Kaposi's sarcoma-associated herpesvirus ORF61 protein sequesters APOBEC3B in filamentous aggregates

Herpesviruses are large DNA viruses that encode homologs of cellular enzymes. The viral ribonucleotide reductase, which consists of large R1 and small R2 subunits, is required for deoxyribonucleotide synthesis. However, herpesviruses have repurposed the R1 subunit for additional non-canonical functions in virus-host interaction and immune evasion. Here, we investigated the R1 proteins of Kaposi's sarcoma-associated herpesvirus (KSHV) and murine gammaherpesvirus 68 (MHV-68), two γ-herpesviruses of the genus Rhadinovirus. We show that the ORF61-encoded viral R1 proteins form elongated cytoplasmic condensates in infected cells, which structurally differ from the previously described R1 condensates of other herpesviruses. Fluorescently labeled ORF61 condensates exhibited the properties of solid aggregates, as determined by fluorescence recovery after photobleaching (FRAP). Correlative light and electron microscopy (CLEM) showed that ORF61 aggregates consist of filamentous bundles. The KSHV ORF61 protein interacted with the cellular cytosine deaminase APOBEC3B in infected cells and translocated it from the nucleus, the site of viral DNA replication, to the cytoplasmic aggregates. Aggregate formation and relocalization of APOBEC3B depended on a conserved Induced Protein Aggregation Motif (IPAM) in the C-terminal part of ORF61. A KSHV ORF61 IPAM mutant was vulnerable to APOBEC3B-mediated deamination and replicated to reduced titers. In contrast, MHV-68 ORF61 did not relocalize human or murine APOBEC3 proteins, suggesting that it engages different target proteins. The results show that rhadinovirus ORF61 proteins form elongated filamentous aggregates in infected cells to sequester and inactivate target proteins, such as APOBEC3B.IMPORTANCEHerpesviruses are large DNA viruses that encode enzymes similar to those in host cells. The R1 subunit of their ribonucleotide reductase is important for DNA synthesis and plays additional roles in immune evasion and virus-host interactions. This study focused on the R1 protein ORF61 of two γ-herpesviruses of the genus Rhadinovirus: KSHV and MHV-68. Unlike their homologs in other herpesviruses, KSHV and MHV-68 R1 proteins form cytoplasmic aggregates consisting of filamentous bundles in infected cells. KSHV ORF61 depletes the mutagenic cellular enzyme APOBEC3B from the nucleus, the site of viral DNA replication, and sequesters it in cytoplasmic aggregates, thereby protecting the viral genome from APOBEC3B-mediated mutations. This process relies on a specific conserved motif in ORF61. However, the MHV-68 ORF61 protein does not redistribute APOBEC3 proteins, suggesting that it binds different targets. These findings reveal how rhadinoviruses use filamentous ORF61 aggregates to manipulate host antiviral defenses.