Characterisation of the SPRY Domain Containing TRIM, TRIM68, as a Regulator of Antiviral Innate Immune Responses

Claire M. Wynne, Royal College of Surgeons in Ireland

Submitted for the Award of Doctor of Philosophy (PhD) to the Royal College of Surgeons, 2012.

Abstract

Innate immune receptors such as Toll-like receptors (TLRs) and RIG-l-like receptors (RLRs) play a key role in viral recognition and activation of transcription factors important for driving the production o f type I interferons (IFNs) and various other pro-inflammatory cytokines. Whilst important for both antiviral and anti-bacterial immunity, IFNs and pro-inflammatroy cytokines can become pathogenic when overproduced, resulting in autoimmune and autoinflammatory diseases. Considering this, proteins that function to turn o ff and limit the production of such cytokines are important immunoregulatory proteins. The tripartite motif-containing (TRIM) protein family are emerging as important anti-viral proteins and thus are compelling targets for modulating the immune response. Using phylogenetic studies, we identify a cluster of SPRY-domain containing TRIM proteins located on chromosome 11, which share a common ancestry and may have arisen from gene duplication events. Here we show that specific TRIM proteins located in this cluster play an important role as negative regulators of type I IFN production. Published data from our laboratory has shown TRIM21, which is also located in this cluster, to be an important negative regulator of IFN production. In this study we identify TRIM68, a known autoantigen in systemic lupus erythematosus (SLE), to be TRIM21's closest homologue.

Results demonstrate TRIM68 to be a novel and potent negative regulator of type I IFN production, whilst proteomic analysis of TRIM68 containing complexes has allowed us to identify TRK-fused gene (TFG) as a bona fide TRIM68 target. The physiological role of TFG is still unknown but it has been shown to interact with IKKy and TANK, two proteins which modulate pro-inflammatory cytokine production. Although TFG has not yet been implicated on the pathway regulating interferon regulatory factor 3 (IRF3) activity, our results strongly suggest that it acts directly on this pathway, potentially acting as an adaptor protein facilitating the interaction between TANK-binding kinase 1 (TBK1) and TRAF family member-associated nuclear factor kappa B activator (TANK). Thus the effects o f TRIM68 on IFN production appear to be via polyubiquitination and degradation of TFG, thereby negatively regulating both nuclear factor kappa B (NFkB) and IRF3 activity. To confirm the critical role of TRIM68 In IFN production we have knocked down TRIM68 in mice via injection of microparticles encapsulating shRNA against TRIM68 and studied the effects of TRIM68 knock down on viral-induced IFN production. TRIM68 knockdown mice display an increase in serum and peritoneum IFN levels post lymphocytic choriomeningitis virus (LCMV) infection, further validating that TRIM68 is a key negative regulator of IFN production. Using NetPhos we have identified two key tyrosines on TRIM68 that are highly conserved between human and mouse. Site directed mutagenesis of these key tyrosines has allowed us to appreciate the important role that tyrosine phosphorylation plays in regulating TRIM68 activity.

We identify TRIM68 to be highly expressed in natural killer (NK) cells and show its expression to increase following LCMV infection. We confirm the presence of TLRs on NK cells and demonstrate that TRIM68 plays an important role in TLRdriven NK cell activation and degranulation. While Investigating the role TRIM68 played in NK cell biology, we observe for the first time that TLR9 activation of NK cells results in interleukin (IL)23pl9 release. Taken together, this work carves the way for the development of novel therapeutics targeting TRIM family members, and TRIM68 specifically, for the treatment of IFN-driven disease such as SLE.