RIOK3 and Its Alternatively Spliced Isoform Have Disparate Roles in the Innate Immune Response to Rift Valley Fever Virus (MP12) Infection.

Rift Valley fever virus (RVFV) is a pathogenic human and livestock RNA virus that poses a significant threat to public health and biosecurity. During RVFV infection, the atypical kinase RIOK3 plays important roles in the innate immune response. Although its exact functions in innate immunity are not completely understood, RIOK3 has been shown to be necessary for mounting an antiviral interferon (IFN) response to RVFV in epithelial cells. Furthermore, after immune stimulation, the splicing pattern for RIOK3 mRNA changes markedly, and RIOK3's dominant alternatively spliced isoform, RIOK3 X2, exhibits an opposite effect on the IFN response by dampening it. Here, we further investigate the roles of RIOK3 and its spliced isoform in other innate immune responses to RVFV, namely the NFκB-mediated inflammatory response. We find that while RIOK3 is important for negatively regulating this inflammatory pathway, its alternatively spliced isoform, RIOK3 X2, stimulates it. Overall, these data demonstrate that both RIOK3 and its X2 isoform have unique roles in separate innate immune pathways that respond to RVFV infection.

The Atypical Kinase RIOK3 Limits RVFV Propagation and Is Regulated by Alternative Splicing.

In recent years, transcriptome profiling studies have identified changes in host splicing patterns caused by viral invasion, yet the functional consequences of the vast majority of these splicing events remain uncharacterized. We recently showed that the host splicing landscape changes during Rift Valley fever virus MP-12 strain (RVFV MP-12) infection of mammalian cells. Of particular interest, we observed that the host mRNA for Rio Kinase 3 (RIOK3) was alternatively spliced during infection. This kinase has been shown to be involved in pattern recognition receptor (PRR) signaling mediated by RIG-I like receptors to produce type-I interferon. Here, we characterize RIOK3 as an important component of the interferon signaling pathway during RVFV infection and demonstrate that RIOK3 mRNA expression is skewed shortly after infection to produce alternatively spliced variants that encode premature termination codons. This splicing event plays a critical role in regulation of the antiviral response. Interestingly, infection with other RNA viruses and transfection with nucleic acid-based RIG-I agonists also stimulated RIOK3 alternative splicing. Finally, we show that specifically stimulating alternative splicing of the RIOK3 transcript using a morpholino oligonucleotide reduced interferon expression. Collectively, these results indicate that RIOK3 is an important component of the mammalian interferon signaling cascade and its splicing is a potent regulatory mechanism capable of fine-tuning the host interferon response.

Tra2beta-Dependent Regulation of RIO Kinase 3 Splicing During Rift Valley Fever Virus Infection Underscores the Links Between Alternative Splicing and Innate Antiviral Immunity.

Rift Valley fever virus (RVFV) is an emerging pathogen that has potential to cause severe disease in humans and domestic livestock. Propagation of RVFV strain MP-12 is negatively impacted by the actions of RIOK3, a protein involved in the cellular immune response to viral infection. During RVFV infection, RIOK3 mRNA is alternatively spliced to produce an isoform that correlates with the inhibition of interferon ß signaling. Here, we identify splicing factor TRA2-ß (also known as TRA2beta and hTRA2-ß) as a key regulator governing the relative abundance of RIOK3 splicing isoforms. Using RT-PCR and minigenes, we determined that TRA2-ß interaction with RIOK3 pre-mRNA was necessary for constitutive splicing of RIOK3 mRNA, and conversely, lack of TRA2-ß engagement led to increased alternative splicing. Expression of TRA2-ß was found to be necessary for RIOK3's antiviral effect against RVFV. Intriguingly, TRA2-ß mRNA is also alternatively spliced during RVFV infection, leading to a decrease in cellular TRA2-ß protein levels. These results suggest that splicing modulation serves as an immune evasion strategy by RVFV and/or is a cellular mechanism to prevent excessive immune response. Furthermore, the results suggest that TRA2-ß can act as a key regulator of additional steps of the innate immune response to viral infection.