Identification of Epstein-Barr virus BORF2 sequences required for APOBEC3B relocalization

Epstein-Barr virus (EBV) is a common herpesvirus that establishes lifetime infections in most people worldwide. To protect the lytically replicating EBV genomes from mutation, the EBV BORF2 protein relocalizes the APOBEC3B cytosine deaminase out of the nucleus, sequestering it in cytoplasmic bodies. This property is conserved in BORF2 homologs in other herpesviruses, including Kaposi's sarcoma-associated herpesvirus ORF61 and herpes simplex virus 1 UL39. Here, we show that a motif conserved in these three proteins (IPAM) is critical for interactions with and relocalization of APOBEC3B. However, the properties of the cytoplasmic bodies formed by BORF2, ORF61, and UL39 differ in that only BORF2 requires APOBEC3B to form cytoplasmic bodies, and only UL39 bodies have properties of aggresomes. We also found that a SUMO-modified site in BORF2 (K741) plays an important role in the formation of bodies with endogenous APOBEC3B, both when expressed on its own and in the context of EBV infection. Additionally, nuclear BORF2-APOBEC3B bodies that formed in early lytic infection contained SUMO, suggesting the importance of SUMOylation in the sequestration of APOBEC3B. Our study provides insights into the mechanisms herpesviruses use to disable APOBEC3B and protect their replicating DNA genomes from undesired editing.IMPORTANCEHerpesviruses must protect their replicating DNA genomes from mutation by APOBEC3B, which they do by relocalizing APOBEC3B from the nucleus into cytoplasmic bodies. This is mediated by Epstein-Barr virus BORF2 and its homologs in Kaposi's sarcoma-associated herpesvirus (ORF61) and herpes simplex virus 1 (UL39). We have shown that a conserved motif in these proteins is critical for this function, and that a SUMO-modified site in BORF2 also plays an important role. This work provides insight into the mechanisms by which BORF2 and its homologs sequester and relocalize APOBEC3B, which is important for maintaining the integrity and infectivity of the respective herpesviruses.