This thesis describes cellular protein TRIM5α as a restriction factor for vaccinia virus (VACV) and two VACV countermeasures. This study followed up a proteomic analysis showing that VACV infection of TERT-immortalised human foetal foreskin fibroblasts (HFFF-TERTs) induced TRIM5 downregulation. This observation was validated in HFFF-TERT and HeLa cells, and TRIM5 degradation was shown to be proteasome-dependent. By using VACV mutants lacking blocks of genes near either genomic terminus, and thereafter mutants lacking individual genes, the gene encoding the viral protein that induces TRIM5α degradation was mapped to gene C6L. Furthermore, the inducible expression of C6 degraded endogenous TRIM5α outwith infection, showing that TRIM5α degradation does not require other viral proteins. Subsequent mechanistic studies showed that C6 interacts directly with the RING domain of TRIM5α. Finally, highly conserved orthologues of C6 in other orthopoxviruses such as cowpox virus (CPXV), rabbitpox virus (RPXV), camelpox virus (CMLV), elephantpox virus, monkeypox virus (MPXV) clades I and II and variola virus (VARV), were shown to co-precipitate and degrade TRIM5α outwith infection.

A hypothesis to explain VACV-mediated TRIM5α degradation was that TRIM5α is an anti-VACV restriction factor, and therefore is removed by VACV to facilitate infection. To test this, cell lines lacking or over-expressing TRIM5 were generated. Whereas VACV replication and spread were enhanced in TRIM5^-/- cell lines, over-expression of TRIM5α had the opposite phenotype. Reintroduction of wildtype (WT) TRIM5α into TRIM5^-/- cells rescued TRIM5α-mediated restriction of VACV, but TRIM5α mutants that lacked E3 ubiquitin ligase activity, the ability to oligomerise or the C-terminal PRY/SPRY domain did not. Notably, during VACV infection, TRIM5α co-localised with virus replication factories, whereas in uninfected cells, TRIM5α was diffuse throughout the cytoplasm.

In addition to C6-mediated degradation of TRIM5α, this thesis reports that VACV exploits the cellular protein, cyclophilin A (CypA), as an additional measure to counteract TRIM5α-mediated restriction. This work was based on prior observations that CypA is utilised by HIV-1 to evade restriction by human TRIM5α, CypA is incorporated into both VACV and HIV-1 virions and the inhibitor of CypA, cyclosporine A (CsA), has anti-viral activity. Whilst VACV replication and spread were reduced in CypA^-/- cells, this phenotype was lost when TRIM5 was also knocked out, showing that CypA is proviral in a TRIM5-dependent manner. Furthermore, CsA restricted virus plaque size in WT but not in TRIM5^-/- cells, showing that the antiviral activity of CsA is also TRIM5-dependent. To determine whether the peptidyl prolyl isomerase activity of CypA is required for its proviral function, two catalytic defective mutants were re-introduced into CypA^-/- cells. Whilst WT CypA rescued virus replication and spread, the mutants did not, showing that CypA enzymatic activity is needed to antagonise TRIM5-mediated restriction.

Next, the binding partner(s) of TRIM5α and CypA was identified by proteomic analysis of proteins that co-precipitated with TRIM5α and CypA during VACV infection. Of the several VACV structural proteins that co-precipitated with TRIM5α, only L3 was also precipitated by CypA, and this co-precipitation was lost in the presence of CsA. These interactions were validated during VACV infection and following ectopic expression of L3, and shown to be direct. The interaction between TRIM5α and L3 was mapped to the C-terminal domains of TRIM5α. During infection in the presence of L3, TRIM5α co-localised to virus factories, whereas when L3 expression was repressed, TRIM5α was elsewhere in the cell. L3 was shown to dimerise and dimerisation was enhanced by TRIM5α and this required its E3 ubiquitin ligase activity, and was antagonised by CypA. Furthermore, TRIM5α and L3 were shown to activate NF-κB synergistically, and this was antagonised by CypA but not a catalytically defective mutant.

Like C6, L3 is highly conserved amongst orthopoxviruses and the L3 from MPXV and VARV co-precipitated with both TRIM5α and CypA, and the latter interaction was disrupted by CsA. Given the 2022 MPXV epidemic and the emergence of tecovirimat-resistant MPXV, the possibility of repurposing CsA as an anti-poxviral drug was tested. Since CsA is an immunosuppressant, two non-immunosuppressive derivatives, NIM811 and alisporivir, were also tested. All three drugs reduced VACV and MPXV replication and spread and disrupted the interaction between VACV and MPXV L3 and CypA. Therefore, NIM811 and alisporivir have anti-poxviral activity and potential clinical applications.