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Papers by Robert Ricciardi

Research paper thumbnail of Tumorigenic adenovirus 12 cells evade NK cell lysis by reducing the expression of NKG2D ligands

Immunology Letters, May 1, 2012

Activation of Natural Killer (NK) cells depends on a balance between signals received from activa... more Activation of Natural Killer (NK) cells depends on a balance between signals received from activation and inhibitory ligands expressed on the surface of target cells. Tumorigenic human adenovirus 12 (Ad12) transformed cells express low levels of the NK cell inhibitory ligand MHC I, but do not exhibit increased sensitivity to NK cell lysis compared to their non-tumorigenic counterparts. Analysis of the expression of activation ligands that bind to the NKG2D receptor revealed that RAE1β and H60 were reduced on the surface of Ad12 mouse cells as well as at the level of transcription. In accord with these results, RAE1 localization to the synapse and sensitivity to NK cell cytotoxicity were also diminished. The reduced transcription of the rat NKG2D ligands, RAEt1L and RRTL, in tumorigenic rat cells compared to non-tumorigenic counterparts implies that both mouse and rat cell lines share a common mechanism of NKG2D ligand activation subverted by Ad12.

Research paper thumbnail of Specificity of the mouse cytotoxic T lymphocyte response to adenovirus 5. E1A is immunodominant in H-2b, but not in H-2d or H-2k mice

The Journal of Immunology

The Ag specificity and MHC restriction of the CTL response to adenovirus 5 (Ad5) in three strains... more The Ag specificity and MHC restriction of the CTL response to adenovirus 5 (Ad5) in three strains of mice, C57BL/10 (H-2b), BALB/c (H-2d), and C3H/HeJ (H-2k), were tested. Polyclonal Ad5-specific CTL were prepared by priming mice in vivo with live Ad5 virus followed by secondary in vitro stimulation of the spleen cells with virus-infected syngeneic cells. The Ad5-specific CTL were Db restricted in C57BL/10 and Kk restricted in C3H/HeJ. In BALB/c mice both Kd- and Dd/Ld-restricted CTL were detected. The polyclonal Ad5-specific CTL response in C57BL/10 mice is directed exclusively against the products of the E1A region, which comprises only 5% of the Ad5 genome. In BALB/c mice E1A is at best a very minor target Ag and in C3H/HeJ mice E1A is not recognized at all. Using the H-2 congenic mouse strains B10.BR (H-2k) and C3H.SW (H-2b) it was shown that the immunodominance of E1A is H-2 dependent. The 19-kDa glycoprotein encoded in the E3 region of Ad5, which binds to class I MHC in the en...

Research paper thumbnail of Murine cell lines stably expressing the influenza virus hemagglutinin gene introduced by a recombinant retrovirus vector are constitutive targets for MHC class I- and class II-restricted T lymphocytes

The Journal of Immunology

A retrovirus vector containing the hemagglutinin (HA) gene of influenza virus was constructed and... more A retrovirus vector containing the hemagglutinin (HA) gene of influenza virus was constructed and used to infect murine cell lines of fibroblast, mastocytoma and B cell lineages which are able to present antigens to MHC-restricted T cells. Stable cell lines were selected in which the retrovirus vector integrated as a single copy in almost all of the individual cell clones examined. The HA mRNA was shown to be of the expected length by Northern blot analysis, but the levels varied among the cell clones. Although the HA transcript was difficult to detect in any of the retrovirus-infected cell clones derived from fibroblasts, HA Ag was easily detected on the cell surface by cytofluorographic analysis. Significantly, retrovirus-infected clones derived from each cell type were recognized by HA-specific class I and class II MHC-restricted T lymphocytes. HA produced in these cells was able to be acquired, processed, and presented to class II-restricted T cells by additional, non-HA-express...

Research paper thumbnail of Complex Class I Transcription Shutoff Is Overcome by Induction of NF-�B and Relief of COUP-TFII Repression

The surface levels of major histocompatibility complex class I antigens are diminished on tumorig... more The surface levels of major histocompatibility complex class I antigens are diminished on tumorigenic adenovirus type 12 (Ad12)-transformed cells, enabling them to escape from immunosurveillant cytotoxic T lymphocytes (CTLs). This is due to the down-regulation of the class I transcriptional enhancer, in which there is strong binding of the repressor COUP-TFII and lack of binding of the activator NF-B. Even though NF-B (p65/p50) translocates to the nuclei of Ad12-transformed cells, it fails to bind to DNA efficiently due to the hypophosphorylation of the p50 subunit. In this study, tumor necrosis factor alpha (TNF-␣) and interleukin 1␤ (IL-1␤) were shown to promote degradation of the NF-B cytoplasmic inhibitor IB␣ and permit the nuclear translocation of a phosphorylated form of NF-B that is capable of binding DNA. Interestingly, when Ad12-transformed cells were treated with TNF-␣ or IL-1␤, class I gene transcription substantially increased when transcriptional repression by COUP-TFII was blocked. This indicates that in cytokine-treated Ad12transformed cells, COUP-TFII is able to repress activation of class I transcription by newly nucleus-localized NF-B. Our results suggest that Ad12 likely employs a "fail-safe" mechanism to ensure that the transcription of class I genes remains tightly repressed under various physiological conditions, thus providing tumorigenic Ad12-transformed cells with a means of escaping CTL recognition and lysis.

Research paper thumbnail of COUP-TFII Is Up-regulated in Adenovirus Type 12 Tumorigenic Cells and Is a Repressor of MHC Class I Transcription

Virology, 2001

Down-regulation of the MHC class I enhancer in tumorigenic Ad12 cells is associated with strong b... more 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-B. By comparison, in nontumorigenic Ad5 cells, class I expression is high due to negligible binding of COUP-TF and strong binding of NF-B. 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-B p50/p52 double-knockout cells enabled us to demonstrate directly that COUP-TFII can completely repress both nonactivated and NF-B-activated MHC class I transcription.

Research paper thumbnail of Association of Histone Deacetylase with COUP-TF in Tumorigenic Ad12-Transformed Cells and Its Potential Role in Shut-Off of MHC Class I Transcription

Virology, 2000

Chicken ovalbumin upstream promoter-transcription factor (COUP-TF) is an orphan nuclear receptor ... more 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.

Research paper thumbnail of Chromatin repression by COUP-TFII and HDAC dominates activation by NF-κB in regulating major histocompatibility complex class I transcription in adenovirus tumorigenic cells

Virology, 2003

In adenovirus type 12 transformed cells, the down-regulation of MHC class I transcription contrib... more In adenovirus type 12 transformed cells, the down-regulation of MHC class I transcription contributes to the tumorigenic phenotype and is solely mediated by Ad12 E1A. Previous in vitro studies with class I enhancer sequences have indicated that there is an increased binding of repressor COUP-TFII and its associated HDAC and a decreased binding of activator NF-B. In this study, we used chromatin immunoprecipitation (ChIP) assay in order to determine in vivo whether these proteins regulate class I transcription by affecting chromatin. The ChIP assay revealed that there is lack of chromatin histone acetylation in the region of the class I enhancer in Ad12-transformed cells. This is regulated by histone deacetylation as it was further demonstrated in vivo that COUP-TFII and HDAC are associated with the class I enhancer chromatin. In agreement with in vitro studies, NF-B could be recruited to the class I enhancer following induction by TNF-␣. However, this enhancer-bound NF-B failed to up-regulate class I expression because the class I enhancer chromatin remained repressed as a result of histone deacetylation by HDAC in association with COUP-TFII. Thus, we have demonstrated for the first time that repression of chromatin through histone deacetylation is a major mechanism in down-regulating class I transcription in Ad12-transformed cells. Finally, Ad12 E1A, a non-DNA binding protein, was shown to be present in the natural protein complex bound to the class I enhancer.

Research paper thumbnail of Expression of histocompatibility antigens H-2K, -D, and -L is reduced in adenovirus-12-transformed mouse cells and is restored by interferon gamma

Proceedings of the National Academy of Sciences, 1985

Primary mouse cells transformed by adenovirus type 12 (Ad12) expressed negligible amounts of clas... more Primary mouse cells transformed by adenovirus type 12 (Ad12) expressed negligible amounts of class I antigens H-2K, -D, and -L on the cell surface and were capable of forming tumors in syngeneic animals, whereas cells transformed by Ad5 continued to express class I antigens and were nontumorigenic. Cells from a tumor, generated by injection of Ad12-transformed mouse cells into a syngeneic mouse, also expressed low levels of H-2 antigens, indicating that this phenotype is maintained in vivo. In all Ad12-transformed cells, synthesis of the H-2 heavy chain was not detected whereas the beta 2-microglobulin light chain was synthesized. Furthermore, the level of cytoplasmic H-2 mRNA in the Ad12 lines was greatly reduced. Reduction of H-2 expression is instructed solely by the transforming region of the viral genome, since this repression occurred in cells transformed by a DNA fragment containing only Ad12 E1A and E1B genes. Addition of recombinant murine interferon gamma strongly stimulat...

Research paper thumbnail of Induction of Neuronal and Tumor-Related Genes by Adenovirus Type 12 E1A

Journal of Virology, 2008

Adenovirus type 12 (Ad12) E1A protein (E1A-12) contains a unique 20-amino-acid spacer region betw... more Adenovirus type 12 (Ad12) E1A protein (E1A-12) contains a unique 20-amino-acid spacer region between the second and third conserved regions. Substitution of a single amino acid in the spacer is able to abrogate Ad12 tumorigenesis. To investigate the function of the spacer, microarray analysis was performed on cells transformed by tumorigenic and nontumorigenic Ad12s that differ only by one amino acid in the spacer. Fewer than 0.8% of approximately 8,000 genes in the microarray exhibited differential expression of threefold and higher. Of these, more than half of the known genes with higher expression in the wild-type Ad12-transformed cells have neuronal-specific functions. Some of the other differentially expressed genes are involved in the regulation of the cell cycle, transcription, cell structure, and tumor invasiveness. Northern blot analyses of a subset of the neuronal genes, including Robo1, N-MYC, and α-internexin, confirmed their strong expression in multiple Ad12 tumorigeni...

Research paper thumbnail of In Adenovirus Type 12 Tumorigenic Cells, Major Histocompatibility Complex Class I Transcription Shutoff Is Overcome by Induction of NF-κB and Relief of COUP-TFII Repression

Journal of Virology, 2002

The surface levels of major histocompatibility complex class I antigens are diminished on tumorig... more The surface levels of major histocompatibility complex class I antigens are diminished on tumorigenic adenovirus type 12 (Ad12)-transformed cells, enabling them to escape from immunosurveillant cytotoxic T lymphocytes (CTLs). This is due to the down-regulation of the class I transcriptional enhancer, in which there is strong binding of the repressor COUP-TFII and lack of binding of the activator NF-κB. Even though NF-κB (p65/p50) translocates to the nuclei of Ad12-transformed cells, it fails to bind to DNA efficiently due to the hypophosphorylation of the p50 subunit. In this study, tumor necrosis factor alpha (TNF-α) and interleukin 1β (IL-1β) were shown to promote degradation of the NF-κB cytoplasmic inhibitor IκBα and permit the nuclear translocation of a phosphorylated form of NF-κB that is capable of binding DNA. Interestingly, when Ad12-transformed cells were treated with TNF-α or IL-1β, class I gene transcription substantially increased when transcriptional repression by COUP...

Research paper thumbnail of Phosphorylation of Serine 337 of NF-κB p50 Is Critical for DNA Binding

Journal of Biological Chemistry, 2003

Research paper thumbnail of DNA Binding of Repressor Nuclear Factor-κB p50/p50 Depends on Phosphorylation of Ser337 by the Protein Kinase A Catalytic Subunit

Journal of Biological Chemistry, 2005

Research paper thumbnail of Transformation by E1A Oncoprotein Involves Ubiquitin-Mediated Proteolysis of the Neuronal and Tumor Repressor REST in the Nucleus

Journal of Virology, 2012

The adenovirus early region 1A (E1A) protein promotes cell immortalization and transformation by ... more The adenovirus early region 1A (E1A) protein promotes cell immortalization and transformation by mediating the activities of key cellular regulators. The repressor element 1-silencing transcription factor (REST), which is a major neuronal and tumor suppressor, was previously found mainly in the cytoplasm rather than in the nuclei of adenovirus-transformed rodent cells (22). We now demonstrate that the loss of REST in the nucleus is due to its rapid degradation by the ubiquitin-proteasome system. Only nuclear REST, but not its cytoplasmic counterpart, was ubiquitinated and degraded. REST degradation was blocked by the ubiquitination inhibitor PYR-41 and the proteasome inhibitor MG-132 but not by the nuclear export inhibitor leptomycin B. REST degradation required both of its two C-terminal degrons that are recognized by the ubiquitin ligase SCF β-TrCP , since deletion or mutation of either degron eliminated degradation. Importantly, E1A was shown to mediate REST ubiquitination and de...

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

Journal of Virology, 1997

The CR3 activation domain of the human adenovirus E1A protein stimulates transcription by forming... more 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.

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

Journal of Virology, 1997

The CR3 activation domain of the human adenovirus E1A protein stimulates transcription by forming... more 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.

Research paper thumbnail of An ATF/CREB site is the major regulatory element in the human herpesvirus 6 DNA polymerase promoter

Journal of Virology, 1994

Human herpesvirus 6 (HHV-6) is a recently described T-cell pathogen whose medical relevance and m... more Human herpesvirus 6 (HHV-6) is a recently described T-cell pathogen whose medical relevance and molecular biology are just beginning to be addressed. As a first look at the regulation of viral genes, control of the HHV-6 DNA polymerase promoter was examined. Polymerase gene transcription in HHV-6-infected cells was found to initiate from a single site located 115 bases upstream of the translation start codon. A polymerase promoter-chloramphenicol acetyltransferase reporter gene construct failed to be expressed in uninfected T cells but was highly active in HHV-6-infected cells. Mutational data indicated that the polymerase promoter is TATA-less. Mutational analysis also revealed that the major upstream promoter regulatory element required for transcriptional activity in HHV-6-infected cells is a palindromic ATF/CREB transcription factor binding site. The significance of this site for promoter induction was further demonstrated by the fact that the polymerase ATF/CREB element, when a...

Research paper thumbnail of An ATF/CREB site is the major regulatory element in the human herpesvirus 6 DNA polymerase promoter

Journal of Virology, 1994

Human herpesvirus 6 (HHV-6) is a recently described T-cell pathogen whose medical relevance and m... more Human herpesvirus 6 (HHV-6) is a recently described T-cell pathogen whose medical relevance and molecular biology are just beginning to be addressed. As a first look at the regulation of viral genes, control of the HHV-6 DNA polymerase promoter was examined. Polymerase gene transcription in HHV-6-infected cells was found to initiate from a single site located 115 bases upstream of the translation start codon. A polymerase promoter-chloramphenicol acetyltransferase reporter gene construct failed to be expressed in uninfected T cells but was highly active in HHV-6-infected cells. Mutational data indicated that the polymerase promoter is TATA-less. Mutational analysis also revealed that the major upstream promoter regulatory element required for transcriptional activity in HHV-6-infected cells is a palindromic ATF/CREB transcription factor binding site. The significance of this site for promoter induction was further demonstrated by the fact that the polymerase ATF/CREB element, when a...

Research paper thumbnail of Cloning and Functional Analysis of Kaposi’s Sarcoma-Associated Herpesvirus DNA Polymerase and Its Processivity Factor

Journal of Virology, 1998

Kaposi’s sarcoma-associated herpesvirus (KSHV), or human herpesvirus 8, is a newly identified vir... more 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.

Research paper thumbnail of Cloning and Functional Analysis of Kaposi’s Sarcoma-Associated Herpesvirus DNA Polymerase and Its Processivity Factor

Journal of Virology, 1998

Kaposi’s sarcoma-associated herpesvirus (KSHV), or human herpesvirus 8, is a newly identified vir... more 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.

Research paper thumbnail of 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

Molecular and Cellular Biology, 1996

Diminished expression of major histocompatibility complex class I antigens on the surface of aden... more 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 ...

Research paper thumbnail of Tumorigenic adenovirus 12 cells evade NK cell lysis by reducing the expression of NKG2D ligands

Immunology Letters, May 1, 2012

Activation of Natural Killer (NK) cells depends on a balance between signals received from activa... more Activation of Natural Killer (NK) cells depends on a balance between signals received from activation and inhibitory ligands expressed on the surface of target cells. Tumorigenic human adenovirus 12 (Ad12) transformed cells express low levels of the NK cell inhibitory ligand MHC I, but do not exhibit increased sensitivity to NK cell lysis compared to their non-tumorigenic counterparts. Analysis of the expression of activation ligands that bind to the NKG2D receptor revealed that RAE1β and H60 were reduced on the surface of Ad12 mouse cells as well as at the level of transcription. In accord with these results, RAE1 localization to the synapse and sensitivity to NK cell cytotoxicity were also diminished. The reduced transcription of the rat NKG2D ligands, RAEt1L and RRTL, in tumorigenic rat cells compared to non-tumorigenic counterparts implies that both mouse and rat cell lines share a common mechanism of NKG2D ligand activation subverted by Ad12.

Research paper thumbnail of Specificity of the mouse cytotoxic T lymphocyte response to adenovirus 5. E1A is immunodominant in H-2b, but not in H-2d or H-2k mice

The Journal of Immunology

The Ag specificity and MHC restriction of the CTL response to adenovirus 5 (Ad5) in three strains... more The Ag specificity and MHC restriction of the CTL response to adenovirus 5 (Ad5) in three strains of mice, C57BL/10 (H-2b), BALB/c (H-2d), and C3H/HeJ (H-2k), were tested. Polyclonal Ad5-specific CTL were prepared by priming mice in vivo with live Ad5 virus followed by secondary in vitro stimulation of the spleen cells with virus-infected syngeneic cells. The Ad5-specific CTL were Db restricted in C57BL/10 and Kk restricted in C3H/HeJ. In BALB/c mice both Kd- and Dd/Ld-restricted CTL were detected. The polyclonal Ad5-specific CTL response in C57BL/10 mice is directed exclusively against the products of the E1A region, which comprises only 5% of the Ad5 genome. In BALB/c mice E1A is at best a very minor target Ag and in C3H/HeJ mice E1A is not recognized at all. Using the H-2 congenic mouse strains B10.BR (H-2k) and C3H.SW (H-2b) it was shown that the immunodominance of E1A is H-2 dependent. The 19-kDa glycoprotein encoded in the E3 region of Ad5, which binds to class I MHC in the en...

Research paper thumbnail of Murine cell lines stably expressing the influenza virus hemagglutinin gene introduced by a recombinant retrovirus vector are constitutive targets for MHC class I- and class II-restricted T lymphocytes

The Journal of Immunology

A retrovirus vector containing the hemagglutinin (HA) gene of influenza virus was constructed and... more A retrovirus vector containing the hemagglutinin (HA) gene of influenza virus was constructed and used to infect murine cell lines of fibroblast, mastocytoma and B cell lineages which are able to present antigens to MHC-restricted T cells. Stable cell lines were selected in which the retrovirus vector integrated as a single copy in almost all of the individual cell clones examined. The HA mRNA was shown to be of the expected length by Northern blot analysis, but the levels varied among the cell clones. Although the HA transcript was difficult to detect in any of the retrovirus-infected cell clones derived from fibroblasts, HA Ag was easily detected on the cell surface by cytofluorographic analysis. Significantly, retrovirus-infected clones derived from each cell type were recognized by HA-specific class I and class II MHC-restricted T lymphocytes. HA produced in these cells was able to be acquired, processed, and presented to class II-restricted T cells by additional, non-HA-express...

Research paper thumbnail of Complex Class I Transcription Shutoff Is Overcome by Induction of NF-�B and Relief of COUP-TFII Repression

The surface levels of major histocompatibility complex class I antigens are diminished on tumorig... more The surface levels of major histocompatibility complex class I antigens are diminished on tumorigenic adenovirus type 12 (Ad12)-transformed cells, enabling them to escape from immunosurveillant cytotoxic T lymphocytes (CTLs). This is due to the down-regulation of the class I transcriptional enhancer, in which there is strong binding of the repressor COUP-TFII and lack of binding of the activator NF-B. Even though NF-B (p65/p50) translocates to the nuclei of Ad12-transformed cells, it fails to bind to DNA efficiently due to the hypophosphorylation of the p50 subunit. In this study, tumor necrosis factor alpha (TNF-␣) and interleukin 1␤ (IL-1␤) were shown to promote degradation of the NF-B cytoplasmic inhibitor IB␣ and permit the nuclear translocation of a phosphorylated form of NF-B that is capable of binding DNA. Interestingly, when Ad12-transformed cells were treated with TNF-␣ or IL-1␤, class I gene transcription substantially increased when transcriptional repression by COUP-TFII was blocked. This indicates that in cytokine-treated Ad12transformed cells, COUP-TFII is able to repress activation of class I transcription by newly nucleus-localized NF-B. Our results suggest that Ad12 likely employs a "fail-safe" mechanism to ensure that the transcription of class I genes remains tightly repressed under various physiological conditions, thus providing tumorigenic Ad12-transformed cells with a means of escaping CTL recognition and lysis.

Research paper thumbnail of COUP-TFII Is Up-regulated in Adenovirus Type 12 Tumorigenic Cells and Is a Repressor of MHC Class I Transcription

Virology, 2001

Down-regulation of the MHC class I enhancer in tumorigenic Ad12 cells is associated with strong b... more 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-B. By comparison, in nontumorigenic Ad5 cells, class I expression is high due to negligible binding of COUP-TF and strong binding of NF-B. 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-B p50/p52 double-knockout cells enabled us to demonstrate directly that COUP-TFII can completely repress both nonactivated and NF-B-activated MHC class I transcription.

Research paper thumbnail of Association of Histone Deacetylase with COUP-TF in Tumorigenic Ad12-Transformed Cells and Its Potential Role in Shut-Off of MHC Class I Transcription

Virology, 2000

Chicken ovalbumin upstream promoter-transcription factor (COUP-TF) is an orphan nuclear receptor ... more 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.

Research paper thumbnail of Chromatin repression by COUP-TFII and HDAC dominates activation by NF-κB in regulating major histocompatibility complex class I transcription in adenovirus tumorigenic cells

Virology, 2003

In adenovirus type 12 transformed cells, the down-regulation of MHC class I transcription contrib... more In adenovirus type 12 transformed cells, the down-regulation of MHC class I transcription contributes to the tumorigenic phenotype and is solely mediated by Ad12 E1A. Previous in vitro studies with class I enhancer sequences have indicated that there is an increased binding of repressor COUP-TFII and its associated HDAC and a decreased binding of activator NF-B. In this study, we used chromatin immunoprecipitation (ChIP) assay in order to determine in vivo whether these proteins regulate class I transcription by affecting chromatin. The ChIP assay revealed that there is lack of chromatin histone acetylation in the region of the class I enhancer in Ad12-transformed cells. This is regulated by histone deacetylation as it was further demonstrated in vivo that COUP-TFII and HDAC are associated with the class I enhancer chromatin. In agreement with in vitro studies, NF-B could be recruited to the class I enhancer following induction by TNF-␣. However, this enhancer-bound NF-B failed to up-regulate class I expression because the class I enhancer chromatin remained repressed as a result of histone deacetylation by HDAC in association with COUP-TFII. Thus, we have demonstrated for the first time that repression of chromatin through histone deacetylation is a major mechanism in down-regulating class I transcription in Ad12-transformed cells. Finally, Ad12 E1A, a non-DNA binding protein, was shown to be present in the natural protein complex bound to the class I enhancer.

Research paper thumbnail of Expression of histocompatibility antigens H-2K, -D, and -L is reduced in adenovirus-12-transformed mouse cells and is restored by interferon gamma

Proceedings of the National Academy of Sciences, 1985

Primary mouse cells transformed by adenovirus type 12 (Ad12) expressed negligible amounts of clas... more Primary mouse cells transformed by adenovirus type 12 (Ad12) expressed negligible amounts of class I antigens H-2K, -D, and -L on the cell surface and were capable of forming tumors in syngeneic animals, whereas cells transformed by Ad5 continued to express class I antigens and were nontumorigenic. Cells from a tumor, generated by injection of Ad12-transformed mouse cells into a syngeneic mouse, also expressed low levels of H-2 antigens, indicating that this phenotype is maintained in vivo. In all Ad12-transformed cells, synthesis of the H-2 heavy chain was not detected whereas the beta 2-microglobulin light chain was synthesized. Furthermore, the level of cytoplasmic H-2 mRNA in the Ad12 lines was greatly reduced. Reduction of H-2 expression is instructed solely by the transforming region of the viral genome, since this repression occurred in cells transformed by a DNA fragment containing only Ad12 E1A and E1B genes. Addition of recombinant murine interferon gamma strongly stimulat...

Research paper thumbnail of Induction of Neuronal and Tumor-Related Genes by Adenovirus Type 12 E1A

Journal of Virology, 2008

Adenovirus type 12 (Ad12) E1A protein (E1A-12) contains a unique 20-amino-acid spacer region betw... more Adenovirus type 12 (Ad12) E1A protein (E1A-12) contains a unique 20-amino-acid spacer region between the second and third conserved regions. Substitution of a single amino acid in the spacer is able to abrogate Ad12 tumorigenesis. To investigate the function of the spacer, microarray analysis was performed on cells transformed by tumorigenic and nontumorigenic Ad12s that differ only by one amino acid in the spacer. Fewer than 0.8% of approximately 8,000 genes in the microarray exhibited differential expression of threefold and higher. Of these, more than half of the known genes with higher expression in the wild-type Ad12-transformed cells have neuronal-specific functions. Some of the other differentially expressed genes are involved in the regulation of the cell cycle, transcription, cell structure, and tumor invasiveness. Northern blot analyses of a subset of the neuronal genes, including Robo1, N-MYC, and α-internexin, confirmed their strong expression in multiple Ad12 tumorigeni...

Research paper thumbnail of In Adenovirus Type 12 Tumorigenic Cells, Major Histocompatibility Complex Class I Transcription Shutoff Is Overcome by Induction of NF-κB and Relief of COUP-TFII Repression

Journal of Virology, 2002

The surface levels of major histocompatibility complex class I antigens are diminished on tumorig... more The surface levels of major histocompatibility complex class I antigens are diminished on tumorigenic adenovirus type 12 (Ad12)-transformed cells, enabling them to escape from immunosurveillant cytotoxic T lymphocytes (CTLs). This is due to the down-regulation of the class I transcriptional enhancer, in which there is strong binding of the repressor COUP-TFII and lack of binding of the activator NF-κB. Even though NF-κB (p65/p50) translocates to the nuclei of Ad12-transformed cells, it fails to bind to DNA efficiently due to the hypophosphorylation of the p50 subunit. In this study, tumor necrosis factor alpha (TNF-α) and interleukin 1β (IL-1β) were shown to promote degradation of the NF-κB cytoplasmic inhibitor IκBα and permit the nuclear translocation of a phosphorylated form of NF-κB that is capable of binding DNA. Interestingly, when Ad12-transformed cells were treated with TNF-α or IL-1β, class I gene transcription substantially increased when transcriptional repression by COUP...

Research paper thumbnail of Phosphorylation of Serine 337 of NF-κB p50 Is Critical for DNA Binding

Journal of Biological Chemistry, 2003

Research paper thumbnail of DNA Binding of Repressor Nuclear Factor-κB p50/p50 Depends on Phosphorylation of Ser337 by the Protein Kinase A Catalytic Subunit

Journal of Biological Chemistry, 2005

Research paper thumbnail of Transformation by E1A Oncoprotein Involves Ubiquitin-Mediated Proteolysis of the Neuronal and Tumor Repressor REST in the Nucleus

Journal of Virology, 2012

The adenovirus early region 1A (E1A) protein promotes cell immortalization and transformation by ... more The adenovirus early region 1A (E1A) protein promotes cell immortalization and transformation by mediating the activities of key cellular regulators. The repressor element 1-silencing transcription factor (REST), which is a major neuronal and tumor suppressor, was previously found mainly in the cytoplasm rather than in the nuclei of adenovirus-transformed rodent cells (22). We now demonstrate that the loss of REST in the nucleus is due to its rapid degradation by the ubiquitin-proteasome system. Only nuclear REST, but not its cytoplasmic counterpart, was ubiquitinated and degraded. REST degradation was blocked by the ubiquitination inhibitor PYR-41 and the proteasome inhibitor MG-132 but not by the nuclear export inhibitor leptomycin B. REST degradation required both of its two C-terminal degrons that are recognized by the ubiquitin ligase SCF β-TrCP , since deletion or mutation of either degron eliminated degradation. Importantly, E1A was shown to mediate REST ubiquitination and de...

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

Journal of Virology, 1997

The CR3 activation domain of the human adenovirus E1A protein stimulates transcription by forming... more 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.

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

Journal of Virology, 1997

The CR3 activation domain of the human adenovirus E1A protein stimulates transcription by forming... more 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.

Research paper thumbnail of An ATF/CREB site is the major regulatory element in the human herpesvirus 6 DNA polymerase promoter

Journal of Virology, 1994

Human herpesvirus 6 (HHV-6) is a recently described T-cell pathogen whose medical relevance and m... more Human herpesvirus 6 (HHV-6) is a recently described T-cell pathogen whose medical relevance and molecular biology are just beginning to be addressed. As a first look at the regulation of viral genes, control of the HHV-6 DNA polymerase promoter was examined. Polymerase gene transcription in HHV-6-infected cells was found to initiate from a single site located 115 bases upstream of the translation start codon. A polymerase promoter-chloramphenicol acetyltransferase reporter gene construct failed to be expressed in uninfected T cells but was highly active in HHV-6-infected cells. Mutational data indicated that the polymerase promoter is TATA-less. Mutational analysis also revealed that the major upstream promoter regulatory element required for transcriptional activity in HHV-6-infected cells is a palindromic ATF/CREB transcription factor binding site. The significance of this site for promoter induction was further demonstrated by the fact that the polymerase ATF/CREB element, when a...

Research paper thumbnail of An ATF/CREB site is the major regulatory element in the human herpesvirus 6 DNA polymerase promoter

Journal of Virology, 1994

Human herpesvirus 6 (HHV-6) is a recently described T-cell pathogen whose medical relevance and m... more Human herpesvirus 6 (HHV-6) is a recently described T-cell pathogen whose medical relevance and molecular biology are just beginning to be addressed. As a first look at the regulation of viral genes, control of the HHV-6 DNA polymerase promoter was examined. Polymerase gene transcription in HHV-6-infected cells was found to initiate from a single site located 115 bases upstream of the translation start codon. A polymerase promoter-chloramphenicol acetyltransferase reporter gene construct failed to be expressed in uninfected T cells but was highly active in HHV-6-infected cells. Mutational data indicated that the polymerase promoter is TATA-less. Mutational analysis also revealed that the major upstream promoter regulatory element required for transcriptional activity in HHV-6-infected cells is a palindromic ATF/CREB transcription factor binding site. The significance of this site for promoter induction was further demonstrated by the fact that the polymerase ATF/CREB element, when a...

Research paper thumbnail of Cloning and Functional Analysis of Kaposi’s Sarcoma-Associated Herpesvirus DNA Polymerase and Its Processivity Factor

Journal of Virology, 1998

Kaposi’s sarcoma-associated herpesvirus (KSHV), or human herpesvirus 8, is a newly identified vir... more 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.

Research paper thumbnail of Cloning and Functional Analysis of Kaposi’s Sarcoma-Associated Herpesvirus DNA Polymerase and Its Processivity Factor

Journal of Virology, 1998

Kaposi’s sarcoma-associated herpesvirus (KSHV), or human herpesvirus 8, is a newly identified vir... more 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.

Research paper thumbnail of 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

Molecular and Cellular Biology, 1996

Diminished expression of major histocompatibility complex class I antigens on the surface of aden... more 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 ...