Regulation of the Transcriptional Activity of the IRF7 Promoter by a Pathway Independent of Interferon Signaling

Genes containing an interferon (IFN)-stimulated response element (ISRE) can be divided into two groups according to their inducibility by IFN and virus infection: one induced only by IFN and the other induced by both IFN and virus infection. Although it is now clear that IFN regulatory factor 7 (IRF7) is a multifunctional gene essential for induction of type I IFNs, regulation of the IRF7 promoter (IRF7p) is poorly understood. The IRF7 gene includes two IFN responsive elements, an IRF-binding element (IRFE) in the promoter region and an ISRE in the first intron, and is induced by the IFN-triggered Jak-STAT pathway by binding of the IFN-stimulated gene factor 3 (ISGF3) complex to the ISRE. In this study, we demonstrate that IRF3 and IRF7, which with the coactivators CREB-binding protein and P300 form the virus-activated factor (VAF) complex upon Sendai virus infection, bind to the IRF7 ISRE and IRFE and can directly activate IRF7 transcription. Promoter reporter assays show that both the ISRE and IRFE are responsive to activation by IRF7 and IRF3. In cells transiently expressing IRF7 or/and IRF3, the VAF level and binding of VAF are clearly increased after Sendai virus infection. Studies with Jak1 kinase inactive 293 cells that were stably transfected with a Jak1 kinase dead dominant negative construct, and the mutant cell lines SAN (IFNalpha-/beta-), U2A (IRF9-), U4A (Jak1-), and DKO (IRF1-/IRF2-) show that the IRF7 transcription activated directly by VAF is distinct from and independent of the IFN signaling pathway. Thus, IRF7 transcription is autoregulated by binding of the IRF7-containing VAF to its own ISRE and IRFE. The results show two distinct mechanisms for the activation of the IRF7 promoter, by IFN and by virus infection. A regulatory network between type I IFNs and IRF7 is proposed. The distinct pathways may reflect special roles for an efficient antiviral response at different stages of virus infection

Differential requirements for cis and trans V(D)J cleavage: effects of substrate length

The assembly of productive synaptic complexes is a critical, but poorly understood, regulatory step in V(D)J recombination. Several lines of evidence suggest that there may be important differences between recombination involving sites situated in cis (on the same DNA molecule) and in trans (on separate molecules). Because biochemical experiments using both purified RAG proteins and crude extracts have failed to detect trans cleavage of plasmid substrates it has been thought that there is a substantial bias against trans synapsis. In conflict with these results are more recent studies showing that purified RAG proteins can catalyze trans cleavage of short oligonucleotide substrates. Furthermore, recent experiments have detected efficient trans cleavage of plasmid substrates in vivo. We sought to investigate why these different systems yield such divergent results. We found that, unexpectedly, the ability of both purified RAG proteins and crude extracts to cleave DNA substrates in trans is a function of substrate length. Our data raise two critical issues: first, oligonucleotides, which are the most commonly used substrates to study V(D)J recombination in vitro, do not mimic the behavior of plasmid substrates; second, in the trans cleavage reaction current purified RAG systems do not accurately reflect the in vivo situation. We propose a unifying model to explain the effects of substrate length and coniguration (cis or trans) on the efficiency of synapsis

Management of patients with non-Hodgkin’s lymphoma: focus on adoptive T-cell therapy

Non-Hodgkin’s lymphoma (NHL) represents a heterogeneous group of malignancies with high diversity in terms of biology, clinical responses, and prognosis. Standard therapy regimens produce a 5-year relative survival rate of only 69%, with the critical need to increase the treatment-success rate of this patient population presenting at diagnosis with a median age of 66 years and many comorbidities. The evidence that an impaired immune system favors the development of NHL has opened the stage for new therapeutics, and specifically for the adoptive transfer of ex vivo-expanded antigen-specific T-cells. In this review, we discuss how T-cells specific for viral-associated antigens, nonviral-associated antigens expressed by the tumor, T-cells redirected through the expression of chimeric antigen receptors, and transgenic T-cell receptors against tumor cells have been developed and used in clinical trials for the treatment of patients with NHLs

Interferon Regulatory Factor 5 Represses Expression of the Epstein-Barr Virus Oncoprotein LMP1: Braking of the IRF7/LMP1 Regulatory Circuit

We have reported evidence for a positive regulatory circuit between interferon regulatory factor 7 (IRF7) and the Epstein-Barr virus (EBV) oncoprotein 1 (LMP1) (S. Ning, A. M. Hahn, and J. S. Pagano, J. Virol. 77:9359-9368, 2003). To explore a possible braking mechanism for this circuit, several type II EBV-infected cell lines that express different levels of LMP1 and IRF7 proteins and therefore are convenient for studying modulation of expression of LMP1 were analyzed. Endogenous levels of IRF7 and LMP1 were directly correlated. Transient expression of an IRF7 dominant-negative mutant decreased LMP1 levels. Endogenous IRF5 and IRF7 proteins were shown to physically associate in EBV-positive cells. Transient expression of IRF5 decreased activation of the LMP1 promoter by IRF7 in a dose-dependent manner. Finally, transfection of either an IRF5 dominant-negative construct or IRF5 small interfering RNA in these cells resulted in increases in endogenous levels of LMP1. These results indicate that IRF5 can downregulate IRF7's induction of expression of LMP1 most likely by interacting with IRF7 and provide a means of modulating a regulatory circuit between IRF7 and LMP1

Interferon Regulatory Factor 7 Is Activated by a Viral Oncoprotein through RIP-Dependent Ubiquitination▿

As a key mediator of type I interferon (IFN) (IFN-α/β) responses, IFN regulatory factor 7 (IRF7) is essential to host immune defenses. Activation of IRF7 generally requires virus-induced C-terminal phosphorylation, which leads to its nuclear accumulation and activation of target genes. Here we use the Epstein-Barr virus (EBV) oncoprotein LMP1, which activates IRF7, to identify factors involved in IRF7 activation. We demonstrate for the first time that RIP activates IRF7 and that RIP and IRF7 interact under physiological conditions in EBV-positive Burkitt's lymphoma cells. We provide evidence that both RIP and IRF7 are ubiquitinated in these cells and that IRF7 preferentially interacts with ubiquitinated RIP. RIP is required for full activation of IRF7 by LMP1, with LMP1 stimulating the ubiquitination of RIP and its interaction with IRF7. Moreover, LMP1 stimulates RIP-dependent K63-linked ubiquitination of IRF7, which regulates protein function rather than proteasomal degradation of proteins. We suggest that RIP may serve as a general activator of IRF7, responding to and transmitting the signals from various stimuli, and that ubiquitination may be a general mechanism for enhancing the activity of IRF7

Interferon Regulatory Factor 7 Regulates Expression of Epstein-Barr Virus Latent Membrane Protein 1: a Regulatory Circuit

We have shown previously that interferon regulatory factor 7 (IRF7), a multifunctional protein intimately involved in latent Epstein-Barr virus (EBV) infection, is induced as well as activated by EBV latent membrane protein 1 (LMP1), the principal EBV oncoprotein. Since the LMP1 promoter (LMP1p) contains an interferon-stimulated response element (ISRE), we hypothesized that IRF7 might be able to regulate LMP1 expression and thus participate in a regulatory circuit between these two genes. In this study, IRF7 was shown first to activate LMP1p in transient transfection assays. Compared with EBV nuclear antigen 2 (EBNA2), the most potent viral transactivator of LMP1p, IRF7 has a lesser effect (approximately 10% that of EBNA2) on induction of LMP1p. Study with IRF7 deletion mutants showed that IRF7 functional domains have similar effects on both the beta interferon (IFN-β) and LMP1 promoters in BJAB and 293 cells, and study with IRF7 phosphomimetic mutants showed that IRF7 phosphorylation may be involved in the activation of these two promoters. Further, the ISRE in LMP1p responds to IRF7 induction and IRF7 binds to this element. In the EBV-positive cell line P3HR1, which lacks the complete EBNA2 and EBV-encoded leader protein genes and hence expresses low-level LMP1, IRF7 alone can notably increase the endogenous LMP1 mRNA and protein levels. These results indicate that LMP1 is regulated by this host cell gene in addition to the viral factor, EBNA2, and may help to explain how LMP1 is expressed in type II latency in the absence of EBNA2. Moreover, IRF7 can regulate a viral gene in addition to a host cellular gene such as the IFN-β gene. Together with the previous data that LMP1 can induce IRF7 expression and facilitate IRF7 phosphorylation and nuclear translocation, these results suggest a positive regulatory circuit between IRF7 and LMP1

Mutational Analysis of All Conserved Basic Amino Acids in RAG-1 Reveals Catalytic, Step Arrest, and Joining-Deficient Mutants in the V(D)J Recombinase

Although both RAG-1 and RAG-2 are required for all steps of V(D)J recombination, little is known about the specific contribution of either protein to these steps. RAG-1 contains three acidic active-site amino acids that are thought to coordinate catalytic metal ions. To search for additional catalytic amino acids and to better define the functional anatomy of RAG-1, we mutated all 86 conserved basic amino acids to alanine and evaluated the mutant proteins for DNA binding, nicking, hairpin formation, and joining. We found several amino acids outside of the canonical nonamer-binding domain that are critical for DNA binding, several step arrest mutants with defects in nicking or hairpin formation, and four RAG-1 mutants defective specifically for joining. Analysis of coding joints formed by some of these mutants revealed excessive deletions, frequent use of short sequence homologies, and unusually long palindromic junctional inserts, known as P nucleotides, that result from aberrant hairpin opening. These features characterize junctions found in scid mice, which are deficient for the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), suggesting that the RAG proteins and DNA-PKcs perform overlapping functions in coding joint formation. Interestingly, the amino acids that are altered in 12 of our mutants are also mutated in human inherited immunodeficiency syndromes. Our analysis of these mutants provides insights into the molecular mechanisms underlying these disorders

The V(D)J Recombinase Efficiently Cleaves and Transposes Signal Joints

V(D)J recombination generates two types of products: coding joints, which constitute the rearranged variable regions of antigen receptor genes, and signal joints, which often form on immunologically irrelevant, excised circular molecules that are lost during cell division. It has been widely believed that signal joints simply convert reactive broken DNA ends into safe, inert products. Yet two curious in vivo observations made us question this assumption: signal ends are far more abundant than coding ends, and signal joints form only after RAG expression is downregulated. In fact, we find that signal joints are not at all inert; they are cleaved quite efficiently in vivo and in vitro by a nick-nick mechanism and form an excellent substrate for RAG-mediated transposition in vitro, possibly explaining how genomic stability in lymphocytes may be compromised