Ashley Campbell, University of Toronto

Ashley Marie Campbell - Department of Molecular Genetics, University of Toronto; Talia Ho - Department of Molecular Genetics, University of Toronto; Kathy Shire - Department of Molecular Genetics, University of Toronto; Zora Sinay - Utrecht University; Edyta Marcon - Donnelly Centre, University of Toronto; Jack Greenblatt - Department of Molecular Genetics, University of Toronto, Donnelly Centre, University of Toronto; Lori Frappier - Department of Molecular Genetics, University of Toronto

Suppressing & Conquering

Epstein-Barr virus (EBV) encodes many proteins that manipulate host processes to promote viral infection. This includes post-transcriptional modulation of host mRNA, through effects on P-bodies, which play important roles in translational repression and mRNA decay. To identify EBV proteins with potential for post-transcriptional regulation, we screened a library of EBV lytic proteins for effects on P-bodies. We identified several EBV proteins that manipulate these granules, and one, BRRF1, that partially localizes to P-bodies, suggesting a role in post-transcriptional regulation. Such a role was demonstrated using luciferase reporter systems, where expression or mRNA tethering of BRRF1 decreased luciferase activity but not luciferase mRNA levels. Through affinity purification mass spectrometry, we identified BRRF1 as interacting with CCR4-NOT, a complex with multiple roles in post-transcriptional mRNA regulation including deadenylation of poly(A) tails. We subsequently showed that BRRF1 interacted with this complex through CNOT9 and that mutation of two tyrosines in BRRF1 (YY mutant) disrupted this interaction. We investigated the importance of CCR4-NOT in EBV lytic infection by silencing the CNOT1 scaffold or CNOT9 proteins, and showed that expression of select EBV late genes were decreased, supporting a proviral role for CCR4-NOT. Finally, mRNA-sequencing was performed on cells with and without BRRF1 expression, revealing that BRRF1 downregulated cellular mRNA encoding CLDN6, partly dependent on the BRRF1-CCR4-NOT interaction. In summary, we have identified novel roles for BRRF1 in post-transcriptional mRNA regulation, some of which involve interaction with CCR4-NOT, and have identified CCR4-NOT as a proviral factor for EBV lytic infection.