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R P Ricciardi

Publications and source records attributed to R P Ricciardi.

At least 19 recordsLinked to original sources

E1A-based determinants of oncogenicity in human adenovirus groups A and C.

A broad spectrum of genetic and molecular investigations carried out with group C, Ad2 and Ad5, and with group A, Ad12, have shown that early region1 (E1) gene products are sufficient for complete transformation of rodent cells in vitro by these viruses. During the past quarter century, the processes by which E1A proteins, in cooperation with E1B proteins, perturb the cell cycle and induce the transformed phenotype, have become well defined. Somewhat less understood is the basis for the differential oncogenicity of these two groups of viruses, and the processes by which the E1A proteins of Ad12 induce a tumorigenic phenotype in transformants resulting from infection of cells in vivo and in vitro. In this chapter we review previous findings and present new evidence which demonstrates that Ad12 E1A possesses two or more independent functions enabling it to induce tumors. One of these functions lies in its capacity to repress transcription of MHC class I genes, allowing the tumor cells to avoid lysis by cytotoxic T lymphocytes. We have shown that class I repression is mediated through increased binding of repressor COUP-TF and decreased binding of NF-kB to the class I enhancer. In addition to mediating immune escape, E1A also determines the susceptibility of transformants to Natural Killer (NK) cell lysis, and in this case, also, Ad12 transformants are not susceptible. By using Ad12 mutants containing chimeric E1A Ad12-Ad5 genes, point mutations, or a specific deletion, we have shown that the unique spacer region of Ad12 E1A is an oncogenic determinant, but is not required for transformation in vitro. Given that the E1A regions responsible for class I repression are first exon encoded, we have examined a set of cell lines transformed by these altered viruses, and have found that while they display greatly reduced tumorigenicity, they maintain a wildtype capacity to repress class I transcription. Whether the spacer contributes to NK evasion remains unresolved. Lastly, we discuss the properties of the Ad2/Ad5 E1A C-terminal negative modulator of tumorigenicity, and examine the effects on transformation, tumor induction and transformant tumorigenicity, when the Ad5 negative modulator is placed by chimeric construction in Ad12 E1A.

Adenovirus E1A Proteins↗

Coup-TFII is up-regulated in adenovirus type 12 tumorigenic cells and is a repressor of MHC class I transcription.

Down-regulation of the MHC class I enhancer in tumorigenic Ad12 cells is associated with strong binding of COUP-TF and negligible binding of activator NF-kappaB. By comparison, in nontumorigenic Ad5 cells, class I expression is high due to negligible binding of COUP-TF and strong binding of NF-kappaB. Here, we show that COUP-TFII, but not COUP-TFI, is expressed in Ad12-transformed cells. The dramatically stronger DNA binding of COUP-TFII to the class I enhancer in Ad12- compared to Ad5-transformed cells correlates with higher COUP-TFII promoter activity and higher levels of COUP-TFII mRNA and protein. Significantly, NF-kappaB p50/p52 double-knockout cells enabled us to demonstrate directly that COUP-TFII can completely repress both nonactivated and NF-kappaB-activated MHC class I transcription.

Adenoviridae↗

The RITE assay: identifying effectors that target the transcription machinery using phage display technology.

We describe an approach using phage display to identify effectors (activators and repressors) of transcription based on the particular component of the general transcription machinery that they target. We refer to this approach as the reverse identification of transcriptional effectors (RITE) assay. A library of phages containing cDNA-encoded peptides displayed on their surfaces is screened using as the target a specific region of one of the general transcription factors (e.g., the C terminus of hTAFII135). The amino acid sequence encoded by the cDNA of an interacting phage is determined and analyzed in a database homology search to identify known or novel factors that may interact with the target protein. Candidate effectors from the homology search are synthesized from recombinant clones and tested for their abilities to bind to the target protein and to functionally modulate transcription in vivo when co-expressed with the transcriptional target protein. Because the RITE assay is a direct measure of the interactions between general transcription proteins and their effectors, it has an advantage over the well-known yeast two-hybrid system, which is not amenable to identifying transcription factor interactions.

Amino Acid Sequence↗

Oligomerization properties of the viral oncoproteins adenovirus E1A and human papillomavirus E7 and their complexes with the retinoblastoma protein.

Human papillomavirus 16 E7 (HPV16 E7) and adenovirus 5 E1A (Ad5 E1A) are encoded by highly divergent viruses yet are functionally similar in their ability to bind the retinoblastoma (pRB) tumor suppressor protein, causing the aberrant displacement of E2F trancription factors. The amino acid residues of HPV16 E7 that are necessary for stability, for inhibition of pRB function, and for cell transformation are also necessary for E7 oligomerization. However, neither the specific oligomerization state of HPV16 E7 nor of Ad5 E1A as a function of pRB-binding has been characterized. To gain insight into HPV16 E7 and Ad5 E1A oligomerization properties, sedimentation equilibrium experiments were performed with recombinant HPV16 E7 and Ad5 E1A proteins. These studies reveal that, despite the overall functional similarities between these proteins, monomers, dimers, and tetramers of HPV16 E7 were detected while only reversible monomer-dimer association was identified for Ad5 E1A. The apparent K(d(monomer)-(dimer)) of HPV16 E7 is approximately 100-fold lower than that of a comparable region of Ad5 E1A, and it is concluded that under physiological protein concentrations HPV16 E7 exists primarily as a dimer. Sedimentation equilibrium experiments of pRB/Ad5 E1A and of pRB/HPV16 E7 complexes demonstrate that the tight association of pRB with the viral oncoproteins does not disturb their inherent oligomerization properties. Taken together, this study demonstrates significant differences between the Ad5 E1A and HPV16 E7 oligomerization states that are potentially related to their distinct structures and specific mechanisms of pRB-inactivation.

Adenovirus E1A Proteins↗

Association of histone deacetylase with COUP-TF in tumorigenic Ad12-transformed cells and its potential role in shut-off of MHC class I transcription.

Chicken ovalbumin upstream promoter-transcription factor (COUP-TF) is an orphan nuclear receptor that represses transcription of many genes. In adenovirus type 12 (Ad12) transformed cells, a high level of binding activity of COUP-TF to the major histocompatibility complex (MHC) class I enhancer correlates with the down-regulation of class I transcription, which, in turn, contributes to tumorigenesis. The mechanism by which COUP-TF represses transcription has yet to be elucidated. Here we show that COUP-TF represses transcription through its association with histone deacetylase. This was demonstrated using reciprocal binding assays that determined that the interaction between COUP-TF and histone deacetylase requires the COUP-TF C-terminal repression domain. Moreover, a histone deacetylase enzymatic activity was found to be associated with COUP-TF in Ad12-transformed cells. Transfection experiments further revealed that exogenous histone deacetylase facilitates transcriptional repression by COUP-TF. Also, supershift assays suggest that the transcriptional corepressor N-CoR, which is known to associate with histone deacetylases, is a part of the COUP-TF complex bound to the MHC class I enhancer R2 site. Finally, we provide evidence that inhibition of histone deacetylases relieves the repression of MHC class I expression in Ad12-transformed cells. Taken together these results support the notion that deacetylation of histones, mediated through COUP-TF, serves to down-regulate MHC class I transcription in Ad12-transformed cells.

Adenoviruses, Human↗

A rapid plate assay for the screening of inhibitors against herpesvirus DNA polymerases and processivity factors.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a newly identified human pathogen with tumorigenic potential. The DNA polymerase (Pol-8) and processivity factor (PF-8) of KSHV were cloned recently. It was shown that PF-8 forms specifically a complex with Pol-8 in vitro and allows it to synthesize fully-extended DNA. Since both Pol-8 and PF-8 are apparently essential for viral DNA replication and since they cannot be substituted by any other cellular or viral proteins, they are potentially excellent antiviral targets. The development of a mechanistic plate assay is now described, which is suitable for rapid high-throughput screening of antiviral agents against Pol-8 and PF-8. The assay allows the measurement of not only total DNA synthesis activity (i.e. nucleotide incorporation) but also processivity (i.e. fully-extended DNA product). In this plate assay, any of the screen-compounds with an inhibitory effect against the total DNA synthesis activity and/or the processivity could be potential antiviral agents that target Pol-8 and/or PF-8. Particularly, since PF-8 is highly specific for Pol-8, the discovery of inhibitory agents against PF-8 may lead to specific antiviral therapies with minimal toxicity to host cells. This assay should be suitable for screening for inhibitory compounds against polymerases and processivity factors of other herpesviruses as well.

Antiviral Agents↗

Mammalian Srb/Mediator complex is targeted by adenovirus E1A protein.

Adenovirus E1A proteins prepare the host cell for viral replication, stimulating cell cycling and viral transcription through interactions with critical cellular regulatory proteins such as RB and CBP. Here we show that the E1A zinc-finger domain that is required to activate transcription of viral early genes binds to a host-cell multiprotein complex containing homologues of yeast Srb/Mediator proteins. This occurs through a stable interaction with the human homologue of Caenorhabditis elegans SUR-2, a protein required for many developmental processes in the nematode. This human Srb/Mediator complex stimulates transcription in vitro in response to both the E1A zinc-finger and the herpes simplex virus VP16 activation domains. Interaction with human Sur-2 is also required for transcription to be activated by the activation domain of a transcription factor of the ETS-family in response to activated mitogen-activated protein (MAP) kinase.

Adenovirus E1A Proteins↗

Reduced phosphorylation of p50 is responsible for diminished NF-kappaB binding to the major histocompatibility complex class I enhancer in adenovirus type 12-transformed cells.

Reduced cell surface levels of major histocompatibility complex class I antigens enable adenovirus type 12 (Ad12)-transformed cells to escape immunosurveillance by cytotoxic T lymphocytes (CTL), contributing to their tumorigenic potential. In contrast, nontumorigenic Ad5-transformed cells harbor significant cell surface levels of class I antigens and are susceptible to CTL lysis. Ad12 E1A mediates down-regulation of class I transcription by increasing COUP-TF repressor binding and decreasing NF-kappaB activator binding to the class I enhancer. The mechanism underlying the decreased binding of nuclear NF-kappaB in Ad12-transformed cells was investigated. Electrophoretic mobility shift assay analysis of hybrid NF-kappaB dimers reconstituted from denatured and renatured p50 and p65 subunits from Ad12- and Ad5-transformed cell nuclear extracts demonstrated that p50, and not p65, is responsible for the decreased ability of NF-kappaB to bind to DNA in Ad12-transformed cells. Hypophosphorylation of p50 was found to correlate with restricted binding of NF-kappaB to DNA in Ad12-transformed cells. The importance of phosphorylation of p50 for NF-kappaB binding was further demonstrated by showing that an NF-kappaB dimer composed of p65 and alkaline phosphatase-treated p50 from Ad5-transformed cell nuclear extracts could not bind to DNA. These results suggest that phosphorylation of p50 is a key step in the nuclear regulation of NF-kappaB in adenovirus-transformed cells.

Adenoviruses, Human↗

The 41-kDa protein of human herpesvirus 6 specifically binds to viral DNA polymerase and greatly increases DNA synthesis.

We previously isolated a 41-kDa early antigen of human herpesvirus 6 (HHV-6), which exhibited nuclear localization and DNA-binding activity (Agulnick et al., 1993). In this study, we observed that a 110-kDa protein was coimmunoprecipitated with p41 from HHV-6-infected cells by an anti-p41 antibody. This 110-kDa protein was identified as the HHV-6 DNA polymerase (Pol-6) by an antibody raised against the N terminus of Pol-6. Reciprocal immunoprecipitation and Western blot analyses confirmed that p41 complexes with Pol-6 in HHV-6-infected cells. In addition, both p41 and Pol-6 were expressed in vitro and shown to form a specific complex. An in vitro DNA synthesis assay using primed M13 single-stranded DNA template demonstrated that p41 not only increased the DNA synthesis activity of Pol-6 but also allowed Pol-6 to synthesize DNA products corresponding to full-length M13 template (7249 nucleotides). By contrast, Pol-6 alone could only synthesize DNA of <100 nucleotides. The functional interaction between Pol-6 and p41 appears to be specific because they could not be physically or functionally substituted in vitro by their herpes simplex virus 1 homologues. Moreover, as revealed by mutational analysis, both the N and C termini of Pol-6 contribute to its binding to p41. In the case of p41, the N terminus is required for increasing DNA synthesis but not binding to Pol-6, whereas the C terminus is totally dispensable.

Cell Line↗

Cloning and functional analysis of Kaposi's sarcoma-associated herpesvirus DNA polymerase and its processivity factor.

Kaposi's sarcoma-associated herpesvirus (KSHV), or human herpesvirus 8, is a newly identified virus with tumorigenic potential. Here, we cloned and expressed the DNA polymerase (Pol-8) of KSHV and its processivity factor (PF-8). Pol-8 bound specifically to PF-8 in vitro. Moreover, the DNA synthesis activity of Pol-8 was shown in vitro to be strongly dependent on PF-8. Addition of PF-8 to Pol-8 allowed efficient synthesis of fully extended DNA products corresponding to the full-length M13 template (7,249 nucleotides), whereas Pol-8 alone could incorporate only several nucleotides. The specificity of PF-8 and Pol-8 for each other was demonstrated by their inability to be functionally replaced by the DNA polymerases and processivity factors of herpes simplex virus 1 and human herpesvirus 6.

Amino Acid Sequence↗

Human colorectal cancer (CRC) antigen CO17-1A/GA733 encoded by adenovirus inhibits growth of established CRC cells in mice.

The human colorectal carcinoma (CRC)-associated Ag CO17-1A/GA733, originally defined by mAbs CO17-1A and GA733, has been a useful target in passive immunotherapy of CRC patients with mAb and in active immunotherapy with anti-idiotypic Abs mimicking the CO17-1A or GA733 epitope. Both approaches have targeted single epitopes. We investigated the capacity of full-length CO17-1A/GA733 Ag expressing multiple potentially immunogenic epitopes and encoded by recombinant adenovirus 5 (Ad5 GA733-2) to induce humoral, cellular, and/or protective immunity in mice. Ad5 GA733-2 induced Ag-specific Abs that reacted predominantly to CO17-1A- and GA733-unrelated epitopes on the Ag and lysed Ag-positive CRC targets in conjunction with effector cells. Ad5 GA733-2-immune mice developed Ag-specific, proliferative lymphocytes of Th1 type and cytolytic lymphocytes. The use of Ad5 GA733-2 to immunize mice bearing established syngeneic CRC cells transfected with the human Ag induced significant and specific tumor regression. Cured mice resisted rechallenge with human CO17-1A/GA733 Ag-negative parental CRC cells, suggesting that targeting the human Ag on the murine transfectants induced protective immunity to other Ag expressed by the parental tumor. These results may explain the high potency of the recombinant vaccine. Thus, rAd5 GA733-2 may have potential as a vaccine for CRC patients.

Adenoviridae↗

The transactivation domain of adenovirus E1A interacts with the C terminus of human TAF(II)135.

The CR3 activation domain of the human adenovirus E1A protein stimulates transcription by forming protein-protein interactions with DNA sequence-specific binding factors and components of the TFIID complex. Here, we demonstrate that CR3 can complex with the extreme C-terminal 105 amino acids of the human TATA box binding-factor-associated protein, hTAF(II)135. Furthermore, the C-terminal region of hTAF(II)135 can block transcriptional stimulation from an E1A-inducible promoter in vivo. This ability of the C terminus of hTAF(II)135 to bind CR3 and to inhibit E1A-inducible activation is highly specific. These results demonstrate for the first time that a discrete fragment of a mammalian TBP-associated factor which targets a specific activator can impair the stimulation of transcription.

Adenovirus E1A Proteins↗

Phosphorylation within the transactivation domain of adenovirus E1A protein by mitogen-activated protein kinase regulates expression of early region 4.

A critical role of the 289-residue (289R) E1A protein of human adenovirus type 5 during productive infection is to transactivate expression of all early viral transcription. Sequences within and proximal to conserved region 3 (CR3) promote expression of these viral genes through interactions with a variety of transcription factors requiring the zinc binding motif in CR3 and in some cases a region at the carboxy-terminal end of CR3, including residues 183 to 188. It is known that 3',5' cyclic AMP (cAMP) reduces the level of phosphorylation of the 289R E1A protein through the activation of protein phosphatase 2A by the E4orf4 protein. This study was designed to identify the E1A phosphorylation sites affected by E4orf4 expression and to determine their importance in regulation of E1A activity. We report here that two previously unidentified sites at Ser-185 and Ser-188 are the targets for decreased phosphorylation in response to cAMP. At least one of these sites, presumably Ser-185, is phosphorylated in vitro by purified mitogen-activated protein kinase (MAPK), and both are hyperphosphorylated in cells which express a constitutively active form of MAPK kinase. Analysis of E1A-mediated transactivation activity indicated that elevated phosphorylation at these sites increased expression of the E4 promoter but not that of E3. We have recently shown that one or more E4 products induce cell death due to p53-independent apoptosis, and thus it seems likely that one role of the E4orf4 protein is to limit production of toxic E4 products by limiting expression of the E4 promoter.

Adenovirus E1A Proteins↗

Testing NF-kappa B1-p50 antibody specificity using knockout mice.

Cell extracts from knockout mice can provide definitive proof of antibody specificity. Two NF-kappa B1-p50 antibodies, sc-114 (a commercial antibody) and NR1157, were observed to recognize proteins having distinct electrophoretic mobilities of 52-55 kD and 50 kD, respectively, by Western blot analysis. In order to discriminate the specificity of these antibodies for NF-kappa B1-p50, whole cell extracts derived from NF-kappa B1-p105 knockout mice were employed. While the NR1157 antibody completely failed to recognize its 50 kD product in p105-/- knockout extracts, the sc-114 antibody still strongly recognized its 52-55 kD product. These data demonstrate that NR1157, but not sc-114, is highly specific for NF-kappa B1-p50 by Western blot analysis. In addition, these results highlight the utility of knockout cell extracts for discerning antibody specificity.

Amino Acid Sequence↗

The first exon of Ad12 E1A excluding the transactivation domain mediates differential binding of COUP-TF and NF-kappa B to the MHC class I enhancer in transformed cells.

The major histocompatibility complex class I enhancer is the target for adenovirus-12 E1A-mediated down-regulation of class I transcription. In Ad12 transformed rodent cells, the class I enhancer is down-regulated through increased binding of the repressor COUP-TF to the R2 element and decreased binding of the activator NF-kappa B (p50/p65) to the R1 element. The reduced surface levels of class I antigens contribute to the tumorigenic potential of Ad12 transformed cells by favoring their immunoescape from cytotoxic T-lymphocytes. Previous studies using transformed cells containing hybrid Ad5/Ad12 E1A (plus Ad12 E1B) genes have indicated that sequences within the first exon of the 266R Ad12 E1A gene are required for class I down-regulation and tumorigenesis. In this study we demonstrate that these same sequences, which exclude the Ad12 CR3 transactivation domain, are also required for increased COUP-TF binding to the R2 element and decreased NF-kappa B binding to the R1 element of the class I enhancer. We further show that diminished NF-kappa B binding is not due to a lack of NF-kappa B1-p50 in the nuclei of Ad12 transformed rat cells.

Adenovirus E1A Proteins↗

Evidence for the involvement of a nuclear NF-kappa B inhibitor in global down-regulation of the major histocompatibility complex class I enhancer in adenovirus type 12-transformed cells.

Diminished expression of major histocompatibility complex class I antigens on the surface of adenovirus type 12 (Ad12)-transformed cells contributes to their high tumorigenic potential by enabling them to escape immune recognition by cytotoxic T lymphocytes. This low class I antigen expression is due to a block in class I transcription, which is mediated by Ad12 E1A. Genetic analysis has shown that the class I enhancer is the target for transcriptional down-regulation. In this study, we show that the ability of the R1 element of the class I enhancer to stimulate transcription is greatly reduced in Ad12-transformed cells. The loss of functional activity by the R1 element was attributed to loss of binding by the NF-kappa B p50-p65 heterodimer. NF-kappa B binding appears to be blocked within the nucleus rather than at the level of nuclear translocation. Significantly, NF-kappa B binding activity could be recovered from the nuclear extracts of Ad12-transformed cells following detergent treatment, suggesting that the block is mediated through a nuclear inhibitor present in the Ad12-transformed cells. These results, taken together with the fact that the R2 element of the class I enhancer exhibits strong binding to the transcriptional repressor COUP-TF, suggest that the class I enhancer is globally down-regulated in Ad12-transformed cells.

Adenoviruses, Human↗

The viral oncoproteins Ad5 E1A, HPV16 E7 and SV40 TAg bind a common region of the TBP-associated factor-110.

A function shared by the adenovirus E1A, papillomavirus E7 and SV40 TAg oncoproteins is their ability to interfere with normal cell growth by interacting with members of the retinoblastoma protein family. In this study, we show that each of these oncoproteins can also bind to the 921 amino acid TBP-associated factor-110 (TAF-110). The significance of the binding is underscored by the observation that each oncoprotein binds to the same 77 amino acid carboxyl region of TAF-110. In the case of E1A and TAg, this finding is consistent with their abilities to stimulate transcription initiation, in part, through their known interactions with TBP. While it is not clear whether E7 can also activate promoters through protein:protein interactions with components of the transcription initiation complex, our demonstration that E7 can bind to TAF-110, as well as TBP, suggests that E7 may modulate the expression of specific promoters which could contribute to the pathogenesis of human papillomavirus.

Adenovirus E1A Proteins↗

Subregions of the adenovirus E1A transactivation domain target multiple components of the TFIID complex.

Transcriptional activation by the adenovirus E1A 289R protein requires direct contacts with the TATA box-binding protein (TBP) and also displays a critical requirement for TBP-associated factors (TAFs) (T.G. Boyer and A. J. Berk, Genes Dev. 7:1810-1823, 1993; J. V. Geisberg, W. S. Lee, A. J. Berk, and R. P. Ricciardi, Proc. Natl. Acad. Sci. USA 91:2488-2492, 1994; W. S. Lee, C. C. Kao, G. O. Bryant, X. Liu, and A. J. Berk, Cell 67:365-376, 1991; and Q. Zhou, P. M. Lieberman, T. G. Boyer, and A. J. Berk, Genes Dev. 6:1964-1974, 1992). In this report, we demonstrate that the activation domain of E1A (CR3) specifically binds to two TAFs, human TAFII250 (hTAFII250) and Drosophila TAFII110 (dTAFII110). These interactions can take place both in vivo and in vitro and require the carboxy-terminal region of CR3; the zinc finger region of CR3, which binds TBP, is not needed to bind these TAFs. We mapped the E1A-binding sites on hTAFII250 to an internal region that contains a number of structural motifs, including an HMG box, a bromodomain, and direct repeats. This represents the first demonstration that hTAFII250 may serve as a target of a transcriptional activator. We also mapped the E1A binding on dTAFII110 to its C-terminal region. This is of significance since, by contrast, Sp1-mediated activation requires binding to the N-terminal domain of dTAFII110. Thus, distinct surfaces of dTAFII110 can serve as target sites for different activators. Our results indicate that E1A may activate transcription, in part, through direct contacts of the CR3 subdomains with selected components of the TFIID complex.

Adenovirus E1A Proteins↗