri E, Jasdave S Chahal, and SJ Flint. “Reduced infectivity of adenovirus type 5 particles and degradation of entering viral genomes associated with incomplete processing of the preterminal protein.”. J Virol 86.24 (2012): , 86, 24, 13554-65. Web.Abstract
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.
Chahal, Jasdave S, and SJ Flint. “Timely synthesis of the adenovirus type 5 E1B 55-kilodalton protein is required for efficient genome replication in normal human cells.”. J Virol 86.6 (2012): , 86, 6, 3064-72. Web.Abstract
Previous studies have indicated that the adenovirus type 5 E1B 55-kDa protein facilitates viral DNA synthesis in normal human foreskin fibroblasts (HFFs) but not in primary epithelial cells. To investigate this apparent difference further, viral DNA accumulation was examined in primary human fibroblasts and epithelial cells infected by the mutant AdEasyE1Δ2347, which carries the Hr6 frameshift mutation that prevents production of the E1B 55-kDa protein, in an E1-containing derivative of AdEasy. Impaired viral DNA synthesis was observed in normal HFFs but not in normal human bronchial epithelial cells infected by this mutant. However, acceleration of progression through the early phase, which is significantly slower in HFFs than in epithelial cells, eliminated the dependence of efficient viral DNA synthesis in HFFs on the E1B 55-kDa protein. These observations suggest that timely synthesis of the E1B 55-kDa protein protects normal cells against a host defense that inhibits adenoviral genome replication. One such defense is mediated by the Mre11-Rad50-Nbs1 complex. Nevertheless, examination of the localization of Mre11 and viral proteins by immunofluorescence suggested that this complex is inactivated similarly in AdEasyE1Δ2347 mutant-infected and AdEasyE1-infected HFFs.
2011
Yatherajam, Gayatri, Wenying Huang, and SJ Flint. “Export of adenoviral late mRNA from the nucleus requires the Nxf1/Tap export receptor.”. J Virol 85.4 (2011): , 85, 4, 1429-38. Web.Abstract
One important function of the human adenovirus E1B 55-kDa protein is induction of selective nuclear export of viral late mRNAs. This protein interacts with the viral E4 Orf6 and four cellular proteins to form an infected-cell-specific E3 ubiquitin ligase. The assembly of this enzyme is required for efficient viral late mRNA export, but neither the relevant substrates nor the cellular pathway that exports viral late mRNAs has been identified. We therefore examined the effects on viral late gene expression of inhibition of the synthesis or activity of the mRNA export receptor Nxf1, which was observed to colocalize with the E1B 55-kDa protein in infected cells. When production of Nxf1 was impaired by using RNA interference, the efficiency of viral late mRNA export was reduced to a corresponding degree. Furthermore, synthesis of a dominant-negative derivative of Nxf1 during the late phase of infection interfered with production of a late structural protein. These observations indicate that the Nxf1 pathway is responsible for export of viral late mRNAs. As the infected-cell-specific E3 ubiquitin ligase targets its known substrates for proteasomal degradation, we compared the concentrations of several components of this pathway (Nxf1, Thox1, and Thoc4) in infected cells that did or did not contain this enzyme. Although the concentration of a well-established substrate, Mre11, decreased significantly in cells infected by adenovirus type 5 (Ad5), but not in those infected by the E1B 55-kDa protein-null mutant Hr6, no E1B 55-kDa protein-dependent degradation of the Nxf1 pathway proteins was observed.
Kato, Sayuri EM, Wenying Huang, and SJ Flint. “Role of the RNA recognition motif of the E1B 55 kDa protein in the adenovirus type 5 infectious cycle.”. Virology 417.1 (2011): , 417, 1, 9-17. Web.Abstract
Although the adenovirus type 5 (Ad5) E1B 55 kDa protein can bind to RNA in vitro, no UV-light-induced crosslinking of this E1B protein to RNA could be detected in infected cells, under conditions in which RNA binding by a known viral RNA-binding protein (the L4 100 kDa protein) was observed readily. Substitution mutations, including substitutions reported to inhibit RNA binding in vitro, did not impair synthesis of viral early or late proteins or alter significantly the efficiency of viral replication in transformed or normal human cells. However, substitutions of conserved residues in the C-terminal segment of an RNA recognition motif specifically inhibited degradation of Mre11. We conclude that, if the E1B 55 kDa protein binds to RNA in infected cells in the same manner as in in vitro assays, this activity is not required for such well established functions as induction of selective export of viral late mRNAs.
2009
Miller, Daniel L, et al.The adenoviral E1B 55-kilodalton protein controls expression of immune response genes but not p53-dependent transcription.”. J Virol 83.8 (2009): , 83, 8, 3591-603. Web.Abstract
The human adenovirus type 5 (Ad5) E1B 55-kDa protein modulates several cellular processes, including activation of the tumor suppressor p53. Binding of the E1B protein to the activation domain of p53 inhibits p53-dependent transcription. This activity has been correlated with the transforming activity of the E1B protein, but its contribution to viral replication is not well understood. To address this issue, we used microarray hybridization methods to examine cellular gene expression in normal human fibroblasts (HFFs) infected by Ad5, the E1B 55-kDa-protein-null mutant Hr6, or a mutant carrying substitutions that impair repression of p53-dependent transcription. Comparison of the changes in cellular gene expression observed in these and our previous experiments (D. L. Miller et al., Genome Biol. 8:R58, 2007) by significance analysis of microarrays indicated excellent reproducibility. Furthermore, we again observed that Ad5 infection led to efficient reversal of the p53-dependent transcriptional program. As this same response was also induced in cells infected by the two mutants, we conclude that the E1B 55-kDa protein is not necessary to block activation of p53 in Ad5-infected cells. However, groups of cellular genes that were altered in expression specifically in the absence of the E1B protein were identified by consensus k-means clustering of the hybridization data. Statistical analysis of the enrichment of genes associated with specific functions in these clusters established that the E1B 55-kDa protein is necessary for repression of genes encoding proteins that mediate antiviral and immune defenses.
2008
LeRoy, Gary, Brenden Rickards, and SJ Flint. “The double bromodomain proteins Brd2 and Brd3 couple histone acetylation to transcription.”. Mol Cell 30.1 (2008): , 30, 1, 51-60. Web.Abstract
Posttranslational histone modifications are crucial for the modulation of chromatin structure and regulation of transcription. Bromodomains present in many chromatin-associated proteins recognize acetylated lysines in the unstructured N-terminal regions of histones. Here, we report that the double bromodomain proteins Brd2 and Brd3 associate preferentially in vivo with hyperacetylated chromatin along the entire lengths of transcribed genes. Brd2- and Brd3-associated chromatin is significantly enriched in H4K5, H4K12, and H3K14 acetylation and contains relatively little dimethylated H3K9. Both Brd2 and Brd3 allowed RNA polymerase II to transcribe through nucleosomes in a defined transcription system. Such activity depended on specific histone H4 modifications known to be recognized by the Brd proteins. We also demonstrate that Brd2 has intrinsic histone chaperone activity and is required for transcription of the cyclin D1 gene in vivo. These data identify proteins that render nucleosomes marked by acetylation permissive to the passage of elongating RNA polymerase II.
2007
Ali, Humayra, et al.The adenovirus L4 33-kilodalton protein binds to intragenic sequences of the major late promoter required for late phase-specific stimulation of transcription.”. J Virol 81.3 (2007): , 81, 3, 1327-38. Web.Abstract
The adenovirus late IVa2 protein is required for maximally efficient transcription from the viral major late (ML) promoter, and hence, the synthesis of the majority of viral late proteins. This protein is a sequence-specific DNA-binding protein that also promotes the assembly of progeny virus particles. Previous studies have established that a IVa2 protein dimer (DEF-B) binds specifically to an intragenic ML promoter sequence necessary for late phase-specific stimulation of ML transcription. However, activation of transcription from the ML promoter correlates with binding of at least one additional infected-cell-specific protein, termed DEF-A, to the promoter. Using an assay for the DNA-binding activity of DEF-A, we identified the unknown protein by using conventional purification methods, purification of FLAG-tagged IVa2-protein-containing complexes, and transient synthesis of viral late proteins. The results of these experiments established that the viral L4 33-kDa protein is the only component of DEF-A: the IVa2 and L4 33-kDa proteins are necessary and sufficient for formation of all previously described complexes in the intragenic control region of the ML promoter. Furthermore, the L4 33-kDa protein binds to the promoter with the specificity characteristic of DEF-A and stimulates transcription from the ML promoter in transient-expression assays.
Miller, Daniel L, et al.Adenovirus type 5 exerts genome-wide control over cellular programs governing proliferation, quiescence, and survival.”. Genome Biol 84 (2007): , 8, 4, R58. Web.Abstract
BACKGROUND: Human adenoviruses, such as serotype 5 (Ad5), encode several proteins that can perturb cellular mechanisms that regulate cell cycle progression and apoptosis, as well as those that mediate mRNA production and translation. However, a global view of the effects of Ad5 infection on such programs in normal human cells is not available, despite widespread efforts to develop adenoviruses for therapeutic applications. RESULTS: We used two-color hybridization and oligonucleotide microarrays to monitor changes in cellular RNA concentrations as a function of time after Ad5 infection of quiescent, normal human fibroblasts. We observed that the expression of some 2,000 genes, about 10% of those examined, increased or decreased by a factor of two or greater following Ad5 infection, but were not altered in mock-infected cells. Consensus k-means clustering established that the temporal patterns of these changes were unexpectedly complex. Gene Ontology terms associated with cell proliferation were significantly over-represented in several clusters. The results of comparative analyses demonstrate that Ad5 infection induces reversal of the quiescence program and recapitulation of the core serum response, and that only a small subset of the observed changes in cellular gene expression can be ascribed to well characterized functions of the viral E1A and E1B proteins. CONCLUSION: These findings establish that the impact of adenovirus infection on host cell programs is far greater than appreciated hitherto. Furthermore, they provide a new framework for investigating the molecular functions of viral early proteins and information relevant to the design of conditionally replicating adenoviral vectors.
Rickards, Brenden, et al.Nucleolin is required for RNA polymerase I transcription in vivo.”. Mol Cell Biol 27.3 (2007): , 27, 3, 937-48. Web.Abstract
Eukaryotic genomes are packaged with histones and accessory proteins in the form of chromatin. RNA polymerases and their accessory proteins are sufficient for transcription of naked DNA, but not of chromatin, templates in vitro. In this study, we purified and identified nucleolin as a protein that allows RNA polymerase II to transcribe nucleosomal templates in vitro. As immunofluorescence confirmed that nucleolin localizes primarily to nucleoli with RNA polymerase I, we demonstrated that nucleolin allows RNA polymerase I transcription of chromatin templates in vitro. The results of chromatin immunoprecipitation experiments established that nucleolin is associated with chromatin containing rRNA genes transcribed by RNA polymerase I but not with genes transcribed by RNA polymerase II or III. Knockdown of nucleolin by RNA interference resulted in specific inhibition of RNA polymerase I transcription. We therefore propose that an important function of nucleolin is to permit RNA polymerase I to transcribe nucleolar chromatin.
2006
Gonzalez, Ramon, et al.Adenovirus E1B 55-kilodalton protein is required for both regulation of mRNA export and efficient entry into the late phase of infection in normal human fibroblasts.”. J Virol 80.2 (2006): , 80, 2, 964-74. Web.Abstract
The human adenovirus type 5 (Ad5) E1B 55-kDa protein is required for selective nuclear export of viral late mRNAs from the nucleus and concomitant inhibition of export of cellular mRNAs in HeLa cells and some other human cell lines, but its contributions(s) to replication in normal human cells is not well understood. We have therefore examined the phenotypes exhibited by viruses carrying mutations in the E1B 55-kDa protein coding sequence in normal human fibroblast (HFFs). Ad5 replicated significantly more slowly in HFFs than it does in tumor cells, a difference that is the result of delayed entry into the late phase of infection. The A143 mutation, which specifically impaired export of viral late mRNAs from the nucleus in infected HeLa cells (R. A. Gonzalez and S. J. Flint, J. Virol. 76:4507-4519, 2002), induced a more severe defect in viral mRNA export in HFFs. This observation indicates that the E1B 55-kDa protein regulates mRNA export during the late phase of infection of normal human cells. Other mutants exhibited phenotypes not observed in HeLa cells. In HFFs infected by the null mutant Hr6, synthesis of viral late mRNAs and proteins was severely impaired. Such defects in late gene expression were the result of inefficient progression into the late phase of infection, for viral DNA synthesis was 10-fold less efficient in Hr6-infected HFFs than in cells infected by Ad5. Similar, but less severe, defects in viral DNA synthesis were induced by the insertion mutation H224, which has been reported to inhibit binding of the E1B 55-kDa protein to p53 (C. C. Kao, P. R. Yew, and A. J. Berk, Virology 179:806-814, 1990).
2005
Flint, SJ, et al.A peptide inhibitor of exportin1 blocks shuttling of the adenoviral E1B 55 kDa protein but not export of viral late mRNAs.”. Virology 337.1 (2005): , 337, 1, 7-17. Web.Abstract
The human subgroup C adenoviral E1B 55 kDa and E4 Orf6 proteins are required for efficient nuclear export of viral late mRNAs, but the cellular pathway that mediates such export has not been identified. As a first step to develop a general approach to address this issue, we have assessed the utility of cell-permeable peptide inhibitors of cellular export receptors. As both E1B and E4 proteins have been reported to contain a leucine-rich nuclear export signal (NES), we synthesized a cell-permeable peptide containing such an NES. This peptide induced substantial inhibition of export of the E1B protein, whereas a control, non-functional peptide did not. However, under the same conditions, the NES peptide had no effect on export of viral late mRNAs. These observations establish that viral late mRNAs are not exported by exportin1, as well as the value of peptide inhibitors in investigation of mRNA export regulation in adenovirus-infected cells.
2004
Iftode, C, and SJ Flint. “Viral DNA synthesis-dependent titration of a cellular repressor activates transcription of the human adenovirus type 2 IVa2 gene.”. Proc Natl Acad Sci U S A 101.51 (2004): , 101, 51, 17831-6. Web.Abstract
Synthesis of progeny DNA genomes in cells infected by human subgroup C adenoviruses leads to several changes in viral gene expression. These changes include transcription from previously silent, late promoters, such as the IV(a2) promoter, and a large increase in the efficiency of major-late (ML) transcription. Some of these changes appear to take place sequentially, because the product of the IV(a2) gene has been implicated in stimulation of ML transcription. Our previous biochemical studies suggested that IV(a2) transcription is regulated by viral DNA synthesis-dependent relief of transcriptional repression by a cellular protein that we termed IV(a2)-RF. To test the relevance of such a repressor-titration mechanism during the viral infectious cycle, we introduced into the endogenous IV(a2) promoter two mutations that impair in vitro-binding of IV(a2)-RF, but introduce no change (Rep7) or one conservative amino acid substitution (Rep6) into the overlapping coding sequence for the viral DNA polymerase. The results of run-on transcription assays indicated that both mutations induced earlier-than-normal and more efficient IV(a2) transcription. Both mutations were also observed to result in modest increases in the efficiency of viral DNA synthesis. However, measurement of the concentration of IV(a2) transcripts as a function of IV(a2) template concentration demonstrated that the Rep mutations increased by up to 60-fold the efficiency with which IV(a2) templates were used during the initial period of the late phase of infection, as predicted by the repressor titration hypothesis. These mutations also increased the efficiency of ML transcription in infected cells.
2003
Flint, SJ, and RA Gonzalez. “Regulation of mRNA production by the adenoviral E1B 55-kDa and E4 Orf6 proteins.”. Curr Top Microbiol Immunol 272 (2003): , 272, 287-330. Print.Abstract
The E1B 55-kDa and E4 Orf6 proteins of human subgroup C adenoviruses both counter host cell defenses mediated by the cellular p53 protein and regulate viral late gene expression. A complex containing the two proteins has been implicated in induction of selective export of viral late mRNAs from the nucleus to the cytoplasm, with concomitant inhibition of export of the majority of newly synthesized cellular mRNAs. The molecular mechanisms by which these viral proteins subvert cellular pathways of nuclear export are not yet clear. Here, we review recent efforts to identify molecular and biochemical functions of the E1B 55-kDa and E4 Orf6 proteins required for regulation of mRNA export, the several difficulties and discrepancies that have been encountered in studies of these viral proteins, and evidence indicating that the reorganization of the infected cell nucleus and production of viral late mRNA at specific intra-nuclear sites are important determinants of selective mRNA export in infected cells. In our view, it is not yet possible to propose a coherent molecular model for regulation of mRNA export by the E1B 55-kDa and E4 Orf6 proteins. However, it should now be possible to address specific questions about the roles of potentially relevant properties of these viral proteins.
Huang, Wenlin, and SJ Flint. “Unusual properties of adenovirus E2E transcription by RNA polymerase III.”. J Virol 77.7 (2003): , 77, 7, 4015-24. Print.Abstract
In adenovirus type 5-infected cells, RNA polymerase III transcription of a gene superimposed on the 5' end of the E2E RNA polymerase II transcription unit produces two small (<100-nucleotide) RNAs that accumulate to low steady-state concentrations (W. Huang, R. Pruzan, and S. J. Flint, Proc. Natl. Acad. Sci. USA 91:1265-1269, 1984). To gain a better understanding of the function of this RNA polymerase III transcription, we have examined the properties of the small E2E RNAs and E2E RNA polymerase III transcription in more detail. The accumulation of cytoplasmic E2E RNAs and the rates of E2E transcription by the two RNA polymerases during the infectious cycle were analyzed by using RNase T(1) protection and run-on transcription assays, respectively. Although the RNA polymerase III transcripts were present at significantly lower concentrations than E2E mRNA throughout the period examined, E2E transcription by RNA polymerase III was found to be at least as efficient as that by RNA polymerase II. The short half-lifes of the small E2E RNAs estimated by using the actinomycin D chase method appear to account for their limited accumulation. The transcription of E2E sequences by RNA polymerase II and III in cells infected by recombinant adenoviruses carrying ectopic E2E-CAT (chloramphenicol transferase) reporter genes with mutations in E2E promoter sequences was also examined. The results of these experiments indicate that recognition of the E2E promoter by the RNA polymerase II transcriptional machinery in infected cells limits transcription by RNA polymerase III, and vice versa. Such transcriptional competition and the properties of E2E RNAs made by RNA polymerase III suggest that the function of this viral RNA polymerase III transcription unit is unusual.
Huang, Wenying, et al.DNA synthesis-dependent relief of repression of transcription from the adenovirus type 2 IVa(2) promoter by a cellular protein.”. Virology 314.1 (2003): , 314, 1, 394-402. Print.Abstract
The promoter of the human adenovirus type 2 IVa(2) gene, which becomes active only during the late phase of infection, is built largely from sequences spanning, and downstream of, the sites of initiation of transcription. These sequences comprise an initiator, an intragenic sequence necessary for efficient transcription from the promoter by RNA polymerase II, and an intragenic binding site for a cellular repressor of IVa(2) transcription. The properties of the latter protein, which is termed IVa(2)-RF, suggested that it might account for the viral DNA synthesis-dependent activation of IVa(2) transcription during the adenoviral productive cycle. Here we report the results of experiments to assess the contributions of DNA template concentration and IVa(2)-RF binding to the activity of the IVa(2) promoter using a transient expression system. When a IVa(2)-EGFP reporter gene was introduced into HeLa cells, in which IVa(2)-RF was identified, no EFGP synthesis could be detected. In contrast, in IVa(2)-RF-containing cells in which the plasmid carrying the chimeric gene replicated, synthesis of both the EGFP protein and the IVa(2)-EGFP mRNA was readily detected. A vector mutation that blocked plasmid replication reduced IVa(2) promoter activity to undetectable levels. In contrast, a IVa(2) promoter substitution that impaired binding of IVa(2)-RF increased IVa(2) promoter activity under all conditions examined. Furthermore, introduction of DNA containing the IV-RF binding site with the chimeric reporter genes resulted in increased transcription from the IVa(2) promoter in the absence of plasmid replication. These properties are consistent with the hypothesis that the relative concentration of the IVa(2) promoter and of the cellular repressor that binds to it governs transcription from this adenoviral promoter.
2002
Gonzalez, Ramon A, and SJ Flint. “Effects of mutations in the adenoviral E1B 55-kilodalton protein coding sequence on viral late mRNA metabolism.”. J Virol 76.9 (2002): , 76, 9, 4507-19. Print.Abstract
The human subgroup C adenoviral E1B 55-kDa protein cooperates with the viral E4 Orf6 protein to induce selective export of viral, late mRNAs from the nucleus to the cytoplasm. Previous studies have suggested that such preferential transport of viral mRNA and the concomitant inhibition of export of cellular mRNAs are the result of viral colonization of specialized microenvironments within the nucleus. However, neither the molecular basis of this phenomenon nor the mechanism by which the E1B 55-kDa protein acts has been elucidated. We therefore examined viral late mRNA metabolism in HeLa cells infected with a series of mutant viruses that carry insertions at various positions in the E1B protein coding sequence (P. R. Yew, C. C. Kao, and A. J. Berk, Virology 179:795-805, 1990). All the mutations examined impaired cytoplasmic accumulation of viral L2 mRNAs and reduced L2 mRNA export efficiency. However, in most cases these defects could be ascribed to reduced E1B 55-kDa protein concentration or the unexpected failure of the altered E1B proteins to enter the nucleus efficiently. The latter property, the pleiotropic defects associated with all the mutations that impaired nuclear entry of the E1B protein, and consideration of its primary sequence suggest that these insertions result in misfolding of the protein. Insertion of four amino acids at residue 143 also inhibited viral mRNA export but resulted in increased rather than decreased accumulation of the E1B 55-kDa protein in the nucleus. This mutation specifically impaired the previously described association of the E1B protein with intranuclear structures that correspond to sites of adenoviral DNA replication and transcription (D. Ornelles and T. Shenk, J. Virol. 65:424-439, 1991) and the colocalization of the E1B and E4 Orf6 proteins. As this insertion has been shown to inhibit the interaction of the E1B with the E4 Orf6 protein in infected cell extracts (S. Rubenwolf, H. Schütt, M. Nevels, H. Wolf, and T. Dobner, J. Virol. 71:1115-1123, 1997), these phenotypes provide direct support for the hypothesis that selective viral mRNA export is determined by the functional organization of the infected cell nucleus.
2001
Brown, LM, et al.Structure of the adenovirus E4 Orf6 protein predicted by fold recognition and comparative protein modeling.”. Proteins 44.2 (2001): , 44, 2, 97-109. Print.Abstract
To facilitate investigation of the molecular and biochemical functions of the adenovirus E4 Orf6 protein, we sought to derive three-dimensional structural information using computational methods, particularly threading and comparative protein modeling. The amino acid sequence of the protein was used for secondary structure and hidden Markov model (HMM) analyses, and for fold recognition by the ProCeryon program. Six alternative models were generated from the top-scoring folds identified by threading. These models were examined by 3D-1D analysis and evaluated in the light of available experimental evidence. The final model of the E4 protein derived from these and additional threading calculations was a chimera, with the tertiary structure of its C-terminal 226 residues derived from a TIM barrel template and a mainly alpha-nonbundle topology for its poorly conserved N-terminal 68 residues. To assess the accuracy of this model, additional threading calculations were performed with E4 Orf6 sequences altered as in previous experimental studies. The proposed structural model is consistent with the reported secondary structure of a functionally important C-terminal sequence and can account for the properties of proteins carrying alterations in functionally important sequences or of those that disrupt an unusual zinc-coordination motif.
Ellsworth, D, RL Finnen, and SJ Flint. “Superimposed promoter sequences of the adenoviral E2 early RNA polymerase III and RNA polymerase II transcription units.”. J Biol Chem 276.1 (2001): , 276, 1, 827-34. Web.Abstract
The human adenovirus type 2 E2 early (E2E) transcriptional control region contains an efficient RNA polymerase III promoter, in addition to the well characterized promoter for RNA polymerase II. To determine whether this promoter includes intragenic sequences, we examined the effects of precise substitutions introduced between positions +2 and +62 on E2E transcription in an RNA polymerase III-specific, in vitro system. Two noncontiguous sequences within this region were necessary for efficient or accurate transcription by this enzyme. The sequence and properties of the functional element proximal to the sites of initiation identified it as an A box. Although a B box sequence could not be unambiguously located, substitutions between positions +42 and +62 that severely impaired transcription also inhibited binding of the human general initiation protein TFIIIC. Thus, this region of the RNA polymerase III E2E promoter contains a B box sequence. We also identified previously unrecognized intragenic sequences of the E2E RNA polymerase II promoter. In conjunction with our previous observations, these data establish that RNA polymerase II and RNA polymerase III promoter sequences are superimposed from approximately positions -30 to +20 of the complex E2E transcriptional control region. The alterations in transcription induced by certain mutations suggest that components of the RNA polymerase II and RNA polymerase III transcriptional machines compete for access to overlapping binding sites in the E2E template.
2000
Lin, HJ, and SJ Flint. “Identification of a cellular repressor of transcription of the adenoviral late IVa(2) gene that is unaltered in activity in infected cells.”. Virology 277.2 (2000): , 277, 2, 397-410. Web.Abstract
The gene encoding the adenovirus type 2 IVa(2) protein, a sequence-specific activator of transcription from the viral major late promoter, is itself transcribed only during the late phase of infection. We previously identified a cellular protein (IVa(2)-RF) that binds specifically to an intragenic sequence of the IVa(2) transcription unit. We now report that precise substitutions within the IVa(2)-RF-binding site that decreased binding affinity increased the efficiency of IVa(2) transcription in in vitro reactions containing IVa(2)-RF. Consistent with the conclusion that this cellular protein represses IVa(2) transcription, mutations that led to more efficient transcription in the presence of IVa(2)-RF were without effect in reactions lacking this cellular protein. No change in the concentration or activity of IVa(2)-RF could be detected in adenovirus-infected cells during the period in which the IVa(2) gene is transcribed. We therefore propose that restriction of IVa(2) transcription to the late phase is the result of titration of this cellular repressor as the number of copies of the IVa(2) promoter increases upon replication of the viral genome.
1998
Huang, W, and SJ Flint. “The tripartite leader sequence of subgroup C adenovirus major late mRNAs can increase the efficiency of mRNA export.”. J Virol 72.1 (1998): , 72, 1, 225-35. Print.Abstract
The subgroup C human adenoviruses induce selective export of newly synthesized viral mRNA from the nucleus to the cytoplasm, with concomitant inhibition of export of the majority of cellular mRNA species. Such posttranscriptional regulation of viral and cellular gene expression in infected cells requires viral E1B and E4 proteins. To facilitate the investigation of parameters that govern selective export in adenovirus-infected cells, we constructed a marked human beta-actin minigene under the control of the glucocorticoid-inducible enhancer-promoter of mouse mammary tumor virus and introduced it into the left end of the adenovirus type 5 (Ad5) genome. Transcription of this reporter gene (designated MA) as well as of a sibling, which differed only in the inclusion of a cDNA copy of the Ad2 major late tripartite leader sequence upstream of beta-actin sequences (termed MtplA), in recombinant virus-infected cells was strictly dependent on the addition of dexamethasone to the medium. When transcription of the MA gene was induced during the late phase of infection, newly synthesized MA RNA entered the cytoplasm. These transcripts, which contain no viral sequences, therefore reproduce the behavior of exceptional cellular mRNA species observed when transcription of their genes is activated during the late phase of infection (U.-C. Yang, W. Huang, and S. J. Flint, J. Virol. 70:4071-4080, 1996). Unexpectedly, however, higher concentrations of newly synthesized RNA accumulated in the cytoplasm when the tripartite leader sequence was present in the reporter RNA, despite equal rates of transcription of the two reporter genes. Examination of the partitioning of both newly synthesized and steady-state populations of MA and MtplA RNAs between nuclear and cytoplasmic compartments indicated that the tripartite leader sequence did not increase RNA stability in the cytoplasm. Comparison of nuclear and cytoplasmic reporter RNA species by Northern blotting, primer extension, and reverse transcription-PCR provided no evidence for altered processing induced by the tripartite leader sequence. We therefore conclude that the tripartite leader sequence, long known to facilitate the translation of mRNAs during the late phase of adenovirus infection, can also modulate mRNA export from the nucleus.
1996
Yang, UC, W Huang, and SJ Flint. “mRNA export correlates with activation of transcription in human subgroup C adenovirus-infected cells.”. J Virol 70.6 (1996): , 70, 6, 4071-80. Print.Abstract
To investigate the mechanisms by which viral mRNA species are distinguished from their cellular counterparts for export to the cytoplasm during the late phase of subgroup C adenovirus infection, we have examined the metabolism of several cellular and viral mRNAs in human cells productively infected by adenovirus type 5 (Ad5). Several cellular mRNAs that were refractory to, or could escape from, adenovirus-induced inhibition of export of mRNA from the nucleus have been identified. This group includes Hsp70 mRNAs synthesized upon heat shock of Ad5-infected 293 or HeLa cells during the late phase of infection. However, successful export in Ad5-infected cells is not a specific response to heat shock, for beta-tubulin and interferon-inducible mRNAs were also refractory to virus-induced export inhibition. The export of these cellular mRNAs, like that of viral late mRNAs, required the E1B 55-kDa protein. Export to the cytoplasm during the late phase of Ad5 infection of several cellular mRNAs, including members of the Hsp70 family whose export was inhibited under some, but not other, conditions, indicates that viral mRNA species cannot be selectively exported by virtue of specific sequence or structural features. Cellular and viral late mRNAs that can be exported from the nucleus to the cytoplasm were expressed from genes whose transcription was induced or activated during the late phase of Ad5 infection. Consistent with the possibility that successful export is governed by transcriptional activation in the late phase of adenovirus infection, newly synthesized viral early E1A mRNA was subject to export inhibition during the late phase of infection.
1995
Smiley, JK, et al.The metabolism of small cellular RNA species during productive subgroup C adenovirus infection.”. Virology 206.1 (1995): , 206, 1, 100-7. Print.Abstract
During the late phase of subgroup C adenovirus infection, export of cellular mRNA from the nucleus to the cytoplasm is inhibited. In one approach to investigate the mechanism whereby viral late mRNAs are selected for export, we have examined the metabolism of small cellular RNA species transcribed by all three RNA polymerases during the late phase of Ad5 infection. No changes in the quantities of [3H]uridine-labeled 5S rRNA or tRNAs entering the cytoplasm were observed in infected cells. Adenovirus type 5 infection reduced the nuclear and cytoplasmic populations of the newly synthesized, snRNP-associated snRNAs U1, U2, U4, U5, and U6. Transcription of a representative snRNA, U1 RNA, was not inhibited, indicating that the post-transcriptional metabolism of snRNAs was perturbed during the late phase of infection. The increased cytoplasmic concentration of newly synthesized U1 RNA in Ad5- compared to mock-infected cells, and the greater reduction of the snRNP-associated compared to the total U1 RNA population, indicated that snRNP assembly in the cytoplasm was impaired. As adenovirus infection does not perturb export from the nucleus of small cellular mRNAs transcribed by RNA polymerases II and III, viral mRNA must be distinguished for selective export at a nuclear step upstream of translocation to the cytoplasm via nuclear pore complexes.
Pruzan, R, and SJ Flint. “Transcription of adenovirus RNA polymerase III genes.”. Curr Top Microbiol Immunol 199 ( Pt 1) (1995): , 199 ( Pt 1), 201-26. Print.
1994
Huang, W, R Pruzan, and SJ Flint. “In vivo transcription from the adenovirus E2 early promoter by RNA polymerase III.”. Proc Natl Acad Sci U S A 91.4 (1994): , 91, 4, 1265-9. Print.Abstract
We have previously reported that the subgroup C adenovirus E2 early (E2E) RNA polymerase II promoter can specify efficient in vitro transcription by RNA polymerase III. We now show that promoter proximal sequences of the E2E transcription unit are also transcribed by RNA polymerase III in nuclei isolated from adenovirus-infected cells. Small E2E RNA species that possessed the same properties as in vitro synthesized RNA polymerase III E2E transcripts were detected in cytoplasmic RNA populations from infected cells by using blotting, primer extension, and RNase protection assays. The 3' termini of these RNAs were mapped to thymidine-rich sequences typical of RNA polymerase III termination sites. These results demonstrate that a single gene can be transcribed by both RNA polymerase II and RNA polymerase III in vivo.
Chen, H, R Vinnakota, and SJ Flint. “Intragenic activating and repressing elements control transcription from the adenovirus IVa2 initiator.”. Mol Cell Biol 14.1 (1994): , 14, 1, 676-85. Print.Abstract
The downstream stimulatory segment of the adenovirus type 2 IVa promoter includes a TA-rich sequence that binds recombinant TATA-binding proteins (TBP) in vitro. We now demonstrate that when placed upstream of the IVa2, initiator, this TA-rich sequence operated as a TATA element but exhibited significantly lower transcriptional and TBP-binding activities than did the TATA box of the adenovirus major late (ML) promoter. In sharp contrast, changing the IVa2 TA-rich sequence in its natural, intragenic context to the ML TATA sequence increased the activity of the IVa2 promoter only slightly. In view of this discrepancy, we examined the effects of single, double, and clustered point mutations in the downstream sequence on the activity of a minimal IVa2 promoter. Mutations between positions +21 and +29 inhibited IVa2 transcription, in some cases to the very low level directed by the IVa2 initiator alone. By contrast, substitutions within the TA-rich sequence increased the efficiency of IVa2 transcription. These results indicated that the downstream, TA-rich sequence does not function as an intragenic TFIID-binding site but rather is included within a negative regulatory element. Electrophoretic mobility shift and methylation interference assays using wild-type and mutated, intragenic promoter sequences identified a HeLa cell component whose binding to the sequence +11 to +27 correlated with repression of IVa2 transcription, suggesting that a negative regulatory element is superimposed upon the intragenic sequence required for efficient transcription from the IVa2 initiator.
1993
Postel, EH, et al.Human c-myc transcription factor PuF identified as nm23-H2 nucleoside diphosphate kinase, a candidate suppressor of tumor metastasis.”. Science 261.5120 (1993): , 261, 5120, 478-80. Print.Abstract
A human gene encoding the c-myc purine-binding transcription factor PuF was identified by screening of a cervical carcinoma cell complementary DNA library with a DNA fragment containing PuF binding sites. The 17-kilodalton bacterially produced PuF was shown to have biological activity and properties similar to that of human PuF. DNA sequence analysis of recombinant PuF revealed perfect identity with the human nm23-H2 nucleoside diphosphate kinase gene, a potential negative regulator of cancer metastasis. These results provide a link between nm23 and the c-myc oncogene and suggest that the nm23 protein can function in vitro in the transcriptional regulation of c-myc expression.
Chatterjee, PK, R Pruzan, and SJ Flint. “Purification of an active TATA-binding protein-containing factor using a monoclonal antibody that recognizes the human TATA-binding protein.”. Protein Expr Purif 45 (1993): , 4, 5, 445-55. Web.Abstract
The human TATA-binding protein was expressed in Escherichia coli as a fusion with an N-terminal hexahistidine sequence, partially purified, and used to raise monoclonal antibodies. More than 50 hybridoma clones producing antibodies that reacted in immunoblot assays with HeLa cell TATA-binding protein and its bacterially synthesized derivative were identified. All antibodies examined recognized epitopes within the N-terminal 159 amino acids of the human TATA-binding protein. Further characterization of one monoclonal antibody, MTBP-6, established that it immunoprecipitates both native HeLa cell TATA-binding protein and TATA-binding protein extracted from cells in the presence of 0.5% SDS. Antibody MTBP-6 immunoprecipitates of native, human cell TATA-binding protein contained the TATA-binding protein and additional polypeptides. Immunoprecipitation of both the TATA-binding protein and several additional polypeptides was specifically blocked by bacterially synthesized, hexahistidine-tagged TATA-binding protein, suggesting that MTBP-6 can efficiently recognize the TATA-binding protein in TFIID and other complexes. Consistent with this conclusion, immunoaffinity chromatography on antibody MTBP-6 permitted purification, in active form, of a TATA-binding protein-containing factor required for transcription by RNA polymerase III. These properties suggest that MTBP-6 will be a useful reagent for the purification and characterization of the multiple TBP-containing complexes present in human cells.
Riley, D, and SJ Flint. “RNA-binding properties of a translational activator, the adenovirus L4 100-kilodalton protein.”. J Virol 67.6 (1993): , 67, 6, 3586-95. Print.Abstract
The adenovirus L4 100-kDa nonstructural protein (100K protein) is required for efficient initiation of translation of viral late mRNA species during the late mRNA species during the late phase of infection (B. W. Hayes, G. C. Telling, M. M. Myat, J. F. Williams, and S. J. Flint, J. Virol. 64:2732-2742, 1990). The RNA-binding properties of this protein were analyzed in an immunoprecipitation assay with the 100K-specific monoclonal antibody 2100K-1 (C. L. Cepko and P. A. Sharp, Virology 129:137-154, 1983). Coprecipitation of the 100K protein and 3H-infected cell RNA was demonstrated. The RNA-binding activity of the 100K protein was inhibited by single-stranded DNA but not by double-stranded DNA, double-stranded RNA, or tRNA. Competition assays were used to investigate the specificity with which the 100K protein binds to RNA in vitro. Although the protein exhibited a strong preference for the ribohomopolymer poly(U) or poly(G), no specific binding to viral mRNA species could be detected; uninfected or adenovirus type 5-infected HeLa cell poly(A)-containing and poly(A)-lacking RNAs were all effective inhibitors of binding of the protein to viral late mRNA. Similar results were obtained when the binding of the 100K protein to a single, in vitro-synthesized L2 mRNA was assessed. The poly(U)-binding activity of the 100K protein was used to compare the RNA-binding properties of the 100K protein prepared from cells infected by adenovirus type 5 and the H5ts1 mutant (B. W. Hayes, G. C. Telling, M. M. Myat, J. F. Williams, and S. J. Flint, J. Virol. 64:2732-2742, 1990). A temperature-dependent decrease in H5ts1 100K protein binding was observed, correlating with the impaired translational function of this protein in vivo. By contrast, wild-type 100K protein RNA binding was unaffected by temperature. These data suggest that the 100K protein acts to increase the translational efficiency of viral late mRNA species by a mechanism that involves binding to RNA.
1992
Kasai, Y, H Chen, and SJ Flint. “Anatomy of an unusual RNA polymerase II promoter containing a downstream TATA element.”. Mol Cell Biol 12.6 (1992): , 12, 6, 2884-97. Print.Abstract
The adenovirus type 2 IVa2 promoter lacks a conventional TATA element yet directs transcription from two closely spaced initiation sites. To define elements required for in vitro transcription of this promoter, IVa2 templates carrying 5' deletions or linker-scanning mutations were transcribed in HeLa whole-cell extracts and the transcripts were analyzed by primer extension. Mutation of the sequence centered on position -47, which is specifically recognized by a cellular factor, reduced the efficiency of IVa2 transcription two- to threefold, whereas mutation of the sequence centered on position -30 selectively impaired utilization of the minor in vivo initiation site. Utilization of the major in vivo site was decreased no more than fivefold by deletion of all sequences upstream of position -15. By contrast, mutation of the region from +13 to +19 or of the initiation region severely impaired IVa2 transcription. The sequence spanning the initiation sites was sufficient to direct accurate initiation by RNA polymerase II from the major in vivo site. Thus, the two initiation sites of the IVa2 promoter are specified by independent elements, and a downstream element is the primary determinant of efficient transcription from both of these sites. The downstream element identified by mutational analysis altered the TATA element-like sequence TATAGAAA lying at positions +21 to +14 in the coding strand. Transcription from the wild-type IVa2 promoter was severely inhibited when endogenous TFIID was inactivated by mild heat treatment. Exogenous human TATA-binding protein (TBP) synthesized in Escherichia coli restored specific IVa2 transcription from both initiation sites when added to such heat-treated extracts. Although efficient IVa2 transcription requires both the downstream TATA sequence and active TFIID, bacterially synthesized TBP also stimulated the low level of IVa2 transcription observed when the TATA sequence was mutated to a sequence that failed to bind TBP.
Li, XC, WL Huang, and SJ Flint. “The downstream regulatory sequence of the adenovirus type 2 major late promoter is functionally redundant.”. J Virol 66.9 (1992): , 66, 9, 5685-90. Print.Abstract
Mutagenesis of promoter sequences and oligonucleotide competition assays have been used to demonstrate the late-phase-specific stimulation of the adenovirus type 2 major late promoter is mediated by functionally redundant elements located between positions +75 and +125. These octamer motif-related sequences are recognized by multiple factors.
Chen, H, and SJ Flint. “Mutational analysis of the adenovirus 2 IVa2 initiator and downstream elements.”. J Biol Chem 267.35 (1992): , 267, 35, 25457-65. Print.Abstract
The initiator element of the adenovirus type 2 IVa2 promoter is sufficient to direct accurate initiation by RNA polymerase II. Analysis of the effects of substitution of specific base pairs on initiator activity in in vitro transcription systems indicated that specific sequences between positions -4 and +5 were essential for initiator activity. Mutations that impaired or eliminated initiator activity altered both base pairs that are conserved in sequence-related initiators and nonconserved sequences. Neither the downstream TA-rich sequence of the IVa2 promoter, nor the adenovirus 2 major late TATA element placed at the same downstream site could overcome the severe inhibitory effects of initiator mutations, indicating that the initiator is the primary determinant of the specificity and direction of IVa2 transcription. By contrast, when the ML TATA element was placed 31 nucleotides upstream of the IVa2 initiator, the precise specificity, but neither the efficiency nor direction of transcription, depended on the presence of a functional initiator. Activity of the IVa2 promoter was relatively insensitive to changes in the orientation or nature of the TA-rich sequence. Furthermore, only a promoter containing the ML TA-TAAAA sequence downstream of the IVa2 initiator was competent to direct both IVa2 transcription and transcription from the opposite strand. The implications of this functional difference for recognition of the downstream element are discussed.
Pruzan, R, PK Chatterjee, and SJ Flint. “Specific transcription from the adenovirus E2E promoter by RNA polymerase III requires a subpopulation of TFIID.”. Nucleic Acids Res 20.21 (1992): , 20, 21, 5705-12. Print.Abstract
The early E2 (E2E) promoter of adenovirus type 2 possesses a TATA-like element and binding sites for the factors E2F and ATF. This promoter is transcribed by RNA polymerase II in high salt nuclear extracts, but by RNA polymerase III in standard nuclear extracts, as judged by sensitivity to low and high, respectively, concentrations of alpha-amanitin. Transcription by the two RNA polymerases initiated at the same site and depended, in both cases, on the TATA-like sequence and upstream elements. However, RNA polymerase III transcripts, unlike those synthesized by RNA polymerase II, terminated at two runs of Ts downstream of the initiation site. Although they are not essential, sequences downstream of the initiation site increased the efficiency of E2E transcription by RNA polymerase III. Such RNA polymerase III dependent transcription required a subpopulation of the general transcription factor, TFIID: TFIID that binds weakly to phosphocellulose (0.3 M eluate) complemented a TFIID-depleted extract to restore RNAp III transcription, whereas TFIID tightly associated with phosphocellulose (1 M eluate) was unable to do so.
1991
Postel, EH, et al.Evidence that a triplex-forming oligodeoxyribonucleotide binds to the c-myc promoter in HeLa cells, thereby reducing c-myc mRNA levels.”. Proc Natl Acad Sci U S A 88.18 (1991): , 88, 18, 8227-31. Print.Abstract
A synthetic 27-base-long oligodeoxyribonucleotide, termed PU1, has been shown to bind to duplex DNA to form a triplex at a single site within the human c-myc P1 promoter. PU1 has been administered to HeLa cells in culture to examine the feasibility of influencing transcription of the c-myc gene in vivo. It is shown that uptake of PU1 into the nucleus of HeLa cells is efficient and that the compound remains intact for at least 4 hr. In nuclei extracted from PU1-treated cells, inhibition of DNase I cleavage is detected within the c-myc P1 promoter at the target site for triplex formation. The inhibition is shown to be both site and oligodeoxyribonucleotide specific. After cellular uptake of PU1, it is shown that steady-state mRNA arising from the c-myc P1 initiation site is selectively reduced relative to total mRNA, relative to mRNA from the alternative c-myc P2 initiation site, and relative to mRNA derived from the beta-actin promoter. Significant mRNA repression is not seen upon treating cells with oligodeoxyribonucleotides that fail to bind to the P1 promoter target. Taken together, these data suggest that triplex formation can occur between an exogenous oligodeoxyribonucleotide and duplex DNA in the nucleus of treated cells.
1990
Hayes, BW, et al.The adenovirus L4 100-kilodalton protein is necessary for efficient translation of viral late mRNA species.”. J Virol 64.6 (1990): , 64, 6, 2732-42. Print.Abstract
When screening a number of adenovirus type 5 (Ad5) temperature-sensitive mutants for defects in viral gene expression, we observed that H5ts1-infected 293 cells accumulated reduced levels of newly synthesized viral late proteins. Pulse-labeling and pulse-chase experiments were used to establish that the late proteins synthesized in H5ts1-infected cells under nonpermissive conditions were as stable as those made in Ad5-infected cells. H5ts1-infected cells contained normal levels of viral late mRNAs. Because these observations implied that translation of viral mRNA species was defective in mutant virus-infected cells, the association of viral late mRNAs with polyribosomes was examined during the late phase of infection at a nonpermissive temperature. In Ad5-infected cells, the majority of the viral L2, L3, L4, pIX, and IVa2 late mRNA species were polyribosome bound. By contrast, these same mRNA species were recovered from H5ts1-infected cells in fractions nearer the top of polyribosome gradients, suggesting that initiation of translation was impaired. During the late phase of infection, neither the polyribosome association nor the translation of most viral early mRNA species was affected by the H5ts1 mutation. This lesion, mapped by marker rescue to the L4 100-kilodalton (kDa) nonstructural protein, has been identified as a single base pair substitution that replaces Ser-466 of the Ad5 100-kDa protein with Pro. A set of temperature-independent revertants of H5ts1 was isolated and characterized. Either true reversion of the H5ts1 mutation or second-site mutation of Pro-466 of the H5ts1 100-kDa protein to Thre, Leu, or His restored both temperature-independent growth and the efficient synthesis of viral late proteins. We therefore conclude that the Ad5 L4 100-kDa protein is necessary for efficient initiation of translation of viral late mRNA species during the late phase of infection.
Smiley, JK, MA Young, and SJ Flint. “Intranuclear location of the adenovirus type 5 E1B 55-kilodalton protein.”. J Virol 64.9 (1990): , 64, 9, 4558-64. Print.Abstract
The intracellular location of the adenovirus type 5 E1B 55-kilodalton (kDa) protein, particularly the question of whether it is associated with nuclear pore complexes, was examined. Fractionation of adenovirus type 5-infected HeLa cell nuclei by an established procedure (N. Dwyer and G. Blobel, J. Cell. Biol. 70:581-591, 1976) yielded one population of E1B 55-kDa protein molecules released by digestion of nuclei with RNase A and a second population recovered in the pore complex-lamina fraction. Free and E1B 55-kDa protein-bound forms of the E4 34-kDa protein (P. Sarnow, C. A. Sullivan, and A. J. Levine, Virology 120:387-394, 1982) were largely recovered in the pore complex-lamina fraction. Nevertheless, the association of E1B 55-kDa protein molecules with this nuclear envelope fraction did not depend on interaction of the E1B 55-kDa protein with the E4 34-kDa protein. Comparison of the immunofluorescence patterns observed with antibodies recognizing the E1B 55-kDa protein or cellular pore complex proteins and of the behavior of these viral and cellular proteins during in situ fractionation suggests that the E1B 55-kDa protein does not become intimately or stably associated with pore complexes in adenovirus-infected cells.
1989
Postel, EH, SE Mango, and SJ Flint. “A nuclease-hypersensitive element of the human c-myc promoter interacts with a transcription initiation factor.”. Mol Cell Biol 911 (1989): , 9, 11, 5123-33. Print.Abstract
Transcription of the human c-myc oncogene is elaborately regulated, but the relevant molecular mechanisms are not yet understood. To begin to define elements and enzyme systems responsible for c-myc transcription in vitro, we partially purified a transcription factor essential for efficient and accurate in vitro initiation from the principal myc promoter, P2. DNA mobility shift assays located the factor binding domain at -142 to -115 with respect to the P1 promoter. This region contains pur/pyr sequences (predominantly purines in one strand), nuclease-hypersensitive sites (U. Siebenlist, L. Henninghausen, J. Battey, and P. Leder, Cell 37:381-391, 1984; C. Boles and M. Hogan, Biochemistry 26:367-376, 1987), and a triple-helix-forming element (M. Cooney, G. Czernuszewicz, E. Postel, S. Flint, and M. Hogan, Science 241:456-459, 1988). Methylation interference mapping established that the factor, termed PuF, directly contacts the repeated palindromic sequence GGGTGGG of the -142/-115 element. The interaction of PuF with this cis-acting element is necessary for P2 transcription in vitro, for (i) deletion of this 5' region from the myc promoter greatly reduced transcription efficiency and (ii) a synthetic duplex oligonucleotide corresponding to the -142/-115 sequence completely repressed c-myc transcription in the presence of the partially purified factor. These observations lend support to the hypothesis that pur/pyr sequences perform important biological roles in the regulation of c-myc gene expression, most likely by serving as transcription factor binding sites.
1988
Chatterjee, PK, et al.DNA-binding properties of an adenovirus 289R E1A protein.”. EMBO J 73 (1988): , 7, 3, 835-41. Print.Abstract
An adenovirus 2 289 amino acid (289R) E1A protein purified from Escherichia coli has been shown to interact with DNA by two independent methods. UV-crosslinking of complexes containing unmodified, uniformly 32P-labelled DNA and purified E1A protein induced efficient labelling of the protein with covalently attached oligonucleotides, indicating that the E1A protein itself contacts DNA. Discrete nucleoprotein species were also observed when E1A protein--DNA complexes were analysed by gel electrophoresis. Although the 289R E1A protein exhibited no significant binding to single-stranded DNA or to RNA, no evidence for its sequence-specific binding to double-stranded DNA was obtained with either assay. Identification of the sites of covalent attachment of 32P-labelled oligonucleotides by partial proteolysis of the crosslinked E1A protein indicated that the interaction of this protein with DNA is mediated via domain(s) in the C-terminal half of the protein. Such previously unrecognized DNA-binding activity is likely to contribute to the regulatory activities of this important adenoviral protein.
Lunt, R, et al.Isolation and characterization of monoclonal antibodies against the adenovirus core proteins.”. Virology 164.1 (1988): , 164, 1, 275-9. Print.Abstract
Monoclonal antibodies have been prepared that recognize adenovirus core proteins V, VII, and mu in ELISA and Western blot assays. Antibodies produced by all of 87 positive hybridoma colonies obtained from a mouse injected with the precursor to protein VII, pVII, produced antibodies that also reacted with purified protein VII in an ELISA assay and all tested recognized denatured protein VII immobilized on nitrocellulose. Such failure to recover antibodies that specifically recognized only protein pVII suggests that epitopes common to the 174 amino acid protein VII and its 197 amino acid precursor were more effective antigenic determinants than the N-terminal 23 amino acid segment unique to pVII. All antibodies raised against protein mu cross-reacted with protein VII in both assays, but only a small fraction of the anti-protein VII or pVII antibodies recognized protein mu. Such cross-reactivity is discussed in relation to an unusual, arginine-rich sequence present in both protein VII and protein mu.
Cooney, M, et al.Site-specific oligonucleotide binding represses transcription of the human c-myc gene in vitro.”. Science 241.4864 (1988): , 241, 4864, 456-9. Print.Abstract
A 27-base-long DNA oligonucleotide was designed that binds to duplex DNA at a single site within the 5' end of the human c-myc gene, 115 base pairs upstream from the transcription origin P1. On the basis of the physical properties of its bound complex, it was concluded that the oligonucleotide forms a colinear triplex with the duplex binding site. By means of an in vitro assay system, it was possible to show a correlation between triplex formation at -115 base pairs and repression of c-myc transcription. The possibility is discussed that triplex formation (site-specific RNA binding to a DNA duplex) could serve as the basis for an alternative program of gene control in vivo.