Novel roles of the Epstein-Barr virus BRRF1 protein in post-transcriptional RNA regulation

In lytic infection, Epstein-Barr virus (EBV) proteins manipulate cellular processes in multiple ways to facilitate virion production and overcome host antiviral responses. Here, we screened a library of EBV lytic proteins for those that manipulate or localize to cellular processing bodies (PBs), known to play important roles in RNA surveillance and miRNA-mediated translational control. One EBV protein, BRRF1, was identified that partially localized to PBs, and affinity purification-mass spectrometry experiments showed that it interacted with the CCR4-NOT complex-a known component of PBs with multiple roles in post-transcriptional mRNA regulation. The CNOT9 subunit of CCR4-NOT was sufficient to bind BRRF1, and its downregulation diminished BRRF1 interaction with the CNOT1 scaffold protein, indicating its importance in mediating the CCR4-NOT interaction with BRRF1. A BRRF1 double-point mutant was generated that failed to bind CNOT9 or interact with CCR4-NOT and was used to assess the importance of the BRRF1-CCR4-NOT interaction. Luciferase reporter assays identified multiple roles for BRRF1 in post-transcriptional regulation, including polyadenylation-dependent destabilization effects and interference with miRNA function, the latter of which was dependent on BRRF1 binding to CCR4-NOT. In addition, mRNA sequencing experiments comparing cells with and without BRRF1 expression identified multiple transcripts affected by BRRF1, some of which were at least partly dependent on the BRRF1-CCR4-NOT interaction. These include Claudin 6, whose transcripts and protein were downregulated by BRRF1 expressed on its own, as well as in lytic infection. Thus, we have identified multiple roles for BRRF1 in post-transcriptional mRNA regulation, some of which involve interaction with CCR4-NOT. IMPORTANCE: To efficiently generate virions, Epstein-Barr virus (EBV) must manipulate cellular processes to optimize the environment and overcome host antiviral defenses. Through unbiased screening methods, we have identified an EBV protein (BRRF1) that localizes to cellular bodies, which play key roles in mRNA regulation, and that interacts with a cellular complex with multiple critical roles in post-transcriptional mRNA regulation. As expected from these interactions, we found that BRRF1 has multiple previously unknown roles in disrupting mRNA levels and translation. Interestingly, one of the transcripts targeted by BRRF1 is Claudin 6, which has multiple roles in cancers, leading to increased cell proliferation and invasion. Our study provides additional insight into how EBV proteins manipulate cells to promote infection and affect oncogenesis.