DUSP11-mediated control of 5'-triphosphate RNA regulates RIG-I sensitivity.

Deciphering the mechanisms that regulate the sensitivity of pathogen recognition receptors is imperative to understanding infection and inflammation. Here we demonstrate that the RNA triphosphatase dual-specificity phosphatase 11 (DUSP11) acts on both host and virus-derived 5'-triphosphate RNAs rendering them less active in inducing a RIG-I-mediated immune response. Reducing DUSP11 levels alters host triphosphate RNA packaged in extracellular vesicles and induces enhanced RIG-I activation in cells exposed to extracellular vesicles. Virus infection of cells lacking DUSP11 results in a higher proportion of triphosphorylated viral transcripts and attenuated virus replication, which is rescued by reducing RIG-I expression. Consistent with the activity of DUSP11 in the cellular RIG-I response, mice lacking DUSP11 display lower viral loads, greater sensitivity to triphosphorylated RNA, and a signature of enhanced interferon activity in select tissues. Our results reveal the importance of controlling 5'-triphosphate RNA levels to prevent aberrant RIG-I signaling and demonstrate DUSP11 as a key effector of this mechanism.

Nucleic acid-protein condensates in innate immune signaling.

The presence of foreign nucleic acids in the cytosol is a marker of infection. Cells have sensors, also known as pattern recognition receptors (PRRs), in the cytosol that detect foreign nucleic acid and initiate an innate immune response. Recent studies have reported the condensation of multiple PRRs including PKR, NLRP6, and cGAS, with their nucleic acid activators into discrete nucleoprotein assemblies. Nucleic acid-protein condensates form due to multivalent interactions and can create high local concentrations of components. The formation of PRR-containing condensates may alter the magnitude or timing of PRR activation. In addition, unique condensates form following RNase L activation or during paracrine signaling from virally infected cells that may play roles in antiviral defense. These observations suggest that condensate formation may be a conserved mechanism that cells use to regulate activation of the innate immune response and open an avenue for further investigation into the composition and function of these condensates. Here we review the nucleic acid-protein granules that are implicated in the innate immune response, discuss general consequences of condensate formation and signal transduction, as well as what outstanding questions remain.

RNase L Reprograms Translation by Widespread mRNA Turnover Escaped by Antiviral mRNAs.

In response to foreign and endogenous double-stranded RNA (dsRNA), protein kinase R (PKR) and ribonuclease L (RNase L) reprogram translation in mammalian cells. PKR inhibits translation initiation through eIF2α phosphorylation, which triggers stress granule (SG) formation and promotes translation of stress responsive mRNAs. The mechanisms of RNase L-driven translation repression, its contribution to SG assembly, and its regulation of dsRNA stress-induced mRNAs are unknown. We demonstrate that RNase L drives translational shut-off in response to dsRNA by promoting widespread turnover of mRNAs. This alters stress granule assembly and reprograms translation by allowing translation of mRNAs resistant to RNase L degradation, including numerous antiviral mRNAs such as interferon (IFN)-ß. Individual cells differentially activate dsRNA responses revealing variation that can affect cellular outcomes. This identifies bulk mRNA degradation and the resistance of antiviral mRNAs as the mechanism by which RNase L reprograms translation in response to dsRNA.

SARS-CoV-2 nsp15 endoribonuclease antagonizes dsRNA-induced antiviral signaling.

Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 has caused millions of deaths since its emergence in 2019. Innate immune antagonism by lethal CoVs such as SARS-CoV-2 is crucial for optimal replication and pathogenesis. The conserved nonstructural protein 15 (nsp15) endoribonuclease (EndoU) limits activation of double-stranded (ds)RNA-induced pathways, including interferon (IFN) signaling, protein kinase R (PKR), and oligoadenylate synthetase/ribonuclease L (OAS/RNase L) during diverse CoV infections including murine coronavirus and Middle East respiratory syndrome (MERS)-CoV. To determine how nsp15 functions during SARS-CoV-2 infection, we constructed a recombinant SARS-CoV-2 (nsp15mut) expressing catalytically inactivated nsp15, which we show promoted increased dsRNA accumulation. Infection with SARS-CoV-2 nsp15mut led to increased activation of the IFN signaling and PKR pathways in lung-derived epithelial cell lines and primary nasal epithelial air-liquid interface (ALI) cultures as well as significant attenuation of replication in ALI cultures compared to wild-type virus. This replication defect was rescued when IFN signaling was inhibited with the Janus activated kinase (JAK) inhibitor ruxolitinib. Finally, to assess nsp15 function in the context of minimal (MERS-CoV) or moderate (SARS-CoV-2) innate immune induction, we compared infections with SARS-CoV-2 nsp15mut and previously described MERS-CoV nsp15 mutants. Inactivation of nsp15 had a more dramatic impact on MERS-CoV replication than SARS-CoV-2 in both Calu3 cells and nasal ALI cultures suggesting that SARS-CoV-2 can better tolerate innate immune responses. Taken together, SARS-CoV-2 nsp15 is a potent inhibitor of dsRNA-induced innate immune response and its antagonism of IFN signaling is necessary for optimal viral replication in primary nasal ALI cultures.

RNA is required for the integrity of multiple nuclear and cytoplasmic membrane-less RNP granules.

Numerous membrane-less organelles, composed of a combination of RNA and proteins, are observed in the nucleus and cytoplasm of eukaryotic cells. These RNP granules include stress granules (SGs), processing bodies (PBs), Cajal bodies, and nuclear speckles. An unresolved question is how frequently RNA molecules are required for the integrity of RNP granules in either the nucleus or cytosol. To address this issue, we degraded intracellular RNA in either the cytosol or the nucleus by the activation of RNase L and examined the impact of RNA loss on several RNP granules. We find the majority of RNP granules, including SGs, Cajal bodies, nuclear speckles, and the nucleolus, are altered by the degradation of their RNA components. In contrast, PBs and super-enhancer complexes were largely not affected by RNA degradation in their respective compartments. RNA degradation overall led to the apparent dissolution of some membrane-less organelles, whereas others reorganized into structures with altered morphology. These findings highlight a critical and widespread role of RNA in the organization of several RNP granules.

dsRNA-induced condensation of antiviral proteins modulates PKR activity.

Mammalian cells respond to dsRNA in multiple manners. One key response to dsRNA is the activation of PKR, an eIF2α kinase, which triggers translational arrest and the formation of stress granules. However, the process of PKR activation in cells is not fully understood. In response to increased endogenous or exogenous dsRNA, we observed that PKR forms novel cytosolic condensates, referred to as dsRNA-induced foci (dRIFs). dRIFs contain dsRNA, form in proportion to dsRNA, and are enhanced by longer dsRNAs. dRIFs enrich several other dsRNA-binding proteins, including ADAR1, Stau1, NLRP1, and PACT. Strikingly, dRIFs correlate with and form before translation repression by PKR and localize to regions of cells where PKR activation is initiated. We hypothesize that dRIF formation is a mechanism that cells use to enhance the sensitivity of PKR activation in response to low levels of dsRNA or to overcome viral inhibitors of PKR activation.

Novel stress granule-like structures are induced via a paracrine mechanism during viral infection.

To rapidly adapt to stresses such as infections, cells have evolved several mechanisms, which include the activation of stress response pathways and the innate immune response. These stress responses result in the rapid inhibition of translation and condensation of stalled mRNAs with RNA-binding proteins and signalling components into cytoplasmic biocondensates called stress granules (SGs). Increasing evidence suggests that SGs contribute to antiviral defence, and thus viruses need to evade these responses to propagate. We previously showed that feline calicivirus (FCV) impairs SG assembly by cleaving the scaffolding protein G3BP1. We also observed that uninfected bystander cells assembled G3BP1-positive granules, suggesting a paracrine response triggered by infection. We now present evidence that virus-free supernatant generated from infected cells can induce the formation of SG-like foci, which we name paracrine granules. They are linked to antiviral activity and exhibit specific kinetics of assembly-disassembly, and protein and RNA composition that are different from canonical SGs. We propose that this paracrine induction reflects a novel cellular defence mechanism to limit viral propagation and promote stress responses in bystander cells.

RNase L activation in the cytoplasm induces aberrant processing of mRNAs in the nucleus.

The antiviral endoribonuclease, RNase L, is activated by the mammalian innate immune response to destroy host and viral RNA to ultimately reduce viral gene expression. Herein, we show that RNase L and RNase L-mediated mRNA decay are primarily localized to the cytoplasm. Consequently, RNA-binding proteins (RBPs) translocate from the cytoplasm to the nucleus upon RNase L activation due to the presence of intact nuclear RNA. The re-localization of RBPs to the nucleus coincides with global alterations to RNA processing in the nucleus. While affecting many host mRNAs, these alterations are pronounced in mRNAs encoding type I and type III interferons and correlate with their retention in the nucleus and reduction in interferon protein production. Similar RNA processing defects also occur during infection with either dengue virus or SARS-CoV-2 when RNase L is activated. These findings reveal that the distribution of RBPs between the nucleus and cytosol is dictated by the availability of RNA in each compartment. Thus, viral infections that trigger RNase L-mediated cytoplasmic RNA in the cytoplasm also alter RNA processing in the nucleus, resulting in an ingenious multi-step immune block to protein biogenesis.

A closer look at mammalian antiviral condensates.

Several biomolecular condensates assemble in mammalian cells in response to viral infection. The most studied of these are stress granules (SGs), which have been proposed to promote antiviral innate immune signaling pathways, including the RLR-MAVS, the protein kinase R (PKR), and the OAS-RNase L pathways. However, recent studies have demonstrated that SGs either negatively regulate or do not impact antiviral signaling. Instead, the SG-nucleating protein, G3BP1, may function to perturb viral RNA biology by condensing viral RNA into viral-aggregated RNA condensates, thus explaining why viruses often antagonize G3BP1 or hijack its RNA condensing function. However, a recently identified condensate, termed double-stranded RNA-induced foci, promotes the activation of the PKR and OAS-RNase L antiviral pathways. In addition, SG-like condensates known as an RNase L-induced bodies (RLBs) have been observed during many viral infections, including SARS-CoV-2 and several flaviviruses. RLBs may function in promoting decay of cellular and viral RNA, as well as promoting ribosome-associated signaling pathways. Herein, we review these recent advances in the field of antiviral biomolecular condensates, and we provide perspective on the role of canonical SGs and G3BP1 during the antiviral response.

Mechanisms and consequences of mRNA destabilization during viral infections.

During viral infection there is dynamic interplay between the virus and the host to regulate gene expression. In many cases, the host induces the expression of antiviral genes to combat infection, while the virus uses "host shut-off" systems to better compete for cellular resources and to limit the induction of the host antiviral response. Viral mechanisms for host shut-off involve targeting translation, altering host RNA processing, and/or inducing the degradation of host mRNAs. In this review, we discuss the diverse mechanisms viruses use to degrade host mRNAs. In addition, the widespread degradation of host mRNAs can have common consequences including the accumulation of RNA binding proteins in the nucleus, which leads to altered RNA processing, mRNA export, and changes to transcription.

DUSP11 activity on triphosphorylated transcripts promotes Argonaute association with noncanonical viral microRNAs and regulates steady-state levels of cellular noncoding RNAs.

RNA silencing is a conserved eukaryotic gene expression regulatory mechanism mediated by small RNAs. In Caenorhabditis elegans, the accumulation of a distinct class of siRNAs synthesized by an RNA-dependent RNA polymerase (RdRP) requires the PIR-1 phosphatase. However, the function of PIR-1 in RNAi has remained unclear. Since mammals lack an analogous siRNA biogenesis pathway, an RNA silencing role for the mammalian PIR-1 homolog (dual specificity phosphatase 11 [DUSP11]) was unexpected. Here, we show that the RNA triphosphatase activity of DUSP11 promotes the RNA silencing activity of viral microRNAs (miRNAs) derived from RNA polymerase III (RNAP III) transcribed precursors. Our results demonstrate that DUSP11 converts the 5' triphosphate of miRNA precursors to a 5' monophosphate, promoting loading of derivative 5p miRNAs into Argonaute proteins via a Dicer-coupled 5' monophosphate-dependent strand selection mechanism. This mechanistic insight supports a likely shared function for PIR-1 in C. elegans Furthermore, we show that DUSP11 modulates the 5' end phosphate group and/or steady-state level of several host RNAP III transcripts, including vault RNAs and Alu transcripts. This study shows that steady-state levels of select noncoding RNAs are regulated by DUSP11 and defines a previously unknown portal for small RNA-mediated silencing in mammals, revealing that DUSP11-dependent RNA silencing activities are shared among diverse metazoans.

ADAR1 limits stress granule formation through both translation-dependent and translation-independent mechanisms.

Stress granules (SGs) are cytoplasmic assemblies of RNA and protein that form when translation is repressed during the integrated stress response. SGs assemble from the combination of RNA-RNA, RNA-protein and protein-protein interactions between messenger ribonucleoprotein complexes (mRNPs). The protein adenosine deaminase acting on RNA 1 (ADAR1, also known as ADAR) recognizes and modifies double-stranded RNAs (dsRNAs) within cells to prevent an aberrant innate immune response. ADAR1 localizes to SGs, and since RNA-RNA interactions contribute to SG assembly and dsRNA induces SGs, we examined how ADAR1 affects SG formation. First, we demonstrate that ADAR1 depletion triggers SGs by allowing endogenous dsRNA to activate the integrated stress response through activation of PKR (also known as EIF2AK2) and translation repression. However, we also show that ADAR1 limits SG formation independently of translation inhibition. ADAR1 repression of SGs is independent of deaminase activity but is dependent on dsRNA-binding activity, suggesting a model where ADAR1 binding limits RNA-RNA and/or RNA-protein interactions necessary for recruitment to SGs. Given that ADAR1 expression is induced during viral infection, these findings have implications for the role of ADAR1 in the antiviral response. This article has an associated First Person interview with the first author of the paper.

SARS-CoV-2 infection triggers widespread host mRNA decay leading to an mRNA export block.

The transcriptional induction of interferon (IFN) genes is a key feature of the mammalian antiviral response that limits viral replication and dissemination. A hallmark of severe COVID-19 disease caused by SARS-CoV-2 is the low presence of IFN proteins in patient serum despite elevated levels of IFN-encoding mRNAs, indicative of post-transcriptional inhibition of IFN protein production. Here, we performed single-molecule RNA visualization to examine the expression and localization of host mRNAs during SARS-CoV-2 infection. Our data show that the biogenesis of type I and type III IFN mRNAs is inhibited at multiple steps during SARS-CoV-2 infection. First, translocation of the interferon regulatory factor 3 (IRF3) transcription factor to the nucleus is limited in response to SARS-CoV-2, indicating that SARS-CoV-2 inhibits RLR-MAVS signaling and thus weakens transcriptional induction of IFN genes. Second, we observed that IFN mRNAs primarily localize to the site of transcription in most SARS-CoV-2 infected cells, suggesting that SARS-CoV-2 either inhibits the release of IFN mRNAs from their sites of transcription and/or triggers decay of IFN mRNAs in the nucleus upon exiting the site of transcription. Lastly, nuclear-cytoplasmic transport of IFN mRNAs is inhibited during SARS-CoV-2 infection, which we propose is a consequence of widespread degradation of host cytoplasmic basal mRNAs in the early stages of SARS-CoV-2 replication by the SARS-CoV-2 Nsp1 protein, as well as the host antiviral endoribonuclease, RNase L. Importantly, IFN mRNAs can escape SARS-CoV-2-mediated degradation if they reach the cytoplasm, making rescue of mRNA export a viable means for promoting the immune response to SARS-CoV-2.

RNase L promotes the formation of unique ribonucleoprotein granules distinct from stress granules.

Stress granules (SGs) are ribonucleoprotein (RNP) assemblies that form in eukaryotic cells as a result of limited translation in response to stress. SGs form during viral infection and are thought to promote the antiviral response because many viruses encode inhibitors of SG assembly. However, the antiviral endoribonuclease RNase L also alters SG formation, whereby only small punctate SG-like bodies that we term RNase L-dependent bodies (RLBs) form during RNase L activation. How RLBs relate to SGs and their mode of biogenesis is unknown. Herein, using immunofluorescence, live-cell imaging, and MS-based analyses, we demonstrate that RLBs represent a unique RNP granule with a protein and RNA composition distinct from that of SGs in response to dsRNA lipofection in human cells. We found that RLBs are also generated independently of SGs and the canonical dsRNA-induced SG biogenesis pathway, because RLBs did not require protein kinase R, phosphorylation of eukaryotic translation initiation factor 2 subunit 1 (eIF2α), the SG assembly G3BP paralogs, or release of mRNAs from ribosomes via translation elongation. Unlike the transient interactions between SGs and P-bodies, RLBs and P-bodies extensively and stably interacted. However, despite both RLBs and P-bodies exhibiting liquid-like properties, they remained distinct condensates. Taken together, these observations reveal that RNase L promotes the formation of a unique RNP complex that may have roles during the RNase L-mediated antiviral response.

The Murine Polyomavirus MicroRNA Locus Is Required To Promote Viruria during the Acute Phase of Infection.

Polyomaviruses (PyVs) can cause serious disease in immunosuppressed hosts. Several pathogenic PyVs encode microRNAs (miRNAs), small RNAs that regulate gene expression via RNA silencing. Despite recent advances in understanding the activities of PyV miRNAs, the biological functions of PyV miRNAs during in vivo infections are mostly unknown. The studies presented here used murine polyomavirus (MuPyV) as a model to assess the roles of the PyV miRNAs in a natural host. This analysis revealed that a MuPyV mutant that is unable to express miRNAs has enhanced viral DNA loads in select tissues at late times after infection. This is consistent with the PyV miRNAs functioning to reduce viral replication during the persistent phase of infection in a natural host. Additionally, the MuPyV miRNA locus promotes viruria during the acute phase of infection as evidenced by a defect in shedding during infection with the miRNA mutant virus. The viruria defect of the miRNA mutant virus could be rescued by infecting Rag2-/- mice. These findings implicate the miRNA locus as functioning in both the persistent and acute phases of infection and suggest a role for MuPyV miRNA in evading the adaptive immune response.IMPORTANCE MicroRNAs are expressed by diverse viruses, but for only a few is there any understanding of their in vivo function. PyVs can cause serious disease in immunocompromised hosts. Therefore, increased knowledge of how these viruses interact with the immune response is of clinical relevance. Here we show a novel activity for a viral miRNA locus in promoting virus shedding. This work indicates that in addition to any role for the PyV miRNA locus in long-term persistence, it also has biological activity during the acute phase. As this mutant phenotype is alleviated by infection of mice lacking an adaptive immune response, our work also connects the in vivo activity of the PyV miRNA locus to the immune response. Given that PyV-associated disease is associated with alterations in the immune response, our findings help to better understand how the balance between PyVs and the immune response becomes altered in pathogenic states.

Expression of short hairpin RNAs using the compact architecture of retroviral microRNA genes.

Short hairpin RNAs (shRNAs) are effective in generating stable repression of gene expression. RNA polymerase III (RNAP III) type III promoters (U6 or H1) are typically used to drive shRNA expression. While useful for some knockdown applications, the robust expression of U6/H1-driven shRNAs can induce toxicity and generate heterogeneous small RNAs with undesirable off-target effects. Additionally, typical U6/H1 promoters encompass the majority of the ∼270 base pairs (bp) of vector space required for shRNA expression. This can limit the efficacy and/or number of delivery vector options, particularly when delivery of multiple gene/shRNA combinations is required. Here, we develop a compact shRNA (cshRNA) expression system based on retroviral microRNA (miRNA) gene architecture that uses RNAP III type II promoters. We demonstrate that cshRNAs coded from as little as 100 bps of total coding space can precisely generate small interfering RNAs (siRNAs) that are active in the RNA-induced silencing complex (RISC). We provide an algorithm with a user-friendly interface to design cshRNAs for desired target genes. This cshRNA expression system reduces the coding space required for shRNA expression by >2-fold as compared to the typical U6/H1 promoters, which may facilitate therapeutic RNAi applications where delivery vector space is limiting.

DUSP11 - An RNA phosphatase that regulates host and viral non-coding RNAs in mammalian cells.

Dual-specificity phosphatase 11 (DUSP11) is a conserved protein tyrosine phosphatase (PTP) in metazoans. The cellular substrates and physiologic activities of DUSP11 remain largely unknown. In nematodes, DUSP11 is required for normal development and RNA interference against endogenous RNAs (endo-RNAi) via molecular mechanisms that are not well understood. However, mammals lack analogous endo-RNAi pathways and consequently, a role for DUSP11 in mammalian RNA silencing was unanticipated. Recent work from our laboratory demonstrated that DUSP11 activity alters the silencing potential of noncanonical viral miRNAs in mammalian cells. Our studies further uncovered direct cellular substrates of DUSP11 and suggest that DUSP11 is part of regulatory pathway that controls the abundance of select triphosphorylated noncoding RNAs. Here, we highlight recent findings and present new data that advance understanding of mammalian DUSP11 during gene silencing and discuss the emerging biological activities of DUSP11 in mammalian cells.

A central role for the primary microRNA stem in guiding the position and efficiency of Drosha processing of a viral pri-miRNA.

Processing of primary microRNA (pri-miRNA) stem-loops by the Drosha-DGCR8 complex is the initial step in miRNA maturation and crucial for miRNA function. Nonetheless, the underlying mechanism that determines the Drosha cleavage site of pri-miRNAs has remained unclear. Two prevalent but seemingly conflicting models propose that Drosha-DGCR8 anchors to and directs cleavage a fixed distance from either the basal single-stranded (ssRNA) or the terminal loop. However, recent studies suggest that the basal ssRNA and/or the terminal loop may influence the Drosha cleavage site dependent upon the sequence/structure of individual pri-miRNAs. Here, using a panel of closely related pri-miRNA variants, we further examine the role of pri-miRNA structures on Drosha cleavage site selection in cells. Our data reveal that both the basal ssRNA and terminal loop influence the Drosha cleavage site within three pri-miRNAs, the Simian Virus 40 (SV40) pri-miRNA, pri-miR-30a, and pri-miR-16. In addition to the flanking ssRNA regions, we show that an internal loop within the SV40 pri-miRNA stem strongly influences Drosha cleavage position and efficiency. We further demonstrate that the positions of the internal loop, basal ssRNA, and the terminal loop of the SV40 pri-miRNA cooperatively coordinate Drosha cleavage position and efficiency. Based on these observations, we propose that the pri-miRNA stem, defined by internal and flanking structural elements, guides the binding position of Drosha-DGCR8, which consequently determines the cleavage site. This study provides mechanistic insight into pri-miRNA processing in cells that has numerous biological implications and will assist in refining Drosha-dependent shRNA design.

Identification, validation, and characterization of noncanonical miRNAs.

Many eukaryotes and some viruses encode microRNAs (miRNAs), small RNAs that post-transcriptionally regulate gene expression. While most miRNAs are generated through the activity of RNA Polymerase II (RNAP II) and subsequent processing by Drosha and Dicer, some viral miRNAs utilize alternative pathways of biogenesis. Some members of the herpesvirus and retrovirus families can direct synthesis of miRNAs through RNAP III transcription rather than RNAP II and can utilize atypical enzymes to generate miRNAs. Though the advantages of alternative miRNA biogenesis remain unclear for herpesviruses, the retroviral miRNA biogenesis routes allow the RNAP II transcribed retroviral genome to escape Drosha cleavage while still expressing abundant, biologically-active miRNAs. These RNAP III-derived miRNAs have unique characteristics that allow for their identification and characterization. In this article, we describe procedures to predict, validate, and characterize RNAP III-transcribed miRNAs and other small RNAs, while providing resources that are also useful for canonical miRNAs.