Arenavirus Nucleoprotein Targets Interferon Regulatory Factor-Activating Kinase IKKε

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

“…1A). In contrast, the NP protein from the lymphocytic choriomeningitis virus (LCMV), which also inhibits IKK function (48), did not interact with TRIM6 (Fig. 1A).…”

Section: Resultsmentioning

confidence: 99%

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

Bharaj

Atkins

Luthra

et al. 2017

“…In this work, we used a more direct approach to engineer rLCMVs in which the majority of the amino acid residues of the viral NP are encoded by the least-preferred codon in mammalian cells, which was predicted to result in reduced protein expression and, therefore, viral attenuation. We selected NP for our initial studies to explore the effect of CD of the arenavirus genome, because NP is the most abundant viral protein present in infected cells and virions (1) and NP also plays many essential roles in the virus life cycle (40,41,44,45,51). We succeeded in generating a variety of rLCMV/NP CD viruses that were highly attenuated in vivo but able to induce protection against a subsequent lethal challenge with rLCMV/WT.…”

Section: Discussionmentioning

confidence: 99%

“…NP is the most abundant viral polypeptide in infected cells and virions and the main component of the virus ribonucleoprotein (vRNP), which directs replication and transcription of the viral genome (1). NP interacts with Z to mediate the incorporation of vRNPs into mature infectious virions (39,40), and NP has also been shown to counteract the cellular host type I interferon (IFN-I) response (41)(42)(43)(44)(45)(46). Because NP has multiple critical functions in the arenavirus life cycle, we chose to investigate if CD of NP resulted in viruses that were highly attenuated in vivo but still able to afford protection against a subsequent challenge with wild-type (WT) recombinant LCMV (rLCMV/WT).…”

mentioning

confidence: 99%

Development of Live-Attenuated Arenavirus Vaccines Based on Codon Deoptimization

Cheng

Ortiz-Riaño

Nogales

et al. 2015

Self Cite

Arenaviruses have a significant impact on public health and pose a credible biodefense threat, but the development of safe and effective arenavirus vaccines has remained elusive, and currently, no Food and Drug Administration (FDA)-licensed arenavirus vaccines are available. Here, we explored the use of a codon deoptimization (CD)-based approach as a novel strategy to develop live-attenuated arenavirus vaccines. We recoded the nucleoprotein (NP) of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) with the least frequently used codons in mammalian cells, which caused lower LCMV NP expression levels in transfected cells that correlated with decreased NP activity in cell-based functional assays. We used reverse-genetics approaches to rescue a battery of recombinant LCMVs (rLCMVs) encoding CD NPs (rLCMV/NP CD ) that showed attenuated growth kinetics in vitro. Moreover, experiments using the well-characterized mouse model of LCMV infection revealed that rLCMV/NP CD1 and rLCMV/NP CD2 were highly attenuated in vivo but, upon a single immunization, conferred complete protection against a subsequent lethal challenge with wild-type (WT) recombinant LCMV (rLCMV/WT). Both rLCMV/NP CD1 and rLCMV/NP CD2 were genetically and phenotypically stable during serial passages in FDA vaccine-approved Vero cells. These results provide proof of concept of the safety, efficacy, and stability of a CD-based approach for developing live-attenuated vaccine candidates against human-pathogenic arenaviruses. IMPORTANCESeveral arenaviruses cause severe hemorrhagic fever in humans and pose a credible bioterrorism threat. Currently, no FDA-licensed vaccines are available to combat arenavirus infections, while antiarenaviral therapy is limited to the off-label use of ribavirin, which is only partially effective and is associated with side effects. Here, we describe the generation of recombinant versions of the prototypic arenavirus LCMV encoding codon-deoptimized viral nucleoproteins (rLCMV/NP CD ). We identified rLCMV/NP CD1 and rLCMV/ NP CD2 to be highly attenuated in vivo but able to confer protection against a subsequent lethal challenge with wild-type LCMV. These viruses displayed an attenuated phenotype during serial amplification passages in cultured cells. Our findings support the use of this approach for the development of safe, stable, and protective live-attenuated arenavirus vaccines.A renaviruses cause chronic infections of rodents across the world, and human infections occur through mucosal exposure to aerosols or by direct contact of abraded skin with infectious materials (1). Several arenaviruses, chiefly Lassa virus (LASV), the causative agent of Lassa fever (LF) in West Africa, and Junín virus (JUNV), the causative agent of Argentine hemorrhagic fever (AHF) in Argentina, cause hemorrhagic fever (HF) disease in humans that is associated with high morbidity and significant mortality and pose important public health problems in their areas of endemicity (1-3). Notably, increased travel has led to the import...

“…NC_018137.1). An expression plasmid carrying green fluorescent protein (GFP)-tagged LC3 (GFP-LC3) (plasmid 21073) was obtained from Addgene (41), and plasmids carrying RIG-I, TRIM25, MAVS, TBK1, interferon regulatory factor 3 (IRF3), constitutively activated IRF3 (IRF3-5D), dominant negative Rab5 (S34N), and the corresponding fusion proteins were previously described (42)(43)(44) or constructed following standard cloning procedures. Plasmids carrying red fluorescent protein (RFP)-tagged B4GalT1 (B4GalT1-RFP), TGOLN-RFP, CALR-C-RFP, Rab5-RFP, and Rab4-RFP were obtained from OriGene.…”

Section: Methodsmentioning

confidence: 99%

Hijacking of RIG-I Signaling Proteins into Virus-Induced Cytoplasmic Structures Correlates with the Inhibition of Type I Interferon Responses

Santiago

Covaleda

Sánchez-Aparicio

et al. 2014

107

114

Recognition of viral pathogens by the retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) family results in the activation of type I interferon (IFN) responses.To avoid this response, most viruses have evolved strategies that target different essential steps in the activation of host innate immunity. In this study, we report that the nonstructural protein NSs of the newly described severe fever with thrombocytopenia syndrome virus (SFTSV) is a potent inhibitor of IFN responses. The SFTSV NSs protein was found to inhibit the activation of the beta interferon (IFN-␤) promoter induced by viral infection and by a RIG-I ligand. Astonishingly, we found that SFTSV NSs interacts with and relocalizes RIG-I, the E3 ubiquitin ligase TRIM25, and TANK-binding kinase 1 (TBK1) into SFTSV NSs-induced cytoplasmic structures. Interestingly, formation of these SFTSV NSs-induced structures occurred in the absence of the Atg7 gene, a gene essential for autophagy. Furthermore, confocal microscopy studies revealed that these SFTSV NSs-induced structures colocalize with Rab5 but not with Golgi apparatus or endoplasmic reticulum markers. Altogether, the data suggest that sequestration of RIG-I signaling molecules into endosome-like structures may be the mechanism used by SFTSV to inhibit IFN responses and point toward a novel mechanism for the suppression of IFN responses. IMPORTANCEThe mechanism by which the newly described SFTSV inhibits host antiviral responses has not yet been fully characterized. In this study, we describe the redistribution of RIG-I signaling components into virus-induced cytoplasmic structures in cells infected with SFTSV. This redistribution correlates with the inhibition of host antiviral responses. Further characterization of the interplay between the viral protein and components of the IFN responses could potentially provide targets for the rational development of therapeutic interventions.