Large-Scale Screen Identifies Nearly 10,000 Viral Ubiquitin Ligases, Revealing Immune-Evasion Mechanisms

A study has identified, on a large scale, ubiquitin ligases that viruses use to evade host immunity.

Caleb Glassman and colleagues reported in a paper published May 26 in the journal Science that they used pooled genetic screening of about 10,000 viral open reading frames (vORFs) to identify viral ubiquitin ligases in a high-throughput, unbiased manner.

The researchers fused a library of about 10,000 vORFs to a nanobody against green fluorescent protein (GFP) and used the loss of GFP fluorescence as an indicator of substrate degradation.

The viral “degradation factors” identified through this approach — factors that induce protein degradation — fell into three broad mechanistic categories: noncanonical ubiquitin ligases, “hijackers” that redirect host CRL complexes, and canonical ubiquitin ligases.

Rotavirus A NSP1 was identified as an example of a noncanonical ligase. Although NSP1 lacks the typical BTB domain of a CUL3 substrate adaptor, it bound CUL3 through ELOC, a CUL2/5 adaptor protein with a BTB-like domain. This mechanism was confirmed by a cryo-electron microscopy structure at 3.3-angstrom (Å) resolution.

Razan virus NSs bypassed adaptor proteins entirely, binding directly to CUL3 through a helical motif in its C-terminal tail, degrading CUL1 and suppressing nuclear factor κB (NF-κB) signaling.

The hijacker proteins Tebiet virus substrate protein (CUL3^BTBD1) and Adana virus NSs (CUL1^β-TrCP) used intact host CRL complexes to target JAK1 and suppress type 1 interferon signaling.

The canonical ubiquitin ligases MGF505 (CUL5^ELOB/C) and MGF360 (CUL2^ELOB/C) from African swine fever virus mediated complex assembly through conserved BC-box motifs and degraded substrates involved in viral restriction and autophagy.

The researchers said all three mechanisms exploit the modularity of the ubiquitin-proteasome system (UPS) by connecting viral ubiquitin ligases to existing host machinery through substrate-recognition modules, expanding the potential functions of the limited viral coding space.

The researchers noted that despite the diversity of degradation mechanisms, viral ubiquitin ligases ultimately converge on immune-related genes known to have antiviral restriction functions. This was interpreted to mean that these pathways represent key bottlenecks in viral transmission.