Antiviral immunity lassoed down by excess RNA

During the past decades, the study of inborn errors of immunity (IEI) has significantly contributed to our understanding of the molecular mechanisms driving severe viral infections in humans (Casanova and Abel, 2021). One of the most profound contributions of IEI research is the identification of specific genetic mutations that impair the function of interferons (IFNs), particularly type I IFNs, which are crucial for antiviral immunity. Rare inborn errors of type I IFN immunity have been linked to severe cases of COVID-19, where deficiencies in this pathway were found to account for ∼15–20% of critical cases in unvaccinated individuals (Casanova and Anderson, 2023). This finding underscores the importance of IFN signaling in controlling viral infections and highlights how genetic predispositions can lead to severe disease manifestations. The exploration of these pathways has not only provided insights into the host’s defense mechanisms but has also suggested potential therapeutic targets for managing severe viral infections (Notarangelo et al., 2020). Furthermore, the ongoing investigation into IEI has revealed that the immune system’s response to viral infections is often characterized by a failure of key immune components, such as leukocytes and signaling pathways, which are essential for mounting an effective antiviral response (Bucciol et al., 2019). This has been particularly evident in cases where patients with IEI exhibit severe reactions to otherwise benign viral infections, emphasizing the critical role of genetic factors in determining disease severity (Ewing and Madan, 2024).

Two recent studies published in the Journal of Experimental Medicine have identified defective RNA processing as a critical factor in impaired antiviral immunity against multiple viruses, particularly in the context of brainstem encephalitis (Chan et al., 2024; Ru et al., 2024). In the first study, Chan et al. reported and clinically and genetically characterized a case of SARS-CoV-2 brainstem encephalitis in a patient with a homozygous pathogenic loss-of-function variant in the RNA lariat debranching enzyme DBR1 (Chan et al., 2024). As a result of the mutation, fibroblasts from the affected individual exhibited reduced levels of DBR1 protein and elevated levels of RNA lariats. The study highlights how accumulation of RNA lariats disrupts intrinsic antiviral immunity, resulting in uncontrolled SARS-CoV-2 replication in DBR1 I120T/I120T human pluripotent stem cell–derived hindbrain neurons, causing severe encephalitis. The findings suggest that DBR1 is crucial for protecting the brainstem from various viruses, including SARS-CoV-2. In a complementary study (Ru et al., 2024), Ru et al. further dissected the molecular mechanisms by which human inherited DBR1 deficiency underlies brainstem viral infections. The authors show that RNA lariat accumulation in DBR1-deficient cells impairs the formation of stress granules (SGs) that are essential for protein kinase R (PKR) activation, a critical step in antiviral immunity. G3BP proteins, important for SG assembly, are degraded by the proteasome in the absence of DBR1, leading to diminished PKR recruitment and antiviral function. As a result, DBR1-deficient cells show increased susceptibility to different viruses such as vesicular stomatitis virus and herpes simplex virus 1 (HSV-1). In their work, the authors show how patient-specific mutations in DBR1 severely compromise PKR activation, linking genetic variations to impaired antiviral responses. Moreover, mutant mice (Dbr1^Y17H/Y17H) display increased viral loads and impaired PKR activation in the brainstem, confirming the essential role of DBR1 in antiviral immunity in vivo. These works collectively suggest that RNA lariats, through their accumulation in DBR1-deficient cells, disrupt G3BP-mediated SG formation and PKR activation, leading to increased viral susceptibility both in vitro and in vivo, underlying brainstem viral infection (see figure).

The RNA lariat debranching enzyme DBR1 is the sole known RNA lariat debranching enzyme in mammals and has been implicated in processes such as class-switch recombination of immunoglobulin genes, and its dysfunction is increasingly associated with several pathologies. DBR1 hydrolyzes the 2–5′ bond in intron lariats produced by the spliceosome (Clark et al., 2023). These intron lariats are rapidly degraded by exonucleases following DBR1 hydrolysis but have also recently been shown to accumulate in the cytosol as stable circular molecules in cells of human, mouse, chicken, frog, and zebrafish origin (Talhouarne and Gall, 2018), suggesting potential roles in evolutionary conserved processes. Previous research has reported that individuals with autosomal recessive mutations in DBR1 exhibit increased susceptibility to encephalitis caused by typically benign viruses, such as HSV-1, influenza B virus, or norovirus (Zhang et al., 2018). The enzyme’s role likely extends beyond RNA processing as it has been linked to immune pathways, including those mediated by Toll-like receptor 3 (TLR3), to regulate central nervous system immunity against viral infections (Zhang et al., 2021). Moreover, DBR1 has been implicated in HIV-1 replication, where it facilitates the reverse transcription of viral RNA by cleaving lariat structures formed during early infection stages (Menees, 2020), potentially helping the virus to evade nucleic acid sensors within the cytoplasm. Inhibition of DBR1 has also been shown to mitigate the toxicity associated with TDP-43, a protein linked to amyotrophic lateral sclerosis, emphasizing the enzyme’s significance in maintaining RNA homeostasis in both viral infections and neurodegenerative diseases (Armakola et al., 2012).

Although traditional understanding of brainstem encephalitis has focused on infectious agents, recent studies indicate that autoimmune processes may also play a significant role (Tan et al., 2013). Indeed, the association of DBR1 with other neurological conditions, such as autoimmune encephalitis, suggests its role extends beyond direct viral defense to encompass broader neuroimmune interactions (Tago et al., 2015). In agreement, recent work shows how the TTDN1/MPLKIP, which is encoded by a gene implicated in non-photosensitive trichothiodystrophy (NP-TTD), functionally links intron lariat processing to spliceosomal function (Townley et al., 2023). The conserved TTDN1 C-terminal region was shown to directly bind DBR1, whereas its N-terminal intrinsically disordered region (IDR) binds the intron-binding complex. Interestingly, TTDN1 loss, or a mutated IDR, caused significant intron lariat accumulation, as well as splicing and gene expression defects, mirroring phenotypes observed in NP-TTD patient cells and a Ttdn1-deficient mouse model recapitulated intron-processing defects and certain neurodevelopmental phenotypes seen in NP-TTD. Whether TTDN1 mutations could be involved in the control of viral infections through mechanisms similar to that identified by Ru et al. for DBR1 loss of function remains to be explored.

Chan et al. demonstrated that SARS-CoV-2 infection leads to elevated levels of specific lariat RNAs in neurons with inherited DBR1 deficiencies (Chan et al., 2024). This accumulation may allow the virus to exploit intronic RNA to enhance its replication or evade host immune responses. Similar patterns have been observed in HSV-1 infections, where stable lariat introns can manipulate splicing pathways to the virus’s advantage (Zhang et al., 2018). In agreement, the unique structural characteristics of lariat RNAs, such as their 2′,5′-phosphodiester linkages, allow for specialized interactions with various cellular factors involved in splicing and RNA degradation (Montemayor et al., 2014). Lariat RNAs have also been shown to sequester components of the RNA interference machinery, potentially modulating host antiviral responses (Li et al., 2016).

In the context of viral brainstem encephalitis, Ru et al. demonstrated that RNA lariat accumulation in DBR1-deficient cells hinders PKR activation, which is essential for controlling viral replication. Interestingly, also circular RNAs (circRNAs), produced through different splicing mechanisms, inhibit PKR activation during homeostasis and infection. Upon viral infection, circRNAs are rapidly degraded by RNase L, restoring PKR activity (Ren et al., 2022). Conversely, low circRNA expression has been linked to aberrant PKR activation in systemic lupus erythematosus, resulting in sterile inflammation (Liu et al., 2019). CircRNAs are also implicated in non-viral diseases, including cancer and neurodegenerative disorders. In cancer, dysregulated circRNAs are associated with tumorigenesis and metastasis, suggesting their potential as biomarkers for diagnosis and prognosis (Wang et al., 2016), and they have been shown to modulate gene expression by acting as miRNA sponges, influencing the availability of miRNAs to target mRNAs (Guo et al., 2018). In neurodegenerative diseases, circRNAs regulate neuronal function, with dysregulation linked to conditions like Alzheimer’s and Parkinson’s disease (Dong et al., 2023; Zhang et al., 2020). As circRNAs may derive from lariat structures that escape debranching, potential links between RNA lariat accumulation and aberrant PKR activity may arise also in the context of these disorders.

In conclusion, the works from Chan et al. and Ru et al. elucidate how DBR1 is a crucial component of the brain’s antiviral defense system against multiple viruses, including SARS-CoV2. Their work also uncovers how the accumulation of RNA lariats due to defective debranching processes can influence viral replication and host immune responses specifically in the brainstem. The interplay between RNA lariat functions and DBR1 mutations, potentially linked also to circRNA biology and other host factors involved in the regulation of splicing, present a complex landscape of which understanding will be key to unveiling the molecular underpinnings of various viral and non-viral pathologies, paving the way for the development of novel therapeutic targets and biomarkers for a broad range of disorders.