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Quaking (QKI) controls RNA metabolism in many biological processes including innate immunity, where its roles remain incompletely understood. To illuminate these roles, we performed genome scale transcriptome profiling in QKI knockout cells with or without poly(I:C) transfection, a double-stranded RNA analog that mimics viral infection. Analysis of RNA-sequencing data shows that QKI knockout upregulates genes induced by interferons, suggesting that QKI is an immune suppressor. Furthermore, differential splicing analysis shows that QKI primarily controls cassette exons, and among these events, we noted that QKI silences splicing of the extra domain A (EDA) exon in fibronectin (FN1) transcripts. QKI knockout results in elevated production and secretion of FN1-EDA protein, which is a known activator of interferons. Consistent with an upregulation of the interferon response in QKI knockout cells, our results show reduced production of dengue virus-2 and Japanese encephalitis virus in these cells. In conclusion, we demonstrate that QKI downregulates the interferon system and attenuates the antiviral state.

Quaking (QKI) is an RNA-binding protein (RBP) involved in multiple aspects of RNA metabolism and many biological processes. Despite a known immune function in regulating monocyte differentiation and inflammatory responses, the degree to which QKI regulates the host interferon (IFN) response remains poorly characterized. Here we show that QKI ablation enhances poly(I:C) and viral infection-induced IFNβ transcription. Characterization of IFN-related signalling cascades reveals that QKI knockout results in higher levels of IRF3 phosphorylation. Interestingly, complementation with QKI-5 isoform alone is sufficient to rescue this phenotype and reduce IRF3 phosphorylation. Further analysis shows that MAVS, but not RIG-I or MDA5, is robustly upregulated in the absence of QKI, suggesting that QKI downregulates MAVS and thus represses the host IFN response. As expected, MAVS depletion reduces IFNβ activation and knockout of MAVS in the QKI knockout cells completely abolishes IFNβ induction. Consistently, ectopic expression of RIG-I activates stronger IFNβ induction via MAVS-IRF3 pathway in the absence of QKI. Collectively, these findings demonstrate a novel role for QKI in negatively regulating host IFN response by reducing MAVS levels.

The four dengue viruses (DENV1-4) are rapidly reemerging infectious RNA viruses. These positive-strand viral genomes contain structured 3′ untranslated regions (UTRs) that interact with various host RNA binding proteins (RBPs). These RBPs are functionally important in viral replication, pathogenesis, and defense against host immune mechanisms. Here, we combined RNA chromatography and quantitative mass spectrometry to identify proteins interacting with DENV1-4 3′ UTRs. As expected, RBPs displayed distinct binding specificity. Among them, we focused on quaking (QKI) because of its preference for the DENV4 3′ UTR (DENV-4/SG/06K2270DK1/2005). RNA immunoprecipitation experiments demonstrated that QKI interacted with DENV4 genomes in infected cells. Moreover, QKI depletion enhanced infectious particle production of DENV4. On the contrary, QKI did not interact with DENV2 3′ UTR, and DENV2 replication was not affected consistently by QKI depletion. Next, we mapped the QKI interaction site and identified a QKI response element (QRE) in DENV4 3′ UTR. Interestingly, removal of QRE from DENV4 3′ UTR abolished this interaction and increased DENV4 viral particle production. Introduction of the QRE to DENV2 3′ UTR led to QKI binding and reduced DENV2 infectious particle production. Finally, reporter assays suggest that QKI reduced translation efficiency of viral RNA. Our work describes a novel function of QKI in restricting viral replication.

As therapeutic recombinant fusion proteins become more widely applicable for the treatment of various types of diseases, there is an increased demand for universal methods such as liquid chromatography (LC)-mass spectrometry (MS) for the determination of their pharmacokinetic properties, particularly their catabolism. The most common approach of analyzing proteins by LC-MS is to digest them into peptides, which can serve as surrogates of the protein. Alternatively, we have developed a novel high-resolution mass spectrometry (HRMS) based approach for analyzing large-molecule proteins at the intact level in biological samples without digestion. We established an immunoaffinity capture LC-HRMS method to quantify the intact parent molecule while simultaneously identifying catabolites for recombinant fusion proteins. We describe this method using dulaglutide, a glucagon-like peptide 1 (GLP1)-Fc fusion protein. Two proteolytic sites within the GLP1 peptide sequence of dulaglutide were identified using this novel LC-HRMS analysis in vivo in mice. These proteolytic sites were identified with the intact molecule being quantified simultaneously. Together with the trypsin digestion based LC-MS/MS analysis using surrogate peptides from different domains of the analyte, an insightful understanding of the pharmacokinetics and in vivo biotransformation of dulaglutide was obtained. Thus, this method enables simultaneous acquisition of both intact drug concentration and important catabolite information for this recombinant fusion protein, providing valuable insight into the integrity of the molecule and its catabolism in vivo. This is critical for designing and screening novel protein therapeutics and for understanding their pharmacokinetics and pharmacodynamics. With continuing advancement of LC-HRMS and software, this method can be very beneficial in drug discovery and development.

Synthetically sulfated hyaluronic acid (HA) has been shown to bind proteins with high affinity through electrostatic interactions. While HA-based hydrogels have been used widely in recent years for drug delivery and tissue engineering applications, incorporation of sulfated HA into these networks to attenuate the release of proteins is yet to be explored. Here, we developed sulfated and methacrylate-modified HA macromers and incorporated them into HA hydrogels through free radical-initiated crosslinking. The sulfated HA macromers bound to a heparin-binding protein (i.e., stromal cell-derived factor-1 alpha, SDF-1α) with an affinity comparable to heparin and did not alter the gelation behavior or network mechanics when copolymerized into hydrogels at low concentrations. Further, these macromers were incorporated into electrospun nanofibrous hydrogels to introduce sulfate groups into macroporous scaffolds. Once incorporated into either uniform or fibrous HA hydrogels, the sulfated HA macromers significantly slowed encapsulated SDF-1α release over 12 days. Thus, these macromers provide a useful way to introduce heparin-binding features into radical-crosslinked hydrogels to alter protein interactions for a range of applications.