Fig 2: Model of interaction between an oncolytic virus and the host type I IFN response and cyclin expression. Host pattern recognition receptors (PRR) will recognize intracellular viral particles and initiate a type I IFN response. Upon viral recognition, IFN signals through the type I IFNα/β receptor (IFNAR) leading to the activation of Janus kinase 1 and/or tyrosine kinase 2 (JAK1/TYK2) and recruitment of signal transducer and activator of transcription 1 (STAT1) for phosphorylation and dimerization. The phosphorylated STAT1 dimer then binds IRF9 to form the interferon-stimulated gene factor 3 complex (ISGF3) complex, which binds IFN-stimulated response elements located in ISGs, resulting in the activation of antiviral genes. Some of the most well-studied antiviral genes include OAS1 and MxA. OAS1 is dsRNA-dependent synthetase that activates the endoribonuclease RNase L to degrade ssRNA while MxA proteins bind target viral proteins to inhibit viral replication. The expression of these ISGs induces an antiviral state. Viruses have developed mechanisms to antagonize the type I IFN response. The ability of E1A protein to decrease IRF9 levels, thereby inhibiting the type I IFN pathway, is shown. Viral E1A protein promotes cyclin E expression, which in turn, binds to and promotes the activation of CDK2. Activated CDK2 promotes viral replication. Palbociclib increases IRF9 expression and promotes cyclin E/CDK2 activation. Thus, the ability of the oncolytic virus to replicate and cause cytotoxicity will be dependent on the level of activation of type I IFN and cyclin E/CDK2.

Fig 3: Mechanisms of U-ISGF3 complex in sorafenib resistance. U-ISGF complex, unphosphorylated interferon-stimulated gene factor-3; U-STAT1, unphosphorylated signal transducer and activator of transcription 1; U-STAT2, unphosphorylated signal transducer and activator of transcription 2; IRF9, interferon regulatory factor 9; U-ISGs, Unphosphorylated interferon-stimulated genes.

Fig 4: Interferon-stimulated genes (ISG) are regulated by EBV miRNAs. (A) 293T cells were cotransfected with individual EBV miRNA expression vectors or an empty vector as control and a reporter plasmid (p6898) containing a luc2 gene under the control of a chimeric promoter consisting of the ISG Mx2 promoter and five canonical ISREs. After 24 h, cells were treated with type I IFN and luciferase signals were measured the next day. As a control, an expression vector encoding human miRNAs (miR-373) was used (p6481). A further control was the IE1 gene from CMV, which is known to inhibit type I IFN signaling (49). P values were calculated by one-way ANOVA test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. (B to G) 293T cells were cotransfected with the indicated wild-type luciferase reporter plasmids (WT) for CBP, IRF9, JAK1, JAK2, MAPK2, and IKKβ or reporter plasmids in which the predicted seed sequences were mutated (mut) together with or without an miRNA-encoding plasmid. The luciferase expression in these cells was assessed and normalized to lysates from cells cotransfected with the wild-type 3′-UTR reporter and empty plasmid (Ø). P values were calculated using the one-way ANOVA test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. (H) Human primary B lymphocytes were infected with wild-type EBV (r_wt/B95.8) or EBV devoid of its miRNAs (r_ΔmiR). Five days postinfection, the transformed EBV-infected cells were counted and seeded at the same density. Four days later, the cells were lysed and the lysates were subjected to quantitative Western blot analysis using the Western blot stain-free TGX Bio-Rad normalization approach (Bio-Rad) and antibodies against IPO7, IFIT1, IRF9, and CBP. The applied protein amounts were analyzed and used to normalize the levels of the specific protein signals. The ratios of protein levels of cells infected with wild-type virus versus cells infected with mutant virus are shown. The figure summarizes the results of four independent replicates. (I) HEK293T cells were cotransfected with a pLCE-based EBV miRNA expression vector, a SIS-inducible element (SIE) responsive firefly luciferase reporter, and a pRL-SV40 renilla luciferase reporter as an internal control. Forty-eight hours after transfection, cells were either mock treated or treated with 10 U per ml universal type I interferon (uIFN) for 6 h and analyzed for luciferase activity. Values are normalized to mock-treated cells and reported relative to the pLCE control. Shown are the averages from three to four independent experiments performed in triplicate. Data were analyzed using Student's t test. *, P < 0.05; **, P < 0.02.

Fig 5: Regulation of viral replication and oncolysis by IFN-regulatory factor 9 (IRF9). Cells were transfected with either control or IRF9-specific siRNA #2 followed by infection with AdGFP or OAdmCherry at a multiplicity of infection (MOI) concentration of five alone or in combination with 500 nM palbociclib for 48 h. (A) Type I IFN signaling and expression of E1A viral protein was assessed by immunoblotting with the indicated antibodies. Quantitative densitometry analysis is shown relative to loading control normalized to palbociclib-sensitive control-treated cells (except for MxA in MDA-MB-231 cells and E1A blots where samples were normalized to OAdmCherry Ctrl). (B) mCherry expression was evaluated by fluorescence microscopy. Bright field images illustrate cytopathic effect (CPE). Scale: 200 µm. Images are representative of three independent experiments. (C) Cell lysates were harvested and combined with collected supernatants to determine OAdmCherry viral titers. (D) Cell number was assessed by crystal violet staining and calculated by measuring the absorbance of solubilized dye at 590 nm. Results are expressed as percent (%) of AdGFP vehicle-treated cells. Error bars, ±SEM of three independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001; compared to the control unless otherwise indicated.