Vertebrate genomes show evidence of repeated exposure to retroviral pathogens, suggesting a prolonged co-evolution between the two¹ . Consequently, cells have developed mechanisms to prevent and contain infection by retroviruses. TRIM5α is one such restriction factor that acts during the early post-entry stages of the retroviral lifecycle, to disrupt retroviral infection by blocking reverse transcription, nuclear import and integration^2 3 . To restrict a particular retrovirus TRIM5α recognises and binds the lattice of capsid protein that forms the inner shell of the retrovirus. As this lattice is conserved across retroviruses, TRIM5α is a pattern recognition receptor for the retroviral capsid⁴ . This recognition is required for restriction and triggers the production of ubiquitin chains which initiate downstream signalling, resulting in a cellular antiviral state.

Ubiquitin chain formation requires an ubiquitin activating enzyme (E1), an ubiquitin conjugating enzyme (E2) and an ubiquitin ligase (E3). To investigate the activation of signalling by TRIM5α and probe the activity of the RING domain, the components necessary for ubiquitylation (an E1, E2 and the TRIM5α RING E3 ligase domain) were expressed and purified, and an in vitro ubiquitin assay was established based upon the formation of K63-ubiquitin chains. Ubiquitin assays showed that the Trim5a RING domain alone had minimal activity. Fusion of the RING domain to GST to promote dimerization resulted in a striking increase in activity.