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SeV DVGs reduce virulence in vivo. (A–C) Mice were infected with 105 TCID50/mouse of SeV Cantell HD (HD) or SeV Cantell LD (LD). (A) Weight loss (***p<0.001, ****p<0.0001; Two-way ANOVA with Bonferroni's post hoc test), (B) Virus titers in the lung, (n = 11 for day 1, n = 6 for day 3), and (C) expression of Ifnb mRNA by RT-qPCR. Gene expression is shown as copy number relative to the housekeeping genes Tuba1b and Rps11 (***p<0.001, Unpaired, two tailed, t student test). (D–F) Mice were infected with 104 TCID50/mouse SeV Cantell LD alone, in the presence of 5,000 HA Units/mouse purified defective particles (DPs) or in the presence of UV-inactivated DPs (UVDP). Mice received DPs or UVDPs immediately following virus inoculation. (D) Weight loss, (†, mice sacrificed due to severe weight loss; ***p<0.001, ****p<0.0001; Two-way ANOVA with Bonferroni's post hoc test), (E) lung lesion score at day 7 post-infection (n = 3)(*p<0.05, Mann-Whitney test). (F) Photos of the lung of a representative mouse at day 7 post-infection. Arrowhead indicates areas of lesions. (G) Lungs from mice infected with SeV Cantell LD alone, or in the presence of DPs or UVDPs were analyzed by flow cytometry for the expression of the SeV NP protein.
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The innate immune response to viruses is initiated when specialized cellular sensors recognize viral danger signals. Here we show that truncated forms of viral genomes that accumulate in infected cells potently trigger the sustained activation of the transcription factors IRF3 and NF-κB and the production type I IFNs through a mechanism independent...
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... Consequently, these truncated forms can only replicate with the assistance of wild viruses, which act as helpers by providing replicase enzymes. They may either interfere with the replication of the parental virus [31] or not, hence they are referred to as defective viral genomes (DVGs) [32,33]. They have a significant functional impact on the pathogenesis and evolution of the wild-type virus. ...
Expanding possibilities for foreign gene expression in cucurbits, we present a novel approach utilising a bipartite vector system based on the cucumber green mottle mosaic virus (CGMMV) genome. Traditional full-length CGMMV vectors face limitations such as a restricted cargo capacity and unstable foreign gene expression. To address these challenges, we developed two 'deconstructed' CGMMV genomes, DG-1 and DG-2. DG-1 features a major internal deletion, resulting in the loss of crucial replicase enzyme domains, rendering it incapable of self-replication. However, a staggered infiltration of DG-1 in CGMMV-infected plants enabled successful replication and movement, facilitating gene-silencing experiments. Conversely, DG-2 was engineered to enhance replication rates and provide multiple cloning sites. Although it exhibited higher replication rates, DG-2 remained localised within infiltrated tissue, displaying trans-replication and restricted movement. Notably, DG-2 demonstrated utility in expressing GFP, with a peak expression observed between 6 and 10 days post-infiltration. Overall, our bipartite system represents a significant advancement in functional genomics, offering a robust tool for foreign gene expression in Nicotiana benthamiana.
... This interaction initiates a signaling cascade involving the activation of various downstream molecules, including TBK1 (TANK-binding kinase 1) and IKKε (I-kappa-B kinase epsilon). MAVS deficiency in cells abrogates the ability of DI RNA to induce antiviral responses to DI RNAs [14]. TBK1 and IKKε subsequently phosphorylate IRF3, IRF7 and NF-κB (Nuclear Factor-kappa B), . ...
... Phosphorylated IRF3, IRF7 and NF-κB translocate to the nucleus and induce the transcription of type I/III IFNs and other proinflammatory cytokines [reviewed in 15]. In general, DI RNAs originating from various RNA viruses have been observed to activate innate immunity and prompt an antiviral response in host cells [14,[16][17][18][19]. This event aids in regulating viral replication and dissemination. ...
... The presence of these VLPs in a virus stock is linked with reduced viral replication and virulence, indicating attenuation [14,[44][45][46]. In this study, we utilized DI290 RNA delivered through DIPs or LNPs to activate cellular innate immune responses capable of counteracting DENV replication. ...
Presently, no approved antiviral drug targets dengue virus (DENV) infection. Treatment mainly relies on supportive measures, while the efficacy of DENV vaccines varies based on factors like vaccine type, circulating DENV serotypes, and the vaccinated population. This study explores using defective interfering particles (DIPs) and lipid nanoparticles (LNPs) to deliver an anti-DENV defective interfering (DI) RNA, known as DI290. Our results showed that both DIPs and DI290 loaded LNPs (LNP-290) effectively suppressed DENV infection in human primary monocyte-derived macrophages (MDMs), THP-1 macrophages and human fibroblasts, representing cell types naturally targeted by DENV. Furthermore, LNP-290 demonstrated inhibition of DENV infection in IFNAR-deficient mice, which lack functional type I interferon (IFN) receptors, leading to decreased viremia and viral replication levels. We then proceeded to examine the involvement of IFN regulatory factor (IRF) 3 and IRF7 in facilitating DI290 RNA-triggered antiviral reactions via retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) using IRF3/IRF7 DKO mice, double knockout for IRF3 and IRF7. LNP-DI290 completely inhibited DENV infection in these DKO mice, indicating that DI290 RNA induced an IFN-mediated cellular antiviral state by a noncanonical pathway. Examination of RNA-Seq data from LNP-treated C57BL/6J mice and IFNAR-deficient mice, along with human MDMs treated with LNPs or DENV DIPs, showed that DI290 RNA treatment notably heightened IFN responses, particularly IFNγ, as well as IFNα/β and IFNλ. Furthermore, administering LNP-290 to mice did not manifest apparent adverse effects on their welfare. In summary, these experiments suggest that DI290 RNA shows potential as a therapeutic approach for combating DENV infection.
... Similar results have been reported for Rosellinia necatrix partitivirus 2 (RnPV2), where a defective virus affects the replication of its parent virus [39]. In animal cells, defective virus genomes may serve the function of interfering with the replication of the WT virus by monopolizing virus polymerases or competing for structural proteins [40,41]. The existence of defective viral dsRNA has also been reported in the family Hypoviridae, but their biological function remains unknown [42][43][44]. ...
In a survey of mycoviruses in Fusarium species that cause sugarcane Pokkah boeng disease, twelve Fusarium strains from three Fusarium species (F. sacchari, F. andiyazi, and F. solani) were found to contain Fusarium sacchari hypovirus 1 (FsHV1), which we reported previously. The genomes of these variants range from 13,966 to 13,983 nucleotides, with 98.6% to 99.9% nucleotide sequence identity and 98.70% to 99.9% protein sequence similarity. Phylogenetic analysis placed these FsHV1 variants within the Alphahypovirus cluster of Hypoviridae. Intriguingly, no clear correlation was found between the geographic origin and host specificity of these viral variants. Additionally, six out of the twelve variants displayed segmental deletions of 1.5 to 1.8 kilobases, suggesting the existence of defective viral dsRNA. The presence of defective viral dsRNA led to a two-thirds reduction in the dsRNA of the wild-type viral genome, yet a tenfold increase in the total viral dsRNA content. To standardize virulence across natural strains, all FsHV1 strains were transferred into a single, virus-free Fusarium recipient strain, FZ06-VF, via mycelial fusion. Strains of Fusarium carrying FsHV1 exhibited suppressed pigment synthesis, diminished microspore production, and a marked decrease in virulence. Inoculation tests revealed varying capacities among different FsHV1 variants to modulate fungal virulence, with the strain harboring the FsHV1-FSA1 showing the lowest virulence, with a disease severity index (DSI) of 3.33, and the FsHV1-FS1 the highest (DSI = 17.66). The identification of highly virulent FsHV1 variants holds promise for the development of biocontrol agents for Pokkah boeng management.
... Both deletion and cbDVGs stimulate interferon (IFN) responses (Fig. 2C). cbDVGs are potent agonists of the types I and III IFN responses, via RIG-I-like receptor (RLR) acti vation, and inducers of pro-inflammatory cytokines IL-6, tumor necrosis factor (TNF), and IL-1β (91)(92)(93)(94)(95)(96)(97)(98). The innate immune response activated by cbDVGs further enhances antigen presentation and dendritic cell maturation (96,97,99). ...
... The innate immune response activated by cbDVGs further enhances antigen presentation and dendritic cell maturation (96,97,99). cbDVGs from Sendai virus more efficiently induced innate IFN responses compared to their homologous full-length viral genomes (91,96,97). Because cbDVGs theoretically form a blunt-ended dsRNA stem motif with a di/triphosphorylated 5′ end lacking 2-O methylation and a 5′-meth ylguanosine cap, cbDVGs theoretically form the canonical RIG-I ligand, which is not present in standard viral genomes. ...
... Multiple contemporary studies have identified correlative roles for DVGs influencing disease severity in humans and have further characterized the functional roles of DVG infection in mouse models. For example, Sendai virus cbDVGs enhanced IFNβ expression, attenuated viral loads, and reduced lung pathology in mouse models (91). Likewise, in mice infected with RSV, cbDVGs rapidly induced IFNβ expression and reduced cellular, primarily neutrophil, infiltration into the alveolus. ...
Defective viral genomes (DVGs) are truncated derivatives of their parental viral genomes generated during an aberrant round of viral genomic replication. Distinct classes of DVGs have been identified in most families of both positive- and negative-sense RNA viruses. Importantly, DVGs have been detected in clinical samples from virally infected individuals and an emerging body of association studies implicates DVGs in shaping the severity of disease caused by viral infections in humans. Consequently, there is growing interest in understanding the molecular mechanisms of de novo DVG generation, how DVGs interact with the innate immune system, and harnessing DVGs as novel therapeutics and vaccine adjuvants to attenuate viral pathogenesis. This minireview focuses on single-stranded RNA viruses (excluding retroviridae), and summarizes the current knowledge of DVG generation, the functions and diversity of DVG species, the roles DVGs play in influencing disease progression, and their application as antivirals and vaccine adjuvants.
... DelVGs arise naturally in human and animal infections (18)(19)(20)(21)23). DelVG abundance is inversely correlated with the severity of influenza disease (24)(25)(26). ...
... DelVG RNAs directly interfere with WT virus by competing for access to the viral replication machinery and possibly for packaging into virions (11,14,30). DelVG RNAs can also be immunogenic, indirectly interfering with viral replication by triggering innate immune sensing pathways that antagonize WT virus replication (23,31,32). ...
... DelVGs arise naturally during influenza virus infections in humans and animals (18)(19)(20)(21)23). ...
Productive infections by RNA viruses require faithful replication of the entire genome. Yet many RNA viruses also produce deletion-containing viral genomes (DelVGs), aberrant replication products with large internal deletions. DelVGs interfere with the replication of wild-type virus and their presence in patients is associated with better clinical outcomes as they. The DelVG RNA itself is hypothesized to confer this interfering activity. DelVGs antagonize replication by out-competing the full-length genome and triggering innate immune responses. Here, we identify an additionally inhibitory mechanism mediated by a new class of viral proteins encoded by DelVGs. We identified hundreds of cryptic viral proteins translated from DelVGs. These DelVG-encoded proteins (DPRs) include canonical viral proteins with large internal deletions, as well as proteins with novel C-termini translated from alternative reading frames. Many DPRs retain functional domains shared with their full-length counterparts, suggesting they may have activity during infection. Mechanistic studies of DPRs derived from the influenza virus protein PB2 showed that they poison replication of wild-type virus by acting as dominant-negative inhibitors of the viral polymerase. These findings reveal that DelVGs have a dual inhibitory mechanism, acting at both the RNA and protein level. They further show that DPRs have the potential to dramatically expand the functional proteomes of diverse RNA viruses.
... RNA viruses produce not only full-length standard viral genomes (stVGs) but also variants, hypermutated RNAs, and nonstandard viral genomes (nsVGs) that provide different functions and advantages to the virus [3,4]. nsVGs produced during RSV and parainfluenza virus infections are critical determinants of infection outcome in vitro and in vivo [5][6][7]. When produced early during infection, nsVGs significantly reduce virus spread and disease severity in mice and humans [5,7]. ...
... nsVGs produced during RSV and parainfluenza virus infections are critical determinants of infection outcome in vitro and in vivo [5][6][7]. When produced early during infection, nsVGs significantly reduce virus spread and disease severity in mice and humans [5,7]. nsVGs impact the infection via stimulation of major signaling pathways that shape the cellular response to the infection. ...
... Identifying cellular pathways and molecular mechanisms by which nsVGs reduce virulence may lead to new strategies to prevent severe disease upon RNA virus infection. One nsVG subpopulation, copy-back viral genomes (cbVGs), has critical roles in inducing the cellular antiviral immune response, controlling the rate of viral replication, and promoting the establishment of persistent infections [6][7][8]. Nonsegmented negative-sense RNA viruses generate cbVGs when the viral polymerase initiates replication at the promoter region, falls off the template, and then reattaches to the nascent strand [3]. The polymerase then uses the nascent strand as a template and continues replicating, copying back the already synthetized RNA (S1A Fig) [3]. ...
Antiviral responses are often accompanied by translation inhibition and formation of stress granules (SGs) in infected cells. However, the triggers for these processes and their role during infection remain subjects of active investigation. Copy-back viral genomes (cbVGs) are the primary inducers of the mitochondrial antiviral signaling (MAVS) pathway and antiviral immunity during Sendai virus (SeV) and respiratory syncytial virus (RSV) infections. The relationship between cbVGs and cellular stress during viral infections is unknown. Here, we show that SG form during infections containing high levels of cbVGs, and not during infections with low levels of cbVGs. Moreover, using RNA fluorescent in situ hybridization to differentiate accumulation of standard viral genomes from cbVGs at a single-cell level during infection, we show that SGs form exclusively in cells that accumulate high levels of cbVGs. PKR activation is increased during high cbVG infections and, as expected, is necessary for virus-induced SG. However, SGs form independent of MAVS signaling, demonstrating that cbVGs induce antiviral immunity and SG formation through 2 independent mechanisms. Furthermore, we show that translation inhibition and SG formation do not affect the overall expression of interferon and interferon stimulated genes during infection, making the stress response dispensable for global antiviral immunity. Using live-cell imaging, we show that SG formation is highly dynamic and correlates with a drastic reduction of viral protein expression even in cells infected for several days. Through analysis of active protein translation at a single-cell level, we show that infected cells that form SG show inhibition of protein translation. Together, our data reveal a new cbVG-driven mechanism of viral interference where cbVGs induce PKR-mediated translation inhibition and SG formation, leading to a reduction in viral protein expression without altering overall antiviral immunity.
... However, around 75 years ago, Preben von Magnus identified IAV defective-interfering (DI) particles, which are incomplete virus particles that impede the replication of fully functional virus particles [4,5]. The IAV DI particles have since been widely observed in laboratory-grown virus cultures [6][7][8] and clinical samples [9,10]. ...
... In addition to DI particle-derived ID vRNAs, which are typically larger than~200 nucleotides, there are other classes of short aberrant vRNAs, namely mini and small vRNAs, with lengths of 50-125 and 22-27 nucleotides, respectively, which are less likely to be encapsulated within influenza virions [27][28][29][30]. ID vRNAs play a vital role in activating the innate immune response by specifically binding to retinoic acid-inducible gene-I (RIG-I) [9,31,32], leading to increased expression of interferon (IFN) and other innate genes in cells [33]. Owing to the potent adjuvant effects of IFNs, ID vRNAs hold great potential as targets for developing LAIVs that can elicit enhanced innate and adaptive immune responses [34][35][36][37][38]. ...
... Previous studies using IAVs enriched for DI particles that carry ID vRNAs revealed their ability to enhance IFN response in vitro [6], promote persistent infections in vitro [79], or reduce virulence in vivo [9], which were generally linked to the ability of ID vRNAs to outcompete the replication of the standard virus genome. However, investigations into the proteins encoded by ID vRNAs are lacking. ...
Influenza A virus (IAV) populations harbor large subpopulations of defective-interfering particles characterized by internally deleted viral genomes. These internally deleted genomes have demonstrated the ability to suppress infectivity and boost innate immunity, rendering them promising for therapeutic and immunogenic applications. In this study, we aimed to investigate the diversity and complexity of the internally deleted IAV genomes within a panel of plaque-purified avian influenza viruses selected for their enhanced interferon-inducing phenotypes. Our findings unveiled that the abundance and diversity of internally deleted viral genomes were contingent upon the viral subculture and plaque purification processes. We observed a heightened occurrence of internally deleted genomes with distinct junctions in viral clones exhibiting enhanced interferon-inducing phenotypes, accompanied by additional truncation in the nonstructural 1 protein linker region (NS1Δ76-86). Computational analyses suggest the internally deleted IAV genomes can encode a broad range of carboxy-terminally truncated and intrinsically disordered proteins with variable lengths and amino acid composition. Further research is imperative to unravel the underlying mechanisms driving the increased diversity of internal deletions within the genomes of viral clones exhibiting enhanced interferon-inducing capacities and to explore their potential for modulating cellular processes and immunity.
... KEYWORDS influenza D efective interfering (DI) virus particles carry defective viral genomes (DVGs) and nor-DVGs in the influenza virus, which is a negative-sense RNA virus with a segmented genome, typically contain internal deletions within the genomic segments while preserving the 59 and 39 termini (4)(5)(6)(7)(8). Naturally occurring DI viruses have been observed during virus propagation (1,2,(9)(10)(11)(12) and in human infections (13)(14)(15), and they are believed to play an important role in modulating infection outcomes (14)(15)(16)(17)(18). DIs presumably compete with the standard virus for components that are essential for replication, can induce antiviral immunity, and facilitate the establishment of persistent infections (reviewed in [5,6,19]). ...
Influenza defective interfering (DI) viruses have long been considered promising antiviral candidates because of their ability to interfere with replication-competent viruses and induce antiviral immunity. However, the mechanisms underlying DI-mediated antiviral immunity have not been extensively explored. Here, we demonstrated the interferon (IFN)-independent protection conferred by the influenza DI virus against homologous virus infection in mice deficient in type I and III IFN signaling. We identified unique host signatures responding to DI coinfection by integrating transcriptional and posttranscriptional regulatory data. DI-treated mice exhibited reduced viral transcription, less intense inflammatory and innate immune responses, and primed multiciliated cell differentiation in their lungs at an early stage of infection, even in the absence of type I or III IFNs. This increased multiciliogenesis could also be detected at the protein level via the immunofluorescence staining of lung tissue from DI-treated mice. Overall, our study provides mechanistic insight into the protection mediated by DIs, implying a unifying theme involving inflammation and multiciliogenesis in maintaining respiratory homeostasis and revealing their IFN-independent antiviral activity. IMPORTANCE During replication, the influenza virus generates genetically defective viruses. These are found in natural infections as part of the virus population within the infected host. Some versions of these defective viruses are thought to have protective effects through their interference with replication-competent viruses and induction of antiviral immunity. To better determine the mechanisms underlying the protective effects of these defective interfering (DI) viruses, we tested a DI that we previously identified in vitro with mice. Mice that were infected with a mix of wild-type influenza and DI viruses had less intense inflammatory and innate immune responses than did mice that were infected with the wild-type virus only, even when type I or III interferons, which are cytokines that play a prominent role in defending the respiratory epithelial barrier, were absent. More interestingly, the DI-infected mice had primed multiciliated cell differentiation in their lungs, indicating the potential promotion of epithelial repair by DIs.
... These components of the RNA virus population provide different functions and advantages to the virus (6,7). One nsVG subpopulation, copy-back viral genomes (cbVGs), has received attention for its important roles in inducing the cellular antiviral responses, controlling the rate of viral replication, and promoting the establishment of persistent infections (4,5,8). Non-segmented negative-sense RNA viruses generate cbVGs when the viral polymerase initiates replication at the promoter region, falls off the template and then reattaches to the nascent strand (7). ...
... The polymerase then uses the nascent strand as a template and continues replicating, copying back the already synthetized RNA (7). The resulting RNA molecules contain highly structured immunostimulatory motifs and lack genes encoding viral proteins (5,9). Although cbVGs can only replicate in the presence of a full-length standard genome that provides essential viral proteins, they are key interactors with the host and drive several cellular responses that determine the infection outcome. ...
... ; https://doi.org/10.1101/2023.05.17.541157 doi: bioRxiv preprint Discussion cbVGs shape the outcome of SeV and RSV infections (3-5, 7, 8, 35). Their importance is highlighted by their involvement in inducing the antiviral response, interfering with virus replication, and establishing persistent viral infections (4,5,8). Here we demonstrate yet another role for cbVGs: to activate PKR signaling and SG formation. ...
Antiviral responses are often accompanied by formation of stress granules (SG) in infected cells. However, the triggers for SG formation and the function of SG during infection remain subjects of active investigation. Copy-back viral genomes (cbVGs) are the primary inducers of the Mitochondrial Antiviral Signaling (MAVS) pathway and antiviral immunity during Sendai Virus (SeV) and Respiratory Syncytial virus (RSV) infections. The relationship between cbVGs and SG formation is unknown. Here we show that SG form during infections containing high levels of cbVGs, and not during infections with low levels of cbVGs. Moreover, using RNA fluorescent in situ hybridization to differentiate accumulation of standard viral genomes from cbVGs at a single-cell level during infection, we show that SG form exclusively in cells that accumulate high levels of cbVGs. As expected, virus-induced SG depend on signaling by Protein Kinase R (PKR), however, they form independent of MAVS signaling. Furthermore, inhibition of SG does not affect the induction of antiviral immunity, demonstrating that cbVGs induce antiviral immunity and SG formation through two independent mechanisms. Using live-cell imaging, we show that SG formation is highly dynamic and correlates with a drastic reduction of viral protein expression even in cells infected for several weeks. Together, our data demonstrate that SG are important agents of cbVG-induced interference by reducing viral protein expression independently of the antiviral response and suggest that SG contribute to sustain persistent infections.
One Sentence Summary
cbVGs trigger SG independent of the antiviral response during RSV and parainfluenza virus infection leading to a reduction of virus protein expression in acute and persisten infections
... However, the capacity of naturally created DVGs to interfere with standard viral genome replication and encapsidation and how they influence the viral disease outcome are unclear. The in vitro relevance of DVGs may depend on their sequence length, production level, and the ratio of DVGs to standard viral genomes within confined intracellular hubs of infection (Tapia et al., 2013;Genoyer and López, 2019). ...
... which is uncoupled from their interference function (Yount et al., 2006;Tapia et al., 2013;López, 2014;Mura et al., 2017). ...
The positive-sense single-stranded (ss) RNA viruses of the Betacoronavirus (beta-CoV) genus can spillover from mammals to humans and are an ongoing threat to global health and commerce, as demonstrated by the current zoonotic pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Current anti-viral strategies focus on vaccination or targeting key viral proteins with antibodies and drugs. However, the ongoing evolution of new variants that evade vaccination or may become drug-resistant is a major challenge. Thus, antiviral compounds that circumvent these obstacles are needed. Here we describe an innovative antiviral modality based on in silico designed fully synthetic mRNA that is replication incompetent in uninfected cells (termed herein PSCT: parasitic anti-SARS-CoV-2 transcript). The PSCT sequence was engineered to include key untranslated cis-acting regulatory RNA elements of the SARS-CoV-2 genome, so as to effectively compete for replication and packaging with the standard viral genome. Using the Vero E6 cell-culture based SARS-CoV-2 infection model, we determined that the intracellular delivery of liposome-encapsulated PSCT at 1 hour post infection significantly reduced intercellular SARS-CoV-2 replication and release into the extracellular milieu as compared to mock treatment. In summary, our findings are a proof-of-concept for the therapeutic feasibility of in silico designed mRNA compounds formulated to hinder the replication and packaging of ssRNA viruses sharing a comparable genomic-structure with beta-CoVs.
