Molecular mechanism of TRIM25 and ZAP mediated antiviral inhibition of arenavirus replication

This joint project bridges across the virology and structural biology research programs in the Foster, Borkar and Dunham groups, focussing on the cellular antiviral mechanisms that that inhibit viral infection and the virus -host interactions that govern these. The Foster group focusses on the largest family of haemorrhagic fever causing viruses known as arenaviruses. These zoonotic viruses are rapidly expanding in their genetic diversity leading to increased annual
outbreaks in endemic regions and to sporadic imported outbreaks in globally, including recent cases in the UK (1). Understanding how host antiviral proteins block arenavirus replication is imperative to addressing current knowledge gaps on the viral lifecycle processes that are key for the development of effective vaccines and treatments (1).
Recent proteomics studies in the Foster lab, identified the interaction between the arenavirus nucleoprotein, NP and the antiviral E3 ligase tripartite motif-containing protein 25 (TRIM25), a key component of the innate immune response that inhibits the replication of a diverse range of pathogenic viruses, but previously unknown for arenaviruses (2). NP is the most abundantly expressed arenavirus protein, is a major orchestrator of host immunosuppression during virus
infection and encapsidates the viral RNA, forming the viral ribonucleocomplexes (vRNPs) needed for infection initiation (1). Using arenavirus infection and CRISPR/Cas9 knockout studies, we have shown that TRIM25 significantly restricts arenavirus replication, that TRIM25 re-localises to NP-containing viral replication sites and that NP- overexpression is sufficient to induce this re-localisation.
TRIM25 was recently identified as key cofactor of the zinc-antiviral protein, ZAP, that targets viral RNAs containing CpG dinucleotides leading to promotion of viral RNA degradation and/or inhibition of viral RNA translation (2). We have demonstrated that ZAP also potently inhibits arenavirus replication and as TRIM25 is also an RNA binding protein, and it remains to be determined how TRIM25 and ZAP activity is co-modulated by their RNA interactions. The molecular structure of TRIM25 is an anti-parallel dimer formed through its coiled-coil domain, that can further multimerise through dimerisation of its N-terminal RING domain, containing E3-ubiquitin ligase activity. The ZAP and other ligand-binding C-terminal PRYSPRY domain is located on either side of the coiled-coil (2).
We hypothesise that TRIM25 may multimerise around the helical vRNP complexes, driving disassembly and exposing RNA sites for ZAP recruitment and subsequent RNA degradation.
Thus, this project aims to use a combination of structural biology (expertise in the Borkar group), biochemistry and molecular virology techniques to:
Characterise how TRIM25 interacts with arenavirus NP protein and with arenavirus viral RNAs with and without ZAP through mutagenesis and arenavirus replication experiments
Recombinantly express and purify TRIM25 and ZAP proteins alone, in complex and in the presence of arenaviral RNA
Visualise by electron microscopy and/or X-ray crystallography apo-structures and TRIM25- NP and TRIM25-NP-RNA-ZAP complexes, incorporating specific mutations that modify RNA and cofactor binding.
Given the molecular tractability of this antiviral mechanism across diverse RNA and DNA viruses, findings from this work will provide key evidence that could transform our understanding of innate immune mechanisms and influence therapeutic design.