The Host E3-Ubiquitin Ligase TRIM6 Ubiquitinates the Ebola Virus VP35 Protein and Promotes Virus Replication

Abstract

Ebola virus (EBOV), a member of the Filoviridae family, is a highly pathogenic virus that causes severe hemorrhagic fever in humans and is responsible for epidemics throughout sub-Saharan, central, and West Africa. The EBOV genome encodes VP35, an important viral protein involved in virus replication by acting as an essential cofactor of the viral polymerase as well as a potent antagonist of the host antiviral type I interferon (IFN-I) system. By using mass spectrometry analysis and coimmunoprecipitation assays, we show here that VP35 is ubiquitinated on lysine 309 (K309), a residue located on its IFN antagonist domain. We also found that VP35 interacts with TRIM6, a member of the E3-ubiquitin ligase tripartite motif (TRIM) family. We recently reported that TRIM6 promotes the synthesis of unanchored K48-linked polyubiquitin chains, which are not covalently attached to any protein, to induce efficient antiviral IFN-I-mediated responses. Consistent with this notion, VP35 also associated noncovalently with polyubiquitin chains and inhibited TRIM6-mediated IFN-I induction. Intriguingly, we also found that TRIM6 enhances EBOV polymerase activity in a minigenome assay and TRIM6 knockout cells have reduced replication of infectious EBOV, suggesting that VP35 hijacks TRIM6 to promote EBOV replication through ubiquitination. Our work provides evidence that TRIM6 is an important host cellular factor that promotes EBOV replication, and future studies will focus on whether TRIM6 could be targeted for therapeutic intervention against EBOV infection.IMPORTANCE EBOV belongs to a family of highly pathogenic viruses that cause severe hemorrhagic fever in humans and other mammals with high mortality rates (40 to 90%). Because of its high pathogenicity and lack of licensed antivirals and vaccines, EBOV is listed as a tier 1 select-agent risk group 4 pathogen. An important mechanism for the severity of EBOV infection is its suppression of innate immune responses. The EBOV VP35 protein contributes to pathogenesis, because it serves as an essential cofactor of the viral polymerase as well as a potent antagonist of innate immunity. However, how VP35 function is regulated by host cellular factors is poorly understood. Here, we report that the host E3-ubiquitin ligase TRIM6 promotes VP35 ubiquitination and is important for efficient virus replication. Therefore, our study identifies a new host factor, TRIM6, as a potential target in the development of antiviral drugs against EBOV.KEYWORDS TRIM6, tripartite motif (TRIM) protein, VP35, viral RNA polymerase, Ebola virus, innate immunity, ubiquitination, unanchored ubiquitin, virus-host interactions

To TRIM or not to TRIM: the balance of host–virus interactions mediated by the ubiquitin system

“…Direct enhancement of EBOV replication by TRIM6 was observed in an EBOV minigenome assay where increasing amounts of TRIM6, but not the catalytically defective C15A RING mutant, enhanced EBOV polymerase activity [180]. Although a K309A mutation on VP35 reduced ubiquitination, the presence of mono-, di-and unanchored ubiquitinated forms of VP35 persisted, indicating VP35 is ubiquitinated at additional residues whose functional roles remain to be determined [180]. Viral co-opting of the host ubiquitin machineries for direct enhancement of replication has been described, with some studies identifying the involvement of different families of E3 ubiquitin ligases.…”

“…This presents itself as a striking mechanism utilized by viruses because hijacking a known antiviral factor and repurposing its functions to enhance replication would give double the advantage. Indeed, we have previously shown that TRIM6 knockout cell lines, which have a reduced IFN-I-mediated antiviral response, do not provide an optimal replication environment for Ebola virus (EBOV) [22,180]. Interestingly, further evidence exists in recent studies that utilized genome-wide siRNA knockdown or CRISPR/Cas9 knockout screens on the potential pro-viral roles of TRIMs (e.g.…”

“…TRIM6 is an important component in IFN production and signalling, although EBOV replication in TRIM6 knockout cells was not increased, suggesting that TRIM6 is required for optimal EBOV replication [22,180]. The EBOV VP35 protein, a known IFN antagonist and cofactor of the viral polymerase, was found to interact with the SPRY domain of TRIM6, implicating VP35 as a target for ubiquitin [180,[188][189][190]. Indeed, MS identified K309 on VP35 as a target for ubiquitination and co-expression of TRIM6 enhanced ubiquitinated forms of VP35 [180].…”

“…The EBOV VP35 protein, a known IFN antagonist and cofactor of the viral polymerase, was found to interact with the SPRY domain of TRIM6, implicating VP35 as a target for ubiquitin [180,[188][189][190]. Indeed, MS identified K309 on VP35 as a target for ubiquitination and co-expression of TRIM6 enhanced ubiquitinated forms of VP35 [180]. Direct enhancement of EBOV replication by TRIM6 was observed in an EBOV minigenome assay where increasing amounts of TRIM6, but not the catalytically defective C15A RING mutant, enhanced EBOV polymerase activity [180].…”

Pleiotropic roles of the ubiquitin-proteasome system during viral propagation

“…The UPS can be manipulated by many viruses to facilitate viral replication, but the proteasome inhibitor MG132 can inhibit viral replication [20,46,47]. For example, the replication of some viruses such as human astrovirus [47], vaccinia virus [48], tombusvirus [49], hepatitis E virus [50,51], porcine circovirus type 2 [52], and Ebola virus [53] are regulated by the UPS, and recent studies revealed that the UPS is necessary for the efficient replication of African swine fever virus and human astrovirus [20,47]. Among these viruses, some encode specific proteins that modify the host Ub machinery.…”