Interferon regulatory factor 7 (IRF7) is an interferoninducible transcription factor required for induction of delayed early interferon ␣ genes and the onset of a potent antiviral state. After induction of IRF7 by autocrine interferon, latent IRF7 is activated by virus-induced phosphorylation on serine residues within the C-terminal regulatory domain. Although it is likely that IRF7 is subjected to a cascade of events responsible for regulating its biological activity, to date no mechanism other than phosphorylation has been reported to modulate IRF7 activity. Here, we report that IRF7 is acetylated in vivo by the histone acetyltransferases p300/CBP-associated factor (PCAF) and GCN5. The single lysine residue target for acetylation, lysine 92, is located in the DNAbinding domain and is conserved throughout the entire IRF family. Mutation of lysine 92 resulted in complete abolition of DNA binding ability. However, a mutant that cannot be acetylated by PCAF due to a change in the surrounding amino acid context of lysine 92 showed increased DNA binding and activity compared with wild type IRF7. Conversely, we showed that acetylated IRF7 displayed impaired DNA binding capability and that over-expression of PCAF led to decreased IRF7 activity. Together, our results strongly suggest that acetylation of lysine 92 negatively modulates IRF7 DNA binding.

Interferon regulatory factor (IRF)7 is a key transcription factor required for establishment of antiviral resistance. In response to infection, IRF7 is activated by phosphorylation through the action of the non-canonical IB kinases, IB kinase-⑀ and TANK-binding kinase 1. Activation leads to nuclear retention, DNA binding, and derepression of transactivation ability. Clusters of serine residues located in the carboxyl-terminal regulatory domain of IRF7 are putative targets of virus-activated kinases. However, the exact sites of phosphorylation have not yet been established. Here, we report a comprehensive structure-activity examination of potential IRF7 phosphorylation sites through analysis of mutant proteins in which specific serine residues were altered to alanine or aspartate. Phosphorylation patterns of these mutants were analyzed by two-dimensional gel electrophoresis, and their transcriptional activity was monitored by reporter assays. Essential phosphorylation events were mapped to amino acids 437-438 and a redundant set of sites at either amino acids 429 -431 or 441. IRF7 recovered from infected cells was heterogeneously phosphorylated at these sites, and greater phosphorylation correlated with increased transactivation. Interestingly, a distinct serine cluster conserved in the related protein IRF3 was also essential for IRF7 activation and distal phosphorylation. However, the essential role of this motif did not appear to be fulfilled by phosphorylation. Rather, these serine residues and an adjacent leucine were required for phosphorylation at distal sites and may determine a conformational element required for function.Production of type I interferon in response to a wide variety of bacterial and viral infections is a major component of innate immunity and is essential to host defense against microbial invasion. Type I interferon (IFN) 1 family consists of a single IFN␤ gene and multiple IFN␣ genes that are clustered on mouse chromosome 4 (1) and transcriptionally activated in virus-infected cells. Transcriptional induction of the IFN␣/␤ genes is a rapid biphasic process in most cell types; initial induction of IFN␣4 and -␤ is dependent on the constitutively expressed transcription factor IRF3 and subsequent induction of the other members of the IFN␣ family (IRF7-dependent) is achieved after positive feedback resulting in IRF7 protein production and activation (2, 3).Similar to many signaling pathways, the activating switch for transcription is achieved by phosphorylation of specific transcription factors. Transcription of the IFN␤ gene is controlled by an enhanceosome composed of at least the AP1 transcription factor (c-jun-ATF2), phosphorylated by the c-Jun kinase, NFB, released from its inhibitor IB after phosphorylation-induced degradation through the action of the IB kinase (composed of IKK␣, IKK␤, and IKK␥/NEMO), and IRF3 and IRF7, activated by phosphorylation. The kinases thought responsible for phosphorylating IRF3 and IRF7 have been recently identified as distant members of the IKK family, T...

Introduction: Congenital hemophilia B is a rare blood disorder, caused by mutations in the F9 gene that lead to dysfunctional, reduced, or no clotting factor IX (FIX), resulting in prolonged bleeding episodes and in severe cases, spontaneous bleeding episodes. Maintaining sufficient FIX activity in the bloodstream through routine prophylactic administration of FIX, is the standard of care for prevention of bleeds in hemophilia B patients in Canada. Breakthrough bleeding (BTB) episodes are treated acutely with additional doses of FIX. In Canada, real-world data for patients with hemophilia B, including clinical outcomes and consumption rates of FIX, are recorded in the Canadian Bleeding Disorders Registry (CBDR). FIX products for patients with hemophilia B are subject to national competitive procurement processes administered by the Canadian Blood Service (CBS) and Héma-Québec. Nonacog beta pegol (N9-GP), an extended half-life (EHL) recombinant FIX concentrate, was recently awarded a CBS contract and subsequently made available across Canada (except Québec) from April 1, 2018 to adult patients. For those patients already on another EHL FIX treatment, a forced switch to N9-GP occurred. The objective of the present study was to estimate the impact on treatment costs of switching from a prior FIX to N9-GP, based on real-world annualized bleed rates (ABRs) and FIX consumption volumes (pre- and post-N9-GP switch) for patients on prophylaxis with their previous treatment and N9-GP, as recorded in the CBDR as of 30 September 2019. Methods: Real-world data from the CBDR for FIX consumption and ABR were used to inform a cost consequence model, developed in Microsoft Excel. Only patients for whom data existed in the CBDR for 6-months pre-switch to N9-GP and who had received ≥3 months of N9-GP treatment were included. Since April 2018, N9-GP replaced eftrenonacog alfa as the EHL product available to adult patients covered by CBS, while nonacog alfa continued to be the recombinant standard half-life (SHL) product available. Based on this, it was assumed that the EHL to N9-GP switches were from eftrenonacog alfa and the SHL switches are from nonacog alfa. For comparison of N9-GP with nonacog alfa and eftrenonacog alfa, treatment of adult males (assumed body weight of 70 kg) with severe hemophilia B was modeled over a 1-year time horizon. Since the competitive procurement process used in Canada results in confidential per-unit FIX prices, a price from a similar market was used for assessment of the cost impact. The German market was selected because all recombinant FIX products available in Canada are reimbursed in Germany. Converting the German per-IU prices, as published in the Lauer-Taxe®, using the Bank of Canada average exchange rate for the previous year, resulted in a price of CAD $2.54, $1.50 and $2.18 per IU for N9-GP, nonacog alfa, and eftrenonacog alfa, respectively. Real-world annualized mean FIX consumption volumes for prophylaxis, per BTB and real-world mean total ABRs for each product were then multiplied by the price per IU for each FIX product to derive estimates of real-world annual treatment costs associated with the use of nonacog alfa and eftrenonacog alfa (pre-switch), and N9-GP (post-switch). Results: Real-world annual prophylaxis consumption volumes, as reported in the CBDR, were reduced following treatment switch to N9-GP (Table 1). The switch to N9-GP was associated with improved ABRs, from 7.38 to 2.56 and 4.76 to 2.68 for patients on prior treatment with nonacog alfa and eftrenonacog alfa, respectively. Comparative treatment costs (for prophylaxis and BTB) based on real-world data were reduced from $643,400 to $412,700 when switching from nonacog alfa to N9-GP and from $486,938 to $358,822 when switching from eftrenonacog alfa to N9-GP. Treatment with N9-GP was therefore associated with a 35.8% and 26.3% reduction in costs following a switch from nonacog alfa, and eftrenonacog alfa, respectively. Conclusion: Real-world FIX consumption and bleeding outcomes data demonstrate that N9-GP is cost-saving compared with nonacog alfa and eftrenonacog alfa (assuming per-IU prices based on German costs converted to Canadian dollars) while also achieving a reduction in ABR, regardless of whether patients previously received an SHL or EHL FIX product. N9-GP can therefore be considered a dominant treatment option compared with nonacog alfa and eftrenonacog alfa for the treatment of hemophilia B. Disclosures MacDonald: Novo Nordisk Canada Inc: Current Employment. Lee:Novo Nordisk A/S: Current Employment. Caillaud:Novo Nordisk Canada Inc: Current Employment. Luckevich:Novo Nordisk Canada Inc.: Current Employment. Bentley:Mtech Access: Consultancy, Other: Consultant for Novo Nordisk.