No abstract

The adenovirus (Adv) oncoprotein E1A stimulates cell proliferation and inhibits differentiation. These activities are primarily linked to the N-terminal region (exon 1) of E1A, which interacts with multiple cellular protein complexes. The C terminus (exon 2) of E1A antagonizes these processes, mediated in part through interaction with C-terminal binding proteins 1 and 2 (CtBP1/2). To identify additional cellular E1A targets that are involved in the modulation of E1A C-terminus-mediated activities, we undertook tandem affinity purification of E1A-associated proteins. Through mass spectrometric analysis, we identified several known E1A-interacting proteins as well as novel E1A targets, such as the forkhead transcription factors, FOXK1/K2. We identified a Ser/Thr-containing sequence motif in E1A that mediated interaction with FOXK1/K2. We demonstrated that the E6 proteins of two beta-human papillomaviruses (HPV14 and HPV21) associated with epidermodysplasia verruciformis also interacted with FOXK1/K2 through a motif similar to that of E1A. The E1A mutants deficient in interaction with FOXK1/K2 induced enhanced cell proliferation and oncogenic transformation. The hypertransforming activity of the mutant E1A was suppressed by HPV21 E6. An E1A-E6 chimeric protein containing the Ser/Thr domain of the E6 protein in E1A interacted efficiently with FOXK1/K2 and inhibited cell transformation. Our results suggest that targeting FOXK1/K2 may be a common mechanism for certain beta-HPVs and Adv5. E1A exon 2 mutants deficient in interaction with the dual-specificity kinases DYRK1A/1B and their cofactor HAN11 also induced increased cell proliferation and transformation. Our results suggest that the E1A C-terminal region may suppress cell proliferation and oncogenic transformation through interaction with three different cellular protein complexes: FOXK1/K2, DYRK(1A/1B)/HAN11, and CtBP1/2. Adenovirus (Adv) is a model DNA tumor virus that has been widely used to decipher critical pathways of oncogenesis. The adenovirus early gene E1A is an intensely investigated viral oncogene and has been instrumental in uncovering common cell cycle-regulatory pathways shared by other DNA tumor virus oncogenes such as human papillomavirus (HPV) E7 (reviewed in reference 43) and simian virus 40 (SV40) T antigen (Ag) (reviewed in reference 9). E1A promotes cellular entry into S phase by deregulating the cell cycle and activates other early viral genes to facilitate viral replication. As a consequence of nonproductive infection, E1A immortalizes rodent cells and also oncogenically transforms these cells in cooperation with other viral or cellular oncogenes (27,32,52,62).The E1A gene encodes two major protein isoforms that are expressed from two mRNAs (13S and 12S) generated by alternative RNA splicing. The 13S mRNA encodes a 289-aminoacid ([aa] 289R) protein while the 12S mRNA encodes a 243-amino-acid (243R) protein. The 289R and 243R proteins differ by a 46-amino-acid region that is unique to 289R. The 243R protein is not required for viral replica...

Organic amine-based buffer compounds such as HEPES (Good's buffers) are commonly applied in experimental systems, including those where the biological effects of peroxynitrite are studied. In such studies 3-morpholinosydnonimine N-ethylcarbamide (SIN-1)

h Hepatitis B virus (HBV) remains a major human pathogen despite the development of both antiviral drugs and a vaccine, in part because the current therapies do not suppress HBV replication far enough to eradicate the virus. Here, we screened 51 troponoid compounds for their ability to suppress HBV RNaseH activity and HBV replication based on the activities of ␣-hydroxytropolones against HIV RNaseH, with the goal of determining whether the tropolone pharmacophore may be a promising scaffold for anti-HBV drug development. Thirteen compounds inhibited HBV RNaseH, with the best 50% inhibitory concentration (IC 50 ) being 2.3 M. Similar inhibition patterns were observed against HBV genotype D and C RNaseHs, implying limited genotype specificity. Six of 10 compounds tested against HBV replication in culture suppressed replication via blocking of viral RNaseH activity, with the best 50% effective concentration (EC 50 ) being 0.34 M. Eighteen compounds inhibited recombinant human RNaseH1, and moderate cytotoxicity was observed for all compounds (50% cytotoxic concentration [CC 50 ] ‫؍‬ 25 to 79 M). Therapeutic indexes ranged from 3.8 to 94. Efficient inhibition required an intact ␣-hydroxytropolone moiety plus one or more short appendages on the tropolone ring, but a wide variety of constituents were permissible. These data indicate that troponoids and specifically ␣-hydroxytropolones are promising lead candidates for development as anti-HBV drugs, providing that toxicity can be minimized. Potential anti-RNaseH drugs are envisioned to be employed in combination with the existing nucleos(t)ide analogs to suppress HBV replication far enough to block genomic maintenance, with the goal of eradicating infection. More than 2 billion people have been infected with hepatitis B virus (HBV) at some time in their lives and up to 350 million remain chronically infected as carriers of HBV (1, 2). Approximately 20% of chronic hepatitis B patients develop liver cirrhosis, leading to hepatic insufficiency and portal hypertension (3). Furthermore, there is a 100-fold higher risk of development of hepatocellular carcinoma in chronic HBV patients than in noncarriers (4). Every year, HBV infection kills more than 500,000 people from cirrhosis, liver failure, and hepatocellular carcinoma (5).The global level of chronic HBV infection still mandates development of new drugs despite the development of excellent vaccines and drugs against the virus. Seven drugs have been approved by the U.S. Food and Drug Administration for treating HBV infection. Interferon alpha and pegylated interferon alpha are immunomodulatory agents. However, the need for subcutaneous administration, the poor long-term responses, the very low cure rates, and the high frequency of adverse side effects make interferon far from an ideal drug (6). The nucleos(t)ide analog drugs lamivudine, adefovir, entecavir, telbivudine, and tenofovir are phosphorylated to their triphosphate derivatives by cellular enzymes and become chain-terminating substrates of the HBV reverse transcr...

Nucleos(t)ide analog drugs profoundly suppress Hepatitis B Virus (HBV) replication but rarely cure the infection, so therapy is usually life-long. The nucleos(t)ide analogs inhibit the viral DNA polymerase and often push HBV to the brink of extinction, so it may be possible to eradicate HBV by suppressing HBV replication further. The HBV ribonuclease H (RNaseH) is a logical new drug target because it is the second of only two viral enzymes essential for viral replication. We recently developed a low throughput screening pipeline for inhibitors of the HBV RNaseH and viral replication. Here, we screened a series of twenty-three nitrogen-based polyoxygenated heterocycles including sixteen 2-hydroxyisoquinoline-1,3(2H,4H)-dione derivatives for anti-HBV RNaseH activity. Nine compounds inhibited the HBV RNaseH, but activity was marginal for eight of them. Compound #1 [2-hydroxyisoquinoline-1,3(2H,4H)-dione, HID] was the best hit with an IC50 of 28.1 µM and an EC50 of 4.2 µM. It preferentially suppressed accumulation of the viral plus-polarity DNA strand in replication inhibition assays, indicating that replication was blocked due to suppression of HBV RNaseH activity. It had a CC50 of 75 µM, yielding a therapeutic index of ~18. The EC50 value was 7-fold lower than the IC50, possibly due to cellular retention or metabolism of the compound, or higher affinity for the full-length enzyme than the recombinant form used for screening. These data indicate that the 2-hydroxyisoquinoline-1,3(2H,4H)-diones will have different structure-activity relationships for the HBV and HIV RNaseHs. Therefore, HID compounds may provide a foundation for development of more effective RNaseH inhibitors of HBV replication.