Hancheng Guan

DNA binding of repressor nuclear factor-κB p50/p50 depends on phosphorylation of Ser337 by the protein kinase A catalytic subunit

The NF-κB p50/p50 homodimer is mainly associated with transcriptional repression. Previously, we demonstrated that phosphorylation of NF-κB p50 Ser337 is critical for DNA binding. Here, we report that p50 Ser337 is constitutively phosphorylated by the protein kinase A catalytic subunit (PKAc) in three different cell types, which may account for the constant binding of p50/p50 to DNA in unstimulated cells. This was demonstrated first by showing that treatment of cells with PKAc-specific inhibitors blocked p50/p50 DNA binding. Second, phosphorylation of p50 by PKAc was prevented by substitution of Ser337 to alanine. Third, both p50 and PKAc proteins as well as kinase activity that phosphorylates p50 were found to co-fractionate following gel filtration chromatography. Finally, PKAc and p50 were shown to be able to reciprocally co-immunoprecipitate one another, and their physical association was blocked by a PKA catalytic site inhibitory peptide. This indicates that phosphorylation of p50 Ser337 involves direct contact with the PKAc catalytic center. In contrast to the dramatic elevation of nuclear p50/p65 heterodimers induced by tumor necrosis factor α, DNA binding of p50/p50 homodimers was not greatly altered. Taken together, these findings reveal for the first time that there is a direct interaction between PKAc and p50 that accounts for constitutive phosphorylation of p50 Ser337 and the existence of DNA bound p50/p50 in the nuclei of most resting cells. This mechanism of DNA binding by p50/p50 following phosphorylation of Ser337 by PKAc may represent an important means for maintaining stable negative regulation of NF-κB gene expression in the absence of extracellular stimulation.

Design of potent poxvirus inhibitors of the heterodimeric processivity factor required for viral replication.

Smallpox constitutes a major bioterrorism threat, which underscores the need to develop antiviral drugs for rapid response in the event of an attack. Viral processivity factors are attractive drug targets in being both specific and essential for their cognate DNA polymerases to synthesize extended strands of DNA. An in silico model of the vacinnia virus processivity factor, comprised of the A20 and D4 heterocomplex, was constructed and used for lead optimization of an indole-based scaffold identified earlier from a high-throughput screening. On the basis of this model, a new class of potent antivirals against vaccinia virus was designed and synthesized, of which two (24a and 24b) exhibited superior improvement over the parent scaffold (IC50 = 42 and 46 vs 82000 nM, respectively). The ability of 24a to suppress vaccinia DNA synthesis is supported by the inhibition of late viral gene expression, as well as by the diminished incorporation of bromodeoxyuridine into viral replication factories.

Identification of genes associated with adenovirus 12 tumorigenesis by microarray

A total of 242 genes were shown to be differentially expressed between haplotypically matched tumorigenic adenovirus 12 (Ad12) and nontumorigenic Ad5-transformed cells using a microarray containing 8734 cDNAs. Eighty-seven of the differentially expressed genes have known roles that include signal transduction, cell growth and proliferation, transcription regulation, protease, and immune functions. The remaining differentially expressed genes are represented by EST cDNAs which have functions that are either completely unknown or proposed, based on sequence similarity to known genes. A subset of 22 differentially expressed genes from the microarray was further examined by Northern blot analyses to verify the identification of new genes associated with Ad12 tumorigenesis. Growth factor receptor binding protein 10 (Grb10) and protease nexin 1 (PN-1) were overexpressed in all of the tumorigenic Ad12-transformed cells examined, whereas expression of these genes was negligible in all of the nontumorigenic Ad5-transformed cells. By contrast, other genes including B cell translocation gene 2 (BTG2) were shown to be significantly up-regulated in Ad5-transformed cells as compared to Ad12-transformed cells.

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

ABSTRACT 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-TFII was blocked. This indicates that in cytokine-treated Ad12-transformed cells, COUP-TFII is able to repress activation of class I transcription by newly nucleus-localized NF-κB. 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.

Tumorigenic Adenovirus Type 12 E1A Inhibits Phosphorylation of NF-κB by PKAc, Causing Loss of DNA Binding and Transactivation

ABSTRACT Human adenovirus type 12 (Ad12) E1A protein (E1A-12) is the key determinant of viral tumorigenesis. E1A-12 mediates major histocompatibility complex class I (MHC-I) shutoff by inhibiting the DNA binding of the transcriptional activator NF-κB (p50/p65) to the class I enhancer. This enables Ad12 tumorigenic cells to avoid class I recognition and lysis by cytotoxic T lymphocytes. In this study, we demonstrate that the phosphorylation of p50 and p65 by the catalytic subunit of protein kinase A (PKAc) is essential for NF-κB DNA binding and transactivation activity. Treatment with H89 and knockdown of PKAc in cells led to the inhibition of phosphorylation at p50 Ser337 and p65 Ser276 and loss of DNA binding by NF-κB. Importantly, NF-κB phosphorylation by PKAc was repressed by tumorigenic E1A-12, but not by nontumorigenic Ad5 E1A (E1A-5). The stable introduction of E1A-12 into Ad5 nontumorigenic cells resulted in a decrease in the phosphorylation of NF-κB, loss of NF-κB DNA binding, and the failure of NF-κB to activate a target promoter, as well as diminution of MHC-I transcription and cell surface expression. Significantly, the amount and enzymatic activity of PKAc were not altered in Ad12 tumorigenic cells relative to its amount and activity in nontumorigenic Ad5 cells. These results demonstrate that E1A-12 specifically prevents NF-κB from being phosphorylated by PKAc.

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

ABSTRACT 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 SCFβ-TrCP, since deletion or mutation of either degron eliminated degradation. Importantly, E1A was shown to mediate REST ubiquitination and degradation by upregulating β-TrCP. Knockdown of E1A in virus-transformed cells reduced both β-TrCP and ubiquitination of nuclear REST. In contrast, when expressed in HeLa cells, E1A enhanced the degradation of nuclear REST. Reconstitution of REST in virus-transformed cells negatively affected E1A-mediated cell proliferation and anchorage-independent growth. These data strongly indicate that E1A stimulates ubiquitination and proteolysis of REST in the nucleus, thereby abolishing the tumor suppressor functions of REST.

The N Terminus of Adenovirus Type 12 E1A Inhibits Major Histocompatibility Complex Class I Expression by Preventing Phosphorylation of NF-κB p65 Ser276 through Direct Binding

ABSTRACT The immune-escape strategy employed by human oncogenic adenovirus type 12 (Ad12) involves downregulation of major histocompatibility complex class I (MHC-I) transcription by disabling the transactivator NF-κB (p50/p65). This is accomplished by the Ad12 E1A protein (E1A-12), which prevents NF-κB from becoming phosphorylated by the protein kinase A catalytic subunit (PKAc). In this study, we examined the interactions between E1A-12 and NF-κB. Our data show that an E1A-12 mutant retaining the N-terminal 66 amino acids was as effective as the wild-type E1A-12 protein (266 amino acids) in binding p65, preventing phosphorylation of p65-Ser276, and inhibiting transactivation. In contrast, the nontumorigenic adenovirus type 5 E1A protein (E1A-5) and other E1A-12 mutants lacking the N-terminal regions were severely defective in these activities. Further studies revealed that an N-terminal peptide consisting of residues 1 to 40 of E1A-12 was able to associate directly with p65 in vitro and prevent PKAc from phosphorylating p65-Ser276. In the absence of the N terminus, there is an almost complete loss of E1A-12 binding to p65. These findings provide solid evidence for the role of the E1A-12 N terminus as an NF-κB binding domain. Significantly, this study indicates that the E1A-12 N terminus prevents PKAc from gaining access to p65 to account for Ser276 hypophosphorylation. The E1A-12 N terminus interaction with p65 serves as a key explanation of how Ad12 downregulates MHC-I transcription and contributes to oncogenesis by escaping cytotoxic T lymphocytes.

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

ABSTRACT 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 tumorigenic cell lines. In contrast, these neuronal genes displayed only minor or negligible expression in cells transformed by spacer-mutated Ad12. Significantly, stable introduction of E1A-12 into nontumorigenic Ad5-transformed cells induced neuronal gene expression. We found that the neuron-restrictive silencer factor, which serves as a master repressor of neuronal genes, was inactivated in both Ad12- and Ad5-transformed cells via cytoplasmic retention, though only Ad12-transformed cells exhibited neuronal gene induction. Mutational analyses of the α-internexin promoter demonstrated that E1A-12-mediated neuronal gene induction further required the activation of neuronal promoter E-box elements. These results indicate that the spacer is involved in mediating neuronal and tumor-related genes.

The processivity factor complex of feline herpes virus-1 is a new drug target.

Feline herpes virus-1 (FHV-1) is ubiquitous in the cat population and is a major cause of blindness for which antiviral drugs, including acyclovir, are not completely effective. Recurrent infections, due to reactivation of latent FHV-1 residing in the trigeminal ganglia, can lead to epithelial keratitis and stromal keratitis and eventually loss of sight. This has prompted the medical need for an antiviral drug that will specifically inhibit FHV-1 infection. A new antiviral target is the DNA polymerase and its associated processivity factor, which forms a complex that is essential for extended DNA strand synthesis. In this study we have cloned and expressed the FHV-1 DNA polymerase (f-UL30) and processivity factor (f-UL42) and demonstrated that both proteins are required to completely synthesize the 7249 nucleotide full-length DNA from the M13 primed-DNA template in vitro. Significantly, a known inhibitor of human herpes simplex virus-1 (HSV-1) processivity complex was shown to inhibit FHV-1 processive DNA synthesis in vitro and block infection of cells. This validates using f-UL42/f-UL30 as a new antiviral drug target to treat feline ocular herpes infection.

A Novel Target and Approach for Identifying Antivirals against Molluscum Contagiosum Virus

ABSTRACT The dermatological disease molluscum contagiosum (MC) presents as lesions restricted solely to the skin. The poxvirus molluscum contagiosum virus (MCV) is responsible for this skin disease that is easily transmitted through casual contact among all populations, with greater frequency in children and immunosuppressed individuals. In addition, sexual transmission of MCV in adolescents and adults is a health concern. Although the skin lesions ultimately resolve in immunocompetent individuals, they can persist for extended periods, be painful, and result in scarring. Treatment is problematic, and there is no drug that specifically targets MCV. The inability of MCV to propagate in cell culture has impeded drug development. To overcome these barriers, we integrated three new developments. First, we identified a new MCV drug target (mD4) that is essential for processive DNA synthesis in vitro. Second, we discovered a small chemical compound that binds to mD4 and prevents DNA synthesis in vitro. Third, and most significant, we engineered a hybrid vaccinia virus (mD4-VV) in which the natural vaccinia D4 (vD4) gene is replaced by the mD4 target gene. This hybrid virus is dependent on mD4 for viral growth in culture and is inhibited by the small compound. This target system provides, for the first time, a platform and approach for the discovery and evaluation of new therapeutics that can be used to treat MC.