MERS-CoV endoribonuclease and accessory proteins jointly evade host innate immunity during infection of lung and nasal epithelial cells

Middle East respiratory syndrome coronavirus (MERS CoV) emerged into humans in 2012, causing highly lethal respiratory disease. The severity of disease may be in part because MERS CoV is adept at antagonizing early innate immune pathways; these include interferon (IFN) production and signaling, protein kinase R (PKR), and oligoadenylate synthetase ribonuclease L (OAS/RNase L), all activated in response to viral double stranded (ds)RNA generated during genome replication. This is in contrast to SARS CoV 2, which we recently reported activates PKR and RNase L and to some extent, IFN signaling. We previously found that MERS-CoV accessory proteins NS4a (dsRNA binding protein) and NS4b (phosphodiesterase) could weakly suppress these pathways, but ablation of each had minimal effect on virus replication. Here we investigated the antagonist effects of the conserved coronavirus endoribonuclease (EndoU), in combination with NS4a or NS4b. Inactivation of EndoU catalytic activity alone in a recombinant MERS-CoV caused little if any effect on activation of the innate immune pathways during infection. However, infection with recombinant viruses containing combined mutations with inactivation of EndoU and deletion of NS4a or inactivation of the NS4b phosphodiesterase promoted robust activation of the dsRNA-induced innate immune pathways. This resulted in ten-fold attenuation of replication in human lung derived A549 and primary nasal cells. Furthermore, replication of these recombinant viruses could be rescued to the level of WT MERS CoV by knockout of host immune mediators MAVS, PKR, or RNase L. Thus, EndoU and accessory proteins NS4a and NS4b together suppress dsRNA induced innate immunity during MERS CoV infection in order to optimize viral replication.

The origin and evolution of loqs2: a gene encoding an antiviral dsRNA binding protein in Aedes mosquitoes

Mosquito borne viruses such as dengue, Zika, yellow fever and Chikungunya cause millions of infections every year. These viruses are mostly transmitted by two urban-adapted mosquito species, Aedes aegypti and Aedes albopictus, that appear to be more permissive to arbovirus infections compared to closely related species. Although mechanistic understanding remains, Aedes mosquitoes may have evolved specialized antiviral mechanisms that potentially contribute to the low impact of viral infection. Recently, we reported the identification of an Aedes specific double-stranded RNA binding protein (dsRBP), named Loqs2, that is involved in the control of infection by dengue and Zika viruses in Ae. aegypti. Loqs2 interacts with two important co-factors of the RNA interference (RNAi) pathway, Loquacious (Loqs) and R2D2, and seems to be a strong regulator of the antiviral defense. However, the origin and evolution of loqs2 remains unclear. Here, we describe that loqs2 likely originated from two independent duplications of the first dsRNA binding domain (dsRBD) of loquacious that occurred before the radiation of the Aedes Stegomya subgenus. After its origin, our analyses suggest that loqs2 evolved by relaxed positive selection towards neofunctionalization. In fact, loqs2 is evolving at a faster pace compared to other RNAi components such as loquacious, r2d2 and Dicer-2 in Aedes mosquitoes. Unlike loquacious, transcriptomic analysis showed that loqs2 expression is tightly regulated, almost restricted to reproductive tissues in Ae. aegypti and Ae. albopictus. Transgenic mosquitoes engineered to ubiquitously express loqs2 show massive dysregulation of stress response genes and undergo developmental arrest at larval stages. Overall, our results uncover the possible origin and neofunctionalization of a novel antiviral gene, loqs2, in Aedes mosquitoes that ultimately may contribute to their effectiveness as vectors for arboviruses.

Immunocapture of dsRNA-bound proteins provides insight into Tobacco rattle virus replication complexes and reveals Arabidopsis DRB2 to be a wide-spectrum antiviral effector

Plant RNA viruses form organized membrane-bound replication complexes to replicate their genomes. This process requires virus- and host-encoded proteins and leads to the production of double-stranded RNA (dsRNA) replication intermediates. Here, we describe the use of Arabidopsis thaliana expressing GFP-tagged dsRNA-binding protein (B2:GFP) to pull down dsRNA and associated proteins in planta upon infection with Tobacco rattle virus (TRV). Mass spectrometry analysis of the dsRNA-B2:GFP-bound proteins from infected plants revealed the presence of viral proteins and numerous host proteins. Among a selection of nine host candidate proteins, eight showed relocalization upon infection, and seven of these colocalized with B2-labeled TRV replication complexes. Infection of A. thaliana T-DNA mutant lines for eight such factors revealed that genetic knockout of dsRNA-BINDING PROTEIN 2 (DRB2) leads to increased TRV accumulation and DRB2 overexpression caused a decrease in the accumulation of four different plant RNA viruses, indicating that DRB2 has a potent and wide-ranging antiviral activity. We propose B2:GFP-mediated pull down of dsRNA to be a versatile method to explore virus replication complex proteomes and to discover key host virus replication factors. Given the universality of dsRNA, development of this tool holds great potential to investigate RNA viruses in other host organisms.

The epigenetic factor FVE orchestrates cytoplasmic SGS3-DRB4-DCL4 activities to promote transgene silencing in Arabidopsis

Posttranscriptional gene silencing (PTGS) is a regulatory mechanism to suppress undesired transcripts. Here, we identified Flowering locus VE (FVE), a well-known epigenetic component, as a new player in cytoplasmic PTGS. Loss-of-function fve mutations substantially reduced the accumulation of transgene-derived small interfering RNAs (siRNAs). FVE interacts with suppressor of gene silencing 3 (SGS3), a master component in PTGS. FVE promotes SGS3 homodimerization that is essential for its function. FVE can bind to single-stranded RNA and double-stranded RNA (dsRNA) with moderate affinities, while its truncated form FVE-8 has a significantly increased binding affinity to dsRNA. These affinities affect the association and channeling of SGS3-RNA to downstream dsRNA binding protein 4 (DRB4)/Dicer-like protein 2/4 (DCL2/4) complexes. Hence, FVE, but not FVE-8, biochemically enhances the DRB4/DCL2/4 activity in vitro. We surmise that FVE promotes production of transgene-derived siRNAs through concertedly tuning SGS3-DRB4/DCL2/4 functions. Thus, this study revealed a noncanonical role of FVE in PTGS.

Cell-free reconstitution reveals the molecular mechanisms for the initiation of secondary siRNA biogenesis in plants

Secondary small interfering RNA (siRNA) production, triggered by primary small RNA targeting, is critical for proper development and antiviral defense in many organisms. RNA-dependent RNA polymerase (RDR) is a key factor in this pathway. However, how RDR specifically converts the targets of primary small RNAs into double-stranded RNA (dsRNA) intermediates remains unclear. Here, we develop an in vitro system that allows for dissection of the molecular mechanisms underlying the production of trans-acting siRNAs, a class of plant secondary siRNAs that play roles in organ development and stress responses. We find that a combination of the dsRNA-binding protein, SUPPRESSOR OF GENE SILENCING3; the putative nuclear RNA export factor, SILENCING DEFECTIVE5, primary small RNA, and Argonaute is required for physical recruitment of RDR6 to target RNAs. dsRNA synthesis by RDR6 is greatly enhanced by the removal of the poly(A) tail, which can be achieved by the cleavage at a second small RNA-binding site bearing appropriate mismatches. Importantly, when the complementarity of the base pairing at the second target site is too strong, the small RNA–Argonaute complex remains at the cleavage site, thereby blocking the initiation of dsRNA synthesis by RDR6. Our data highlight the light and dark sides of double small RNA targeting in the secondary siRNA biogenesis.

Low dose/dose rate γ irradiation protects Drosophila melanogaster chromosomes from double strand breaks and telomere fusions by modulating the expression of Loquacious.

It is still continuously debated whether the low-dose/dose-rate (LDDR) of ionizing radiation represents a hazard for humans. Model organisms, such as fruit flies, are considered valuable systems to reveal insights into this issue. We found that, in wild-type Drosophila melanogaster larval neuroblasts, the frequency of Chromosome Breaks (CBs), induced by acute γ-radiation, is significantly reduced when flies are previously exposed to a protracted dose of 0.4 Gy delivered at a dose rate of 2.5 mGy/h. This indicates that this exposure, which is associated with an increased expression of DNA damage response proteins, induces a radioadaptive response (RAR) that protects Drosophila from extensive DNA damage. Interestingly, the same exposure reduces the frequency of telomere fusions (TFs) from Drosophila telomere capping mutants suggesting that the LDDR can generally promote a protective response on chromatin sites that are recognized as DNA breaks. Deep RNA sequencing revealed that RAR is associated with a modulation of genes involved in RNA processing and the reduction of expression of Loquacious D (Loqs-RD) gene that encodes a well-conserved dsRNA binding protein required for esiRNAs biogenesis. Remarkably, loss of Loqs mimics the LDDR-mediated chromosome protection as it decreases the IR-induced CBs and TF frequency. Thus, our work identifies Loqs as a key factor in mediating the cellular response to LDDR and reveals that the study of the molecular bases underlying RAR is instrumental for unraveling novel and conserved epigenetic routes that have evolved to preserve genome integrity following robust insults to DNA.

Single-molecule analysis of processive double-stranded RNA cleavage by Drosophila Dicer-2

Drosophila Dicer-2 (Dcr-2) produces small interfering RNAs from long double-stranded RNAs (dsRNAs), playing an essential role in antiviral RNA interference. The dicing reaction by Dcr-2 is enhanced by Loquacious-PD (Loqs-PD), a dsRNA-binding protein that partners with Dcr-2. Previous biochemical analyses have proposed that Dcr-2 uses two distinct—processive or distributive—modes of cleavage by distinguishing the terminal structures of dsRNAs and that Loqs-PD alters the terminal dependence of Dcr-2. However, the direct evidence for this model is lacking, as the dynamic movement of Dcr-2 along dsRNAs has not been traced. Here, by utilizing single-molecule imaging, we show that the terminal structures of long dsRNAs and the presence or absence of Loqs-PD do not essentially change Dcr-2’s cleavage mode between processive and distributive, but rather simply affect the probability for Dcr-2 to undergo the cleavage reaction. Our results provide a refined model for how the dicing reaction by Dcr-2 is regulated.

Noncanonical immune response to the inhibition of DNA methylation by Staufen1 via stabilization of endogenous retrovirus RNAs

DNA-methyltransferase inhibitors (DNMTis), such as azacitidine and decitabine, are used clinically to treat myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Decitabine activates the transcription of endogenous retroviruses (ERVs), which can induce immune response by acting as cellular double-stranded RNAs (dsRNAs). Yet, the posttranscriptional regulation of ERV dsRNAs remains uninvestigated. Here, we find that the viral mimicry and subsequent cell death in response to decitabine require the dsRNA-binding protein Staufen1 (Stau1). We show that Stau1 directly binds to ERV RNAs and stabilizes them in a genome-wide manner. Furthermore, Stau1-mediated stabilization requires a long noncoding RNA TINCR, which enhances the interaction between Stau1 and ERV RNAs. Analysis of a clinical patient cohort reveals that MDS and AML patients with lower Stau1 and TINCR expressions exhibit inferior treatment outcomes to DNMTi therapy. Overall, our study reveals the posttranscriptional regulatory mechanism of ERVs and identifies the Stau1-TINCR complex as a potential target for predicting the efficacy of DNMTis and other drugs that rely on dsRNAs.

Determination of dsRNA interactome upon Sindbis virus infection in human cells identifies SFPQ as a critical proviral factor

Viruses are obligate intracellular parasites, which depend on the host cellular machineries to replicate their genome and complete their infectious cycle. Long double stranded (ds)RNA is a common viral by-product originating during RNA virus replication and is universally sensed as a danger signal to trigger the antiviral response. As a result, viruses hide dsRNA intermediates into viral replication factories and have evolved strategies to hijack cellular proteins for their benefit. The characterization of the host factors associated to viral dsRNA and involved in viral replication remains a major challenge to develop new antiviral drugs against RNA viruses. Here, we performed anti-dsRNA immunoprecipitation followed by mass spectrometry to fully characterize the dsRNA interactome in Sindbis virus (SINV) infected human HCT116 cells. Among the validated factors, we characterized SFPQ (Splicing factor, proline-glutamine rich) as a new dsRNA-associated factor upon SINV infection. We proved that SFPQ is able to directly bind to dsRNAs in vitro, that its association to dsRNA is independent of single-stranded (ss)RNA flanking regions in vivo and that it is able to bind to the viral genome upon infection. Furthermore, we showed that both knock-down and knock-out of SFPQ reduce SINV infection in human HCT116 and SK-N-BE(2) cells, suggesting that SFPQ could enhance viral replication. Overall, this study not only represents a resource to further study SINV dsRNA-associated factors upon infection but also identifies SFPQ as a new proviral dsRNA binding protein.