E1B Protein

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S. J. Flint - One of the best experts on this subject based on the ideXlab platform.

  • the human adenovirus type 5 E1B 55 kda Protein obstructs inhibition of viral replication by type i interferon in normal human cells
    PLOS Pathogens, 2012
    Co-Authors: Jasdave S Chahal, S. J. Flint
    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.

  • Role of the RNA recognition motif of the E1B 55 kDa Protein in the adenovirus type 5 infectious cycle
    Virology, 2011
    Co-Authors: Sayuri E.m. Kato, Wenying Huang, S. J. Flint
    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.

  • the adenoviral E1B 55 kilodalton Protein controls expression of immune response genes but not p53 dependent transcription
    Journal of Virology, 2009
    Co-Authors: D Miller, Wenying Huang, Brenden Rickards, Michael Mashiba, S. J. Flint
    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.

  • a peptide inhibitor of exportin1 blocks shuttling of the adenoviral E1B 55 kda Protein but not export of viral late mrnas
    Virology, 2005
    Co-Authors: S. J. Flint, Wenying Huang, Joseph Goodhouse, Saw Kyin
    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.

  • effects of mutations in the adenoviral E1B 55 kilodalton Protein coding sequence on viral late mrna metabolism
    Journal of Virology, 2002
    Co-Authors: Ramon A Gonzalez, S. J. Flint
    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. Schutt, 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.

David A. Ornelles - One of the best experts on this subject based on the ideXlab platform.

  • p53 status does not determine outcome of E1B 55 kilodalton mutant adenovirus lytic infection
    Journal of Virology, 1998
    Co-Authors: Felicia Goodrum, David A. Ornelles
    Abstract:

    The early region 1B (E1B) of adenovirus type 5 (Ad5) encodes overlapping functions essential for virus mediated cellular transformation in cooperation with E1A by suppressing p53-mediated apoptosis or a G1 growth arrest (52, 62). The E1A Proteins are potent transactivators that relieve cellular growth suppression and induce quiescent cells to enter S phase by binding members of the retinoblastoma Protein family and transcription factors such as p300 (reviewed in reference 17). This action of E1A results in the accumulation of p53. p53 is a cellular growth suppressor that acts as a G1 checkpoint control (reviewed in references 37 and 74). In response to viral challenge, p53 may induce a G1 growth arrest by inducing genes such as the cyclin-dependent kinase inhibitor p21/WAF1/Cip1 gene (16, 69) or apoptosis by inducing genes such as bax1 (43). Either response by p53 is expected to severely hinder Ad replication and transformation (38). The E1B 55-kDa and 19-kDa Proteins are required to fully transform cells (4, 19, 23, 56, 64). The E1B 19-kDa Protein is a functional homologue of the proto-oncogene-encoded Bcl-2 and prevents apoptosis by similar mechanisms (12, 52). The E1B 55-kDa Proteins of Ad5 and Ad12 have been hypothesized to permit E1A-induced DNA synthesis and transactivation by preventing a p53-mediated G1 growth arrest (51, 60, 61). The 55-kDa Protein complexes with the amino-terminal end of p53 and inhibits its activity as a transcription factor (33, 72, 73). This inhibition of p53-mediated transactivation by the large E1B Protein is required for transformation by both the weakly oncogenic group C and highly oncogenic group A Ads (33, 72, 75, 76). E1B 55-kDa Protein-mediated inactivation of p53 has been hypothesized to be required for viral replication in the lytic infection (7); however, this has not been demonstrated. The Ad E4orf6 Protein was recently shown to bind p53 and block transcriptional activation mediated by p53 (14, 46). Subsequently, the E4orf6 Protein was shown to cooperate with the E1A and E1B Proteins to transform baby rat kidney cells (46), to convert the nontumorigenic 293 human cell line (24) into a tumorigenic cell line in nude mice, and to block p53-dependent apoptosis (44). In both transformed and productively infected cells, coexpression of the E1B 55-kDa and the E4orf6 Proteins decreased the stability of p53 (44, 46, 50). These findings suggest that the E4orf6 Protein may encode some overlapping and redundant functions with the E1B 55-kDa Protein with regard to transformation. At late times in the lytic infection, the E1B 55-kDa Protein facilitates the transport of viral late mRNA while inhibiting the transport of most cellular mRNA in association with the E4orf6 Protein (3, 8, 26, 36, 49). Ad mutants that fail to express the E1B 55-kDa Protein are defective for expression of late viral Proteins and replicate poorly. The functional interaction between the E1B 55-kDa and E4orf6 Proteins may be mediated by primate-specific cellular factors (22, 47). The transport of several cellular messages, including the heat shock Protein 70, β-tubulin, and interferon-inducible Mx-A and 6-16 mRNAs, requires the E1B 55-kDa Protein late in Ad infection. This effect correlates with activation of their transcription during the late phase (45, 71). In addition to selectively blocking transport of most cellular mRNA, the E1B 55-kDa Protein further modulates host cell shutoff by inhibiting host Protein synthesis by mechanisms unrelated to the inhibition of mRNA transport (2). We have recently demonstrated that the E1B 55-kDa Protein functions in promoting Ad replication independently of the cell cycle. E1B 55-kDa mutant Ads produce virus most efficiently when cells are infected during S phase and are restricted from replication in cells infected during G1 (20). These findings suggest that the E1B 55-kDa Protein plays a role in deregulating the cell cycle to the advantage of the lytic infection. Perhaps this function represents a link between the functions of the E1B 55-kDa Protein in the lytic infection and transformation. Because the E1B 55-kDa Protein inhibits the function of p53, it has been hypothesized that an E1B 55-kDa mutant Ad can replicate only in cells lacking a functional p53. Evidence supporting this hypothesis includes the finding that the E1B 55-kDa mutant Ad dl1520 failed to lyse U2OS cells which contain wild-type p53. Furthermore, the E1B 55-kDa mutant virus induced more severe cytopathic effect in the RKO human colon cancer cell line transfected with a dominant negative p53 gene than in the parental cell line containing a wild-type p53 gene (7). However, p53 null tumor cells were not more susceptible to lysis by the E1B 55-kDa mutant virus than some cancer cells containing wild-type p53 (28). Furthermore, the relative ability of the E1B 55-kDa mutant virus to suppress tumor growth compared to the wild-type Ad was unaffected by the status of p53 (7). By contrast, Ridgway et al. (54) suggest that the interaction between p53 and the E1B 55-kDa Protein is necessary for efficient Ad replication and that neither the wild-type nor E1B 55-kDa mutant virus replicated in p53-mutant cell lines. These authors reported that the wild-type Ad failed to efficiently shut off α-actin synthesis, synthesize viral DNA, and induce cytopathic effect in the p53 mutant cell lines T98G and 143B. They suggest that p53 may mediate the interaction between the E1B 55-kDa and E4orf6 Proteins in the lytic infection (54). However, Rubenwolf et al. (55) demonstrated that p53 is not required for coimmunoprecipitation of the E1B 55-kDa and E4orf6 Proteins although the two Proteins have been shown to bind p53 independently (14, 55, 57). The work presented here demonstrates that the inability of the E1B mutant virus to replicate efficiently and produce virus in all infected cells is not strictly due to the failure to abrogate p53 function. Among a variety of cell lines analyzed, the capacity of the E1B 55-kDa mutant virus to replicate, synthesize viral DNA, and produce virus in all infected cells did not correlate with the status of p53. However, the ability of the E1B 55-kDa mutant virus to replicate differed among the cell types studied. Furthermore, the ability of the E1B 55-kDa mutant virus to induce cell killing correlated with permissivity to virus growth and not the status of p53. In a cell line expressing a temperature-sensitive p53 allele, active p53 only moderately affected the replication of the E1B 55-kDa mutant virus. The defect in E1B 55-kDa mutant virus replication due to reduced temperature was as much as 10-fold greater than the defect due to p53 function. Indeed, the E1B 55-kDa mutant virus produced equivalent yields of virus and synthesized equivalent levels of late viral Proteins compared to the wild-type virus in cells maintained at 39°C but not at 32°C. The cell cycle restriction of the E1B 55-kDa mutant virus was also partially overcome at 39°C, and progeny virus were produced in a larger fraction of infected cells. By contrast, the apparent cell cycle restriction was exacerbated at 32°C. Since both the cell cycle restriction and the defect in late gene expression exhibited a cold-sensitive phenotype, we speculate that the functions of the E1B 55-kDa Protein in promoting mRNA transport and cell cycle-independent viral replication may be linked.

  • Adenovirus early region 4 34-kilodalton Protein directs the nuclear localization of the early region 1B 55-kilodalton Protein in primate cells.
    Journal of virology, 1996
    Co-Authors: Felicia Goodrum, Thomas Shenk, David A. Ornelles
    Abstract:

    The localization of the adenovirus type 5 34-kDa E4 and 55-kDa E1B Proteins was determined in the absence of other adenovirus Proteins. When expressed by transfection in human, monkey, hamster, rat, and mouse cell lines, the E1B Protein was predominantly cytoplasmic and typically was excluded from the nucleus. When expressed by transfection, the E4 Protein accumulated in the nucleus. Strikingly, when coexpressed by transfection in human, monkey, or baby hamster kidney cells, the E1B Protein colocalized in the nucleus with the E4 Protein. A complex of the E4 and E1B Proteins was identified by coimmunoprecipitation in transfected HeLa cells. By contrast to the interaction observed in primate and baby hamster kidney cells, the E4 Protein failed to direct the E1B Protein to the nucleus in rat and mouse cell lines as well as CHO and V79 hamster cell lines. This failure of the E4 Protein to direct the nuclear localization of the E1B Protein in REF-52 rat cells was overcome by fusion with HeLa cells. Within 4 h of heterokaryon formation and with Protein synthesis inhibited, a portion of the E4 Protein present in the REF-52 nuclei migrated to the HeLa nuclei. Simultaneously, the previously cytoplasmic E1B Protein colocalized with the E4 Protein in both human and rat cell nuclei. These results suggest that a primate cell-specific factor mediates the functional interaction of the E1B and E4 Proteins of adenovirus.

  • localization of the adenovirus early region 1b 55 kilodalton Protein during lytic infection association with nuclear viral inclusions requires the early region 4 34 kilodalton Protein
    Journal of Virology, 1991
    Co-Authors: David A. Ornelles, Thomas Shenk
    Abstract:

    Abstract The distribution of the adenovirus early region 1B 55-kDa Protein (E1B-55kDa) in lytically infected HeLa cells was determined. At the time of infection, when the E1B-55kDa Protein facilitates the cytoplasmic accumulation of viral mRNA while simultaneously restricting the accumulation of most cellular mRNA, five distinct intracellular localizations of the Protein were observed. Only one of these was disrupted when cells were infected with a mutant virus that fails to produce a second viral Protein encoded by early region 4 (E4-34kDa). This Protein normally forms a complex with the E1B-55kDa polypeptide, enabling it to influence RNA metabolism. This key localization of the E1B Protein was within and about the periphery of nuclear viral inclusion bodies believed to be the site of viral DNA replication and transcription. In the absence of the E4-34kDa Protein, the coincidence of E1B-55kDa-specific immunofluorescence and phase-dense viral inclusions was reduced compared with that in a wild-type infection. Similarly, by immunoelectron microscopy, the relative number of E1B-55kDa-specific immunogold particles associated with the clear fibrillar inclusion bodies was reduced. However, the E4-34kDa Protein was not required for the close association of the early region 2A DNA binding Protein with the viral inclusions. We propose that the viral 55-kDa-34-kDa Protein complex interacts with a cellular factor required for cytoplasmic accumulation of mRNAs and directs it to the periphery of the transcriptionally active viral inclusion bodies. This model provides an explanation for the ability of these viral Proteins to simultaneously enhance accumulation of viral mRNAs and inhibit accumulation of cellular mRNAs.

Thomas Shenk - One of the best experts on this subject based on the ideXlab platform.

  • Adenovirus early region 4 34-kilodalton Protein directs the nuclear localization of the early region 1B 55-kilodalton Protein in primate cells.
    Journal of virology, 1996
    Co-Authors: Felicia Goodrum, Thomas Shenk, David A. Ornelles
    Abstract:

    The localization of the adenovirus type 5 34-kDa E4 and 55-kDa E1B Proteins was determined in the absence of other adenovirus Proteins. When expressed by transfection in human, monkey, hamster, rat, and mouse cell lines, the E1B Protein was predominantly cytoplasmic and typically was excluded from the nucleus. When expressed by transfection, the E4 Protein accumulated in the nucleus. Strikingly, when coexpressed by transfection in human, monkey, or baby hamster kidney cells, the E1B Protein colocalized in the nucleus with the E4 Protein. A complex of the E4 and E1B Proteins was identified by coimmunoprecipitation in transfected HeLa cells. By contrast to the interaction observed in primate and baby hamster kidney cells, the E4 Protein failed to direct the E1B Protein to the nucleus in rat and mouse cell lines as well as CHO and V79 hamster cell lines. This failure of the E4 Protein to direct the nuclear localization of the E1B Protein in REF-52 rat cells was overcome by fusion with HeLa cells. Within 4 h of heterokaryon formation and with Protein synthesis inhibited, a portion of the E4 Protein present in the REF-52 nuclei migrated to the HeLa nuclei. Simultaneously, the previously cytoplasmic E1B Protein colocalized with the E4 Protein in both human and rat cell nuclei. These results suggest that a primate cell-specific factor mediates the functional interaction of the E1B and E4 Proteins of adenovirus.

  • localization of the adenovirus early region 1b 55 kilodalton Protein during lytic infection association with nuclear viral inclusions requires the early region 4 34 kilodalton Protein
    Journal of Virology, 1991
    Co-Authors: David A. Ornelles, Thomas Shenk
    Abstract:

    Abstract The distribution of the adenovirus early region 1B 55-kDa Protein (E1B-55kDa) in lytically infected HeLa cells was determined. At the time of infection, when the E1B-55kDa Protein facilitates the cytoplasmic accumulation of viral mRNA while simultaneously restricting the accumulation of most cellular mRNA, five distinct intracellular localizations of the Protein were observed. Only one of these was disrupted when cells were infected with a mutant virus that fails to produce a second viral Protein encoded by early region 4 (E4-34kDa). This Protein normally forms a complex with the E1B-55kDa polypeptide, enabling it to influence RNA metabolism. This key localization of the E1B Protein was within and about the periphery of nuclear viral inclusion bodies believed to be the site of viral DNA replication and transcription. In the absence of the E4-34kDa Protein, the coincidence of E1B-55kDa-specific immunofluorescence and phase-dense viral inclusions was reduced compared with that in a wild-type infection. Similarly, by immunoelectron microscopy, the relative number of E1B-55kDa-specific immunogold particles associated with the clear fibrillar inclusion bodies was reduced. However, the E4-34kDa Protein was not required for the close association of the early region 2A DNA binding Protein with the viral inclusions. We propose that the viral 55-kDa-34-kDa Protein complex interacts with a cellular factor required for cytoplasmic accumulation of mRNAs and directs it to the periphery of the transcriptionally active viral inclusion bodies. This model provides an explanation for the ability of these viral Proteins to simultaneously enhance accumulation of viral mRNAs and inhibit accumulation of cellular mRNAs.

Phillip H Gallimore - One of the best experts on this subject based on the ideXlab platform.

  • the high levels of p53 present in adenovirus early region 1 transformed human cells do not cause up regulation of mdm2 expression
    Virology, 1995
    Co-Authors: Roger J A Grand, Arnold J. Levine, Philip S Lecane, Darerca Owen, Michael L Grant, Sally Roberts, Phillip H Gallimore
    Abstract:

    Abstract The cellular Protein MDM2 can bind to the tumor suppressor gene product p53 and abrogate its transcriptional activity. In addition, p53 can regulate expression of the mdm2 gene. We and others have previously shown that p53 is present at high levels in adenovirus-transformed cells which express the larger E1B Protein. In view of these observations the expression of MDM2 in a panel of adenovirus transformed human cell lines has been examined. Two major species (98K and 80K) were detected, together with a number of minor species of higher and lower molecular weight. While there was little variation in levels of 9BK Protein between cell lines, appreciable differences in the expression of the 80K component were apparent. There was no correlation between MDM2 and p53 expression in any of the adenovirus transformants, nor with the viral Proteins expressed. The pattern and level of MDM2 detected was similar to that seen in human tumor cell lines and in human fetal tissue. Northern blot analysis suggested that MDM2 expression was regulated at the transcriptional level. Stable interactions were observed between p53 and MDM2 in the adenovirus-transformed cell lines and in Ad5 E1 HEK 293 cells a ternary complex of p53, MDM2, and the Ad5 E1B 58K Protein was demonstrated. In view of the lack of correlation between the level of p53 and MDM2 in adenovirus E1-transformed cells, the capacity of p53 to cause transcriptional activation was assessed using transfected CAT constructs linked to p53 responsive elements. p53 transcriptional activity was similar in all of the cell lines examined and did not correlate with Protein expression. It is concluded, on the basis of all of these data, that the high concentrations of p53 found in adenovirus transformants are not transcriptionally active and have no influence on MDM2 expression. However, when expression of p53 was increased following infection with mutant adenoviruses, which do not express the larger E1B Proteins, there was an appreciable increase in p53 transcriptional activity and in the levels of all of the MDM2 components.

  • the quaternary structure of the adenovirus 12 early region 1b 54k Protein
    Virology, 1995
    Co-Authors: Roge J A Grand, Sally Roberts, Tracey Mustoe, Phillip H Gallimore
    Abstract:

    The quaternary structure of the adenovirus early region 1B 54K Protein has been examined under denaturing and nondenaturing conditions. In the presence of SDS the Protein has a strong tendency to form disulfide-linked high-molecular-weight polymers. Under nondenaturing, but reducing, conditions the in vitro-translated 54K polypeptide forms oligomers (probably tetramers) of molecular weight approximately 2 x 10(5). After subcellular fractionation of Ad12 early region 1-transformed cells, the 54K E1B Protein present in the cytoplasm had a molecular weight similar to that determined for the in vitro-translated material. However, two populations of the viral Protein could be distinguished in the nucleus-one of a size similar to that seen in the cytoplasm and the other of appreciably higher molecular weight (approximately 2 x 10(6)). No difference in migration pattern was observed after treatment of the nuclear extract with DNase I or RNase. A proportion of the Ad12 E1B 54K Protein in both the high- and the low-molecular-weight populations in the nucleus was found to form a complex with p53, and it is therefore concluded that the very high molecular weight derives from interaction with an, as yet, unidentified component.

  • overexpression of wild type p53 and c myc in human fetal cells transformed with adenovirus early region 1
    Virology, 1993
    Co-Authors: Roger J A Grand, Philip S Lecane, Michael L Grant, Sally Roberts, David P Lane, Lawrence S Young, Christopher W Dawson, Phillip H Gallimore
    Abstract:

    The expression of p53 in a large panel of adenovirus (Ad) 2/5- and 12-transformed human, rat, and mouse cells has been examined. In all cases, in the absence of the larger Ad E1B Protein, the level of p53 is very low. In human and rat cells when the Ad 12 E1B 54K polypeptide is expressed, p53 is much more abundant, although this is not the case in Ad 12 E1-transformed mouse cells. We conclude that expression of p53 is determined by virus serotype, host cell type, and viral Proteins expressed. p53 in Ad 12 E1-transformed human cells is wild type but has an extended half-life. Stabilization is not through Protein-Protein interaction with the Ad E1B Protein. The level of expression of c-Myc is also elevated in Ad-transformed human cells but this does not correlate with the presence of the E1B Protein or with p53. However, Northern blot analysis indicates a direct correlation between mRNA and Protein levels. We conclude that c-Myc is regulated at the transcriptional level, whereas p53 is regulated at the post-translational level in adenovirus transformants.

Ramon A Gonzalez - One of the best experts on this subject based on the ideXlab platform.

  • the biology of the adenovirus E1B 55k Protein
    FEBS Letters, 2019
    Co-Authors: Paloma Hidalgo, Thomas Dobner, Ramon A Gonzalez
    Abstract:

    The adenovirus E1B 55K (E1B) Protein plays major roles in productive adenoviral infection and cellular transformation. Interest in E1B increased because of the potential of adenoviruses as therapeutic vectors, and the E1B gene is commonly deleted from adenovirus vectors for anticancer therapy. E1B activities are spatiotemporally regulated through SUMOylation and phosphorylation, and through interactions with multiple partners that occur presumably at different intracellular sites and times postinfection. E1B is implicated in the formation of viral replication compartments and regulates viral genome replication and transcription, transcriptional repression, degradation of cellular Proteins, and several intranuclear steps of viral late mRNA biogenesis. Here, we review advances in our understanding of E1B during productive adenovirus replication and discuss fundamental aspects that remain unresolved.

  • an early function of the adenoviral E1B 55 kda Protein is required for the nuclear relocalization of the cellular p53 Protein in adenovirus infected normal human cells
    Virology, 2008
    Co-Authors: F M Cardoso, Sayuri E.m. Kato, Wenying Huang, Jane S Flint, Ramon A Gonzalez
    Abstract:

    It is well established that the human subgroup C adenovirus type 5 (Ad5) E1B 55 kDa Protein can regulate the activity and concentration of the cellular tumor suppressor, p53. However, the contribution(s) of these functions of the E1B Protein to viral reproduction remains unclear. To investigate this issue, we examined properties of p53 in normal human cells infected by E1B mutant viruses that display defective entry into the late phase or viral late mRNA export. The steady-state concentrations of p53 were significantly higher in cells infected by the E1B 55 kDa null mutant Hr6 or three mutants carrying small insertions in the E1B 55 kDa Protein coding sequence than in Ad5-infected cells. Nevertheless, none of the mutants induced apoptosis in infected cells. Rather, the localization of p53 to E1B containing nuclear sites observed during infection by Ad5 was prevented by mutations that impair interaction of the E1B Protein with p53 and/or with the E4 Orf6 Protein. These results indicate that the E1B Protein fulfills an early function that correlates efficient entry into the late phase with the localization of E1B and p53 in the nucleus of Ad5-infected normal human cells.

  • effects of mutations in the adenoviral E1B 55 kilodalton Protein coding sequence on viral late mrna metabolism
    Journal of Virology, 2002
    Co-Authors: Ramon A Gonzalez, S. J. Flint
    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. Schutt, 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.