Mechanism Underlying The Nuclear Import Of Interferon Regulatory Factor 3 (IRF3)

Innate immune response contributes to the first line of host defense against microbial infection. Upon viral infection, cellular pathogen recognition receptors (PRRs) sense viral pathogen-associated molecular patterns (PAMPs) and trigger a series of downstream signaling cascades to stimulate the expression of type Ⅰ interferons (IFNs), inflammatory factors and antiviral effector proteins. These downstram cytokines and effector proteins act together to inhibit viral replication, eradicate virus-invaded cells, and facilitate the initiation of adaptive immune response.Accurate cellular localization plays a crucial role in the effective function of most signaling proteins, and nuclear trafficking is central to the function of transcription factors. The interferon regulatory factor 3 (IRF3) is a master transcription factor responsible for the induction of type Ⅰ interferon (IFN), which plays a crucial role in host antiviral innate immune responses. Upon virus infection, IRF3 undergoes C-terminal phosphorylation, dimerization, and then translocates into the nucleus, subsequently forming a complex with the coactivators CBP/p300 and binding to the positive regulatory domain (PRD) and PRD-LEs of the IFN-α/β promoters and interferon-stimulated response element (ISRE) of targeted genes, including cytokines RANTE and IP 10. The biological effect of IRF3 transcriptional activity requires its ability to translocate into the nucleus. For this reason, the nuclear-cytoplasmic shuttling is a critical aspect of its regulation.In our study, we demonstrated that IRF3 contains a bipartite NLS that controls both DNA binding activity and nuclear import. First, analysis of green fluorescent protein (GFP)-IRF3 fusion proteins shows that IRF3 contains the necessary sequences within or flanking the DBD (amino acids 64-130) to specify its nuclear import. Further mutagenesis determined the basic amino acids responsible for IRF3 nuclear import.Our studies showed that the bipartite NLS of IRF3 is characterized by the basic amino acidclusters 77KR78and 86RK87,which occupy theminor site and major binding site for importin alpha, respectively. Mutation of 77KR78 partially abrogates the nulcear import ability, whereas mutation of 86RK87 totally disrupts the nuclear import ability. Immunoflurescence imaging assays furthermore reinforced our results. Our study further demonstrated that the bipartite NLS of IRF3 is also critical for IRF3 DNA-binding activity, indicating that the two functions of this region are integrated, which is unique in the IRF family proteins. Finally, we demonstrated that IRF3 NLS is essential for virus-induced IFN-β production and cellular antiviral immune response through viral infection and IFN bioassay.Overall, our results revealed a previously unrecognized bipartite nuclear localization signal for IRF3 that contains both DNA-binding and nuclear import ability, which is of great interest to characterization of IRF3 regulation and IRF3-mediated antiviral immune responses.