Regulated Nuclear-Cytoplasmic Localization of Interferon Regulatory Factor 3, a Subunit of Double-Stranded RNA-Activated Factor 1

Abstract

Viral double-stranded RNA (dsRNA) generated during the course of infection leads to the activation of a latent transcription factor, dsRNA-activated factor 1 (DRAF1). DRAF1 binds to a DNA target containing the type I interferon-stimulated response element and induces transcription of responsive genes. DRAF1 is a multimeric transcription factor containing the interferon regulatory factor 3 (IRF-3) protein and one of the histone acetyl transferases, CREB binding protein (CBP) or p300 (CBP/p300). In uninfected cells, the IRF-3 component of DRAF1 resides in the cytoplasm. The cytoplasmic localization of IRF-3 is dependent on a nuclear export signal, and we demonstrate IRF-3 recognition by the chromosome region maintenance 1 (CRM1) (also known as exportin 1) shuttling receptor. Following infection and specific phosphorylation, IRF-3 accumulates in the nucleus where it associates with CBP and p300. We identify a nuclear localization signal (NLS) in IRF-3 that is critical for nuclear accumulation. Mutation of the NLS abrogates nuclear localization even following infection. The NLS appears to be active constitutively, but it is recognized by only a subset of importin-α shuttling receptors. Evidence is presented to support a model in which IRF-3 normally shuttles between the nucleus and the cytoplasm but cytoplasmic localization is dominant prior to infection. Following infection, phosphorylated IRF-3 can bind to the CBP/p300 proteins resident in the nucleus. We provide the evidence of a role for CBP/p300 binding in the nuclear sequestration of a transcription factor that normally resides in the cytoplasm.

ACKNOWLEDGMENTS

We thank the members of our laboratory who have provided helpful suggestions and Jennifer Gallub for her technical assistance. Our thanks extend to Richard Goodman for the gifts of CBP and p300 expression plasmids, Gerard Grosveld for the gift of CRM1 expression plasmid, David Livingston for the gift of p300 cDNA plasmids, John Hiscott for the gift of the IRF-3/5D plasmid, Barbara Wolff for the gift of leptomycin B, Connie Nguyen and Susan Taylor for the gift of the PKI clone, Karsten Weis for the gift of the hSRP1α clone, Takemi Enomoto for the gift of the Qip1 clone, Particia Cortes for the gift of the hSRP1 clone, and Y. Hirai for the gift of the KPNA3 clone.

This work was supported by grants from the National Institutes of Health (RO1CA50773 and PO1CA28146) to N.C.R. and by a scholarship from the Council for Tobacco Research to C.D.