VIRAL HIJACKING OF UBIQUITIN LIGASES: LESSONS FROM HUMAN CYTOMEGALOVIRUS pUL145

Viruses evolve mechanisms to exploit cellular pathways that increase viral fitness, e.g., enhance viral replication or evade the host cell immune response. The ubiquitin-proteosome system, a fundamental pathway regulating protein fate in eukaryotes, is hijacked by all seven classes of viruses. The Cullin-RING family of ubiquitin (Ub) ligases are frequently coopted by diverged viruses because they can target a broad array of substrates by forming multisubunit assemblies comprised of a variety of adapters and substrate receptors. For example, the linker subunit DDB1 in the cullin 4-RING Ub ligase (CRL4DDB1) interacts with an H-box motif found in several unrelated viral proteins, including V protein of simian virus 5 (SV5-V), the HBx protein of hepatitis B virus (HBV), and the recently identified pUL145 protein of cytomegalovirus (HCMV). In HCMV-infected cells, pUL145 repurposes CRL4DDB1 to target STAT2, a protein vital to the antiviral immune response. However, the details of how these diverged viral sequences hijack DDB1 is not well understood. We used a combination of binding assays, X-ray crystallography, alanine scanning, cell-based assays, and computational analysis to reveal that viral H-box motifs seem to bind to DDB1 with a higher affinity than the H-box motifs from host proteins DCAF1 and DDB2. This analysis reveals that viruses maintain native hot-spot residues in the H-box motif of host DCAFs and are also able to evolve favorable interactions at neighboring residues within the H-box through either direct or solvent-mediated interactions. Overall, these studies reveal how viruses can evolve strategies to produce high-affinity binding and quality interactions with DDB1 to repurpose its Ub ligase machinery.