Degradation of nucleoproteins by TRIM21-based synthetic restriction factors

Nucleproteins are essential for multiple parts of the virus life cycle, including genome compaction for post-entry shielding of the viral genome from host pattern recognition receptors and for packaging during the assembly of new virions. Due to their vital role in infection, nucleoprotein sequences are also usually well conserved within viral families. Such factors make nucleoproteins an ideal antiviral target. We therefore sought to selectively target and degrade the nucleoproteins of different viruses through various synthetic restriction approaches as a proof-of-principal for an antiviral strategy. One of these approaches utilises TRIM21, an intracellular antibody receptor known to neutralise adenovirus (AdV) by clustering around antibody-coated AdV capsids and targeting the virus for degradation. We fused the RING E3 ligase domain of TRIM21 either to the nucleoprotein directly or to nanobodies that bind the nucleoprotein. We tested the ability of these constructs to restrict model RNA viruses including influenza A virus (IAV) and Hazara virus (HAZV) through the expression of these synthetic restriction factors in target cells and via electroporation-dependent neutralisation assays. RING-nanobodies, which bind IAV nucleoprotein, were able to potently restrict infection. Similarly, the expression of HAZV nucleoprotein fused directly to RING was also able to restrict subsequent HAZV infection. The current work aims to understand the underlying molecular mechanisms behind how these synthetic restriction factors function to prevent infection and whether they can be adapted to restrict other RNA viruses. Overall, the targeted degradation of viral nucleoproteins could serve as a novel therapeutic approach against a multitude of pathogenic viruses.