Abstract
The physiologic function of tripartite motif protein 56 (TRIM56), a ubiquitously expressed E3 ligase classified within the large TRIM protein family, remains elusive. Gene knockdown studies have suggested TRIM56 as a positive regulator of the type I interferon (IFN-I) antiviral response elicited via the Toll-like receptor 3 (TLR3) and cyclic GMP–AMP synthase (cGAS)–stimulator of interferon genes (STING) pathways, which detect and respond to danger signals—extracellular double-stranded (ds) RNA and cytosolic dsDNA, respectively. However, to what extent these pathways depend on TRIM56 in human cells is unclear. In addition, it is debatable whether TRIM56 plays a part in controlling the expression of IFN-stimulated genes (ISGs) resulting from IFN-I based antiviral treatment. In this study, we created HeLa-derived TRIM56 null cell lines by gene editing and used these cell models to comprehensively examine the impact of endogenous TRIM56 on innate antiviral responses. Our results showed that TRIM56 knockout severely undermined the upregulation of ISGs by extracellular dsRNA and that loss of TRIM56 weakened the response to cytosolic dsDNA. ISG induction and ISGylation following IFN-α stimulation, however, were not compromised by TRIM56 deletion. Using a vesicular stomatitis virus-based antiviral bioactivity assay, we demonstrated that IFN-α could efficiently establish an antiviral state in TRIM56 null cells, providing direct evidence that TRIM56 is not required for the general antiviral action of IFN-I. Altogether, these data ascertain the contributions of TRIM56 to TLR3- and cGAS–STING-dependent antiviral pathways in HeLa cells and add to our understanding of the roles this protein plays in innate immunity.
Highlights
Tripartite motif protein 56 (TRIM56) is a member of the large TRIM protein family of E3 ligases that are involved in a broad array of host processes, including, but not limited to, proliferation, differentiation, development and, recently, immunity [1,2,3,4,5,6,7]
Gene knockdown experiments in HEK293 [4], HeLa [5], and THP-1 [6] cells have demonstrated that TRIM56 contributes to the cytosolic DNA-sensing pathway, it is controversial whether the substrate for the TRIM56 E3 ligase is cyclic GMP–AMP synthase [6] or its downstream adaptor, stimulator of interferon genes (STING) [4]
Experiments based on RNA interference (RNAi)-mediated depletion of TRIM56 in HEK293 and Huh7.5 cells reconstituted with Toll-like receptor 3 (TLR3) expression and in HeLa cells harboring a physiologic level of TLR3 had suggested a critical role of TRIM56 in this viral double-stranded RNA-sensing pathway [5]
Summary
Tripartite motif protein 56 (TRIM56) is a member of the large TRIM protein family of E3 ligases that are involved in a broad array of host processes, including, but not limited to, proliferation, differentiation, development and, recently, immunity [1,2,3,4,5,6,7]. In addition to its reported antiviral activities, TRIM56 has been implicated in regulating innate immune-signaling pathways that culminate in the induction of type I interferon (IFN-I) response, a hallmark of the intrinsic, immediate defense mechanisms of mammalian hosts against viral infections. In contrast to the mechanism proposed for the TRIM56 regulation of the cGAS-STING pathway that hinges on the E3 ubiquitin ligase activity, TRIM56 promotes IFN-I and chemokine production via the TLR3 pathway in a non-canonical, E3 ligase-independent fashion. Rather, such capacity correlates with an interaction of TRIM56 with Toll/interleukin-1-receptor-domaincontaining adapter-inducing interferon-β (TRIF), the adaptor for TLR3. Our data do not support a significant role, if any, of TRIM56 in regulating ISG induction downstream of the IFN-I receptors or impacting the establishment of a general antiviral state by IFN-I
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