Degradation of a novel DNA damage response protein, tankyrase 1 binding protein 1, following adenovirus infection

ABSTRACT Infection by most DNA viruses activates a cellular DNA damage response (DDR), which may be to the detriment or advantage of the virus. In the case of adenoviruses, they neutralize antiviral effects of DDR activation by targeting a number of proteins for rapid proteasome-mediated degradation. We have now identified a novel DDR protein, tankyrase 1 binding protein 1 (TNKS1BP1) (also known as Tab182), which is degraded during infection by adenovirus serotype 5 and adenovirus serotype 12. In both cases, degradation requires the action of the early region 1B55K (E1B55K) and early region 4 open reading frame 6 (E4orf6) viral proteins and is mediated through the proteasome by the action of cullin-based cellular E3 ligases. The degradation of Tab182 appears to be serotype specific, as the protein remains relatively stable following infection with adenovirus serotypes 4, 7, 9, and 11. We have gone on to confirm that Tab182 is an integral component of the CNOT complex, which has transcriptional regulatory, deadenylation, and E3 ligase activities. The levels of at least 2 other members of the complex (CNOT3 and CNOT7) are also reduced during adenovirus infection, whereas the levels of CNOT4 and CNOT1 remain stable. The depletion of Tab182 with small interfering RNA (siRNA) enhances the expression of early region 1A proteins (E1As) to a limited extent during adenovirus infection, but the depletion of CNOT1 is particularly advantageous to the virus and results in a marked increase in the expression of adenovirus early proteins. In addition, the depletion of Tab182 and CNOT1 results in a limited increase in the viral DNA level during infection. We conclude that the cellular CNOT complex is a previously unidentified major target for adenoviruses during infection. IMPORTANCE Adenoviruses target a number of cellular proteins involved in the DNA damage response for rapid degradation. We have now shown that Tab182, which we have confirmed to be an integral component of the mammalian CNOT complex, is degraded following infection by adenovirus serotypes 5 and 12. This requires the viral E1B55K and E4orf6 proteins and is mediated by cullin-based E3 ligases and the proteasome. In addition to Tab182, the levels of other CNOT proteins are also reduced during adenovirus infection. Thus, CNOT3 and CNOT7, for example, are degraded, whereas CNOT4 and CNOT1 are not. The siRNA-mediated depletion of components of the complex enhances the expression of adenovirus early proteins and increases the concentration of viral DNA produced during infection. This study highlights a novel protein complex, CNOT, which is targeted for adenovirus-mediated protein degradation. To our knowledge, this is the first time that the CNOT complex has been identified as an adenoviral target. </jats:p

Amisulpride and olanzapine combination treatment versus each monotherapy in acutely ill patients with schizophrenia in Germany (COMBINE): a double-blind randomised controlled trial

Background Combining antipsychotics is common in schizophrenia treatment, despite evidence-based guidelines generally not recommending such practice. Otherwise, evidence remains inconclusive, especially regarding specific combinations. The trial aimed to test whether a combination of amisulpride plus olanzapine is more effective than either intervention as a monotherapy. Methods A multicentre, 16-week, randomised, double-blind, controlled trial was done at 16 psychiatric in-patient centres throughout Germany. Inclusion criteria were adults aged 18-65 years with non-first episode schizophrenia or schizoaffective disorder and with a Positive and Negative Syndrome Scale (PANSS) total score of at least 70 and at least two items of the positive symptoms subscale rated at least 4. Patients were randomly assigned to receive 16 weeks of treatment with either amisulpride plus olanzapine, amisulpride plus placebo, or olanzapine plus placebo (1:1:1), and block randomisation was stratified by study site. To keep patients and investigators masked throughout the duration of the trial, amisulpride, olanzapine, and placebo were administered as identical capsules. Flexibly dosed monotherapy of oral amisulpride (amisulpride plus placebo, 200-800 mg per day) or olanzapine (olanzapine plus placebo, 5-20 mg per day) was compared with a combination of amisulpride plus olanzapine. The primary outcome was symptom reduction measured by the PANSS total score after 8 weeks, in the modified intention-to-treat population (all patients randomly assigned to an intervention and receiving at least one study drug dose). As determined a priori, group differences were examined by t tests (Bonferroni-Holm-adjustment) followed by pre-planned Bayesian analyses as well as imputation methods based on mixed models to account for missing values and post-hoc ANCOVA adjusting for PANSS baseline scores. The study was registered on ClinicalTrials.gov, NCT01609153; the German Clinical Trials Register, DRKS00003603; and the European Union Drug Regulating Authorities Clinical Trials Database, EudraCT-No. 2011-002463-20. Findings Between June 15, 2012, and Dec 15, 2018, 13 692 patients were assessed for eligibility. 13 364 patients were excluded (including for not meeting inclusion criteria, declining to participate, or inappropriate reasons for changing pharmacological treatment), and 328 were then randomly assigned to an intervention group. 112 patients were randomly assigned to receive amisulpride plus olanzapine, 109 were randomly assigned to receive amisulpride plus placebo, and 107 were randomly assigned to receive olanzapine plus placebo. 321 patients were analysed for the primary outcome in the modified intention-to-treat population after exclusion of screening failures and patients who did not receive the intervention (110 for amisulpride plus olanzapine, 109 for amisulpride plus placebo, and 102 for olanzapine plus placebo). Among the 321 patients who were randomly assigned to intervention groups and analysed for the primary outcome, 229 (71%) were male, 92 (29%) were female; the mean age was 40.2 years (SD 11.7); and 296 (92%) were White and 25 (8%) were classified as other ethnicity. PANSS total score improved significantly more at 8 weeks in the amisulpride plus olanzapine group (-29.6 [SD 14.5]) than in the olanzapine plus placebo group (-24.1 [13.4], p=0.049, Cohen's d=0.396). A significant difference was not observed in reduction of PANSS total score between the amisulpride and olanzapine group compared with the amisulpride and placebo group (-25.2 [SD 15 .9], p=0.095, Cohen's d=0.29). After 8 weeks and 16 weeks, sexual dysfunction, weight, and waist circumference increase were significantly higher for patients receiving amisulpride plus olanzapine than for those receiving amisulpride plus placebo, with no differences in serious adverse events. Two patients died during study participation; one randomly assigned to the amisulpride plus olanzapine group, and one assigned to the olanzapine plus placebo group (both assessed with no relation to treatment). Interpretation The advantages of amisulpride plus olanzapine have to be weighed against a higher propensity for side-effects. The use of this specific combination therapy could be an alternative to monotherapy in certain clinical situations, but side-effects should be considered. Copyright (C) 2022 Elsevier Ltd. All rights reserved