Recognition Of Viral RNA Research Articles

Helicase protein DDX11 as a novel antiviral factor promoting RIG-I-MAVS-mediated signaling pathway.

Innate immunity is the first and most rapid host defense against virus infection. Recognition of viral RNA by the retinoic acid-inducible gene 1 (RIG-I)-like receptors (RLRs) initiates innate antiviral immune responses. How the binding of viral RNA to and activation of the RLRs are regulated remains enigmatic. In this study, we identified DEAD/H-box helicase 11 (DDX11) as a positive regulator of the RIG-I-mitochondrial antiviral signaling protein (MAVS)-mediated signaling pathways. Mechanistically, we demonstrated that DDX11 bound to viral RNA, interacted with RIG-I, and promoted their binding to viral RNA. DDX11 also promoted the interaction between RIG-I and MAVS and activation of RIG-I-MAVS signaling. Overall, our results elucidate the role of DDX11 in RIG-I-MAVS-dependent signaling pathways and may shed light on innate immune gene regulation.

1mΨ influences the performance of various positive-stranded RNA virus-based replicons

Self-amplifying RNAs (saRNAs) are versatile vaccine platforms that take advantage of a viral RNA-dependent RNA polymerase (RdRp) to amplify the messenger RNA (mRNA) of an antigen of interest encoded within the backbone of the viral genome once inside the target cell. In recent years, more saRNA vaccines have been clinically tested with the hope of reducing the vaccination dose compared to the conventional mRNA approach. The use of N1-methyl-pseudouridine (1mΨ), which enhances RNA stability and reduces the innate immune response triggered by RNAs, is among the improvements included in the current mRNA vaccines. In the present study, we evaluated the effects of this modified nucleoside on various saRNA platforms based on different viruses. The results showed that different stages of the replication process were affected depending on the backbone virus. For TNCL, an insect virus of the Alphanodavirus genus, replication was impaired by poor recognition of viral RNA by RdRp. In contrast, the translation step was severely abrogated in coxsackievirus B3 (CVB3), a member of the Picornaviridae family. Finally, the effects of 1mΨ on Semliki forest virus (SFV), were not detrimental in in vitro studies, but no advantages were observed when immunogenicity was tested in vivo.

Microbial Matryoshka: Addressing the Relationship between Pathogenic Flagellated Protozoans and Their RNA Viral Endosymbionts (Family Totiviridae).

Three genera of viruses of the family Totiviridae establish endosymbiotic associations with flagellated protozoa responsible for parasitic diseases of great impact in the context of One Health. Giardiavirus, Trichomonasvirus, and Leishmaniavirus infect the protozoa Giardia sp., Trichomonas vaginalis, and Leishmania sp., respectively. In the present work, we review the characteristics of the endosymbiotic relationships established, the advantages, and the consequences caused in mammalian hosts. Among the common characteristics of these double-stranded RNA viruses are that they do not integrate into the host genome, do not follow a lytic cycle, and do not cause cytopathic effects. However, in cases of endosymbiosis between Leishmaniavirus and Leishmania species from the Americas, and between Trichomonasvirus and Trichomonas vaginalis, it seems that it can alter their virulence (degree of pathogenicity). In a mammalian host, due to TLR3 activation of immune cells upon the recognition of viral RNA, uncontrolled inflammatory signaling responses are triggered, increasing pathological damage and the risk of failure of conventional standard treatment. Endosymbiosis with Giardiavirus can cause the loss of intestinal adherence of the protozoan, resulting in a benign disease. The current knowledge about viruses infecting flagellated protozoans is still fragmentary, and more research is required to unravel the intricacies of this three-way relationship. We need to develop early and effective diagnostic methods for further development in the field of translational medicine. Taking advantage of promising biotechnological advances, the aim is to develop ad hoc therapeutic strategies that focus not only on the disease-causing protozoan but also on the virus.

Lysosomal endonuclease RNase T2 and PLD exonucleases cooperatively generate RNA ligands for TLR7 activation

Toll-like receptor 7 (TLR7) is essential for recognition of RNA viruses and initiation of antiviral immunity. TLR7 contains two ligand-binding pockets that recognize different RNA degradation products: pocket 1 recognizes guanosine, while pocket 2 coordinates pyrimidine-rich RNA fragments. We found that the endonuclease RNase T2, along with 5' exonucleases PLD3 and PLD4, collaboratively generate the ligands for TLR7. Specifically, RNase T2 generated guanosine 2',3'-cyclic monophosphate-terminated RNA fragments. PLD exonuclease activity further released the terminal 2',3'-cyclic guanosine monophosphate (2',3'-cGMP) to engage pocket 1 and was also needed to generate RNA fragments for pocket 2. Loss-of-function studies in cell lines and primary cells confirmed the critical requirement for PLD activity. Biochemical and structural studies showed that PLD enzymes form homodimers with two ligand-binding sites important for activity. Previously identified disease-associated PLD mutants failed to form stable dimers. Together, our data provide a mechanistic basis for the detection of RNA fragments by TLR7.

Elevated expression of Toll-like receptor 7 and its correlation with clinical features in patients with primary Sjögren’s syndrome

BackgroundThe labial salivary glands (LSGs) are important for the diagnosis, evaluation of therapeutic efficacy, and genetic analyses of primary Sjögren’s syndrome (pSS). In autoimmune diseases, the recognition of self nucleic acids and viral RNA and DNA through endogenous Toll-like receptor(TLR) triggers the production of type I IFN and pro-inflammatory cytokines, leading to the occurrence and progression of the disease. Here, we detected the expression of TLR7 in LSGs and analyse its correlation with clinical features and serum cytokines in pSS patients.MethodsLSGs and serum samples were obtained from 56 pSS patients and 19 non-SS patients (non-pSS patients). The expression of TLR7 in the LSGs was evaluated with immunohistochemistry. The serum levels of interferon-α (IFN-α) and interleukin-6 (IL-6) were quantified by ELISA. Laboratory parameters were measured by clinical standard laboratory techniques.ResultsTLR7-positive cells in pSS were localized in the ductal epithelial cells and lymphocytes of LSGs. The expression of TLR7 was upregulated in pSS patients compared with controls. Patients with anti-Ro52 antibody positivity showed higher TLR7 levels than those who were negative but not those with anti-Ro60 and anti-SSB. TLR7 levels were positively associated with the levels of IgG, IgA, ANA, IL-6, IFN-α and serum globulin but were not associated with IgM, C3, C4, or rheumatoid factor (RF) in serum.ConclusionTLR7 may be involved in the inflammatory response and the production of antibodies in pSS and plays an important role in local and systemic pSS manifestations. This research showed that TLR7 is involved in pSS pathogenesis.

TF-FVIIa PAR2-β-Arrestin Signaling Sustains Organ Dysfunction in Coxsackievirus B3 Infection of Mice.

Accumulating evidence implicates the activation of G-protein-coupled PARs (protease-activated receptors) by coagulation proteases in the regulation of innate immune responses. Using mouse models with genetic alterations of the PAR2 signaling platform, we have explored contributions of PAR2 signaling to infection with coxsackievirus B3, a single-stranded RNA virus provoking multiorgan tissue damage, including the heart. We show that PAR2 activation sustains correlates of severe morbidity-hemodynamic compromise, aggravated hypothermia, and hypoglycemia-despite intact control of the virus. Following acute viral liver injury, canonical PAR2 signaling impairs the restoration process associated with exaggerated type I IFN (interferon) signatures in response to viral RNA recognition. Metabolic profiling in combination with proteomics of liver tissue shows PAR2-dependent reprogramming of liver metabolism, increased lipid droplet storage, and gluconeogenesis. PAR2-sustained hypodynamic compromise, reprograming of liver metabolism, as well as imbalanced IFN responses are prevented in β-arrestin coupling-deficient PAR2 C-terminal phosphorylation mutant mice. Thus, wiring between upstream proteases and immune-metabolic responses results from biased PAR2 signaling mediated by intracellular recruitment of β-arrestin. Importantly, blockade of the TF (tissue factor)-FVIIa (coagulation factor VIIa) complex capable of PAR2 proteolysis with the NAPc2 (nematode anticoagulant protein c2) mitigated virus-triggered pathology, recapitulating effects seen in protease cleavage-resistant PAR2 mice. These data provide insights into a TF-FVIIa signaling axis through PAR2-β-arrestin coupling that is a regulator of inflammation-triggered tissue repair and hemodynamic compromise in coxsackievirus B3 infection and can potentially be targeted with selective coagulation inhibitors.

The RNA-binding protein ZFP36 strengthens innate antiviral signaling by targeting RIG-I for K63-linked ubiquitination.

Innate immunity is the first line of defense against infections, which functions as a significant role in resisting pathogen invasion. Rapid immune response is initiated by pattern recognition receptors (PRRs) quickly distinguishing "self" and "non-self." Upon evolutionarily conserved pathogen-associated molecular pattern (PAMP) is recognized by PRRs, innate immune response against infection is triggered via an orchestration of molecular interaction, cytokines cascades, and immune cells. RIG-I plays a critical role in type I interferon (IFN-I) production by direct recognition of cytoplasmic double-stranded viral RNA. However, the activation mechanism of RIG-I is incompletely understood. In this study, we reported RNA-binding protein ZFP36 as a positive regulator of RIG-I-mediated IFN-I production. ZFP36 is a member of Zinc finger proteins (ZFPs) characterized by the zinc finger (ZnF) motif, which broadly involved gene transcription and signal transduction. However, its role in regulating antiviral innate immune signaling is still unclear. We found that ZFP36 associates with RIG-I and potentiates the FN-β production induced by SeV. Mechanistically, ZFP36 promotes K63-linked polyubiquitination of RIG-I, mostly at K154/K164/K172, thereby facilitating the activation of RIG-I during infection. While the mutant ZFP36 (C118S/C162S) failed to increase polyubiquitination of RIG-I and SeV induced FN-β. Our findings collectively demonstrated that ZFP36 acts as a positive regulator in antiviral innate immunity by targeting RIG-I for K63-linked ubiquitination, thus improving our understanding of the activation mechanism of RIG-I.

Characterization of RNA driven structural changes in full length RIG-I leading to its agonism or antagonism.

RIG-I (retinoic acid inducible gene-I) can sense subtle differences between endogenous and viral RNA in the cytoplasm, triggering an anti-viral immune response through induction of type I interferons (IFN) and other inflammatory mediators. Multiple crystal and cryo-EM structures of RIG-I suggested a mechanism in which the C-terminal domain (CTD) is responsible for the recognition of viral RNA with a 5'-triphoshate modification, while the CARD domains serve as a trigger for downstream signaling, leading to the induction of type I IFN. However, to date contradicting conclusions have been reached around the role of ATP in the mechanism of the CARD domains ejection from RIG-I's autoinhibited state. Here we present an application of NMR spectroscopy to investigate changes induced by the binding of 5'-triphosphate and 5'-OH dsRNA, both in the presence and absence of nucleotides, to full length RIG-I with all its methionine residues selectively labeled (Met-[ϵ-13CH3]). With this approach we were able to identify residues on the CTD, helicase domain, and CARDs that served as probes to sense RNA-induced conformational changes in those respective regions. Our results were analyzed in the context of either agonistic or antagonistic RNAs, by and large supporting a mechanism proposed by the Pyle Lab in which CARD release is primarily dependent on the RNA binding event.

Mapping of long stretches of highly conserved sequences in over 6 million SARS-CoV-2 genomes.

We identified 11 conserved stretches in over 6.3 million SARS-CoV-2 genomes including all the major variants of concerns. Each conserved stretch is ≥100 nucleotides in length with ≥99.9% conservation at each nucleotide position. Interestingly, six of the eight conserved stretches in ORF1ab overlapped significantly with well-folded experimentally verified RNA secondary structures. Furthermore, two of the conserved stretches were mapped to regions within the S2-subunit that undergo dynamic structural rearrangements during viral fusion. In addition, the conserved stretches were significantly depleted for zinc-finger antiviral protein (ZAP) binding sites, which facilitated the recognition and degradation of viral RNA. These highly conserved stretches in the SARS-CoV-2 genome were poorly conserved at the nucleotide level among closely related β-coronaviruses, thus representing ideal targets for highly specific and discriminatory diagnostic assays. Our findings highlight the role of structural constraints at both RNA and protein levels that contribute to the sequence conservation of specific genomic regions in SARS-CoV-2.

Translation Arrest: A Key Player in Plant Antiviral Response.

Plants evolved several mechanisms to protect themselves against viruses. Besides recessive resistance, where compatible host factors required for viral proliferation are absent or incompatible, there are (at least) two types of inducible antiviral immunity: RNA silencing (RNAi) and immune responses mounted upon activation of nucleotide-binding domain leucine-rich repeat (NLR) receptors. RNAi is associated with viral symptom recovery through translational repression and transcript degradation following recognition of viral double-stranded RNA produced during infection. NLR-mediated immunity is induced upon (in)direct recognition of a viral protein by an NLR receptor, triggering either a hypersensitive response (HR) or an extreme resistance response (ER). During ER, host cell death is not apparent, and it has been proposed that this resistance is mediated by a translational arrest (TA) of viral transcripts. Recent research indicates that translational repression plays a crucial role in plant antiviral resistance. This paper reviews current knowledge on viral translational repression during viral recovery and NLR-mediated immunity. Our findings are summarized in a model detailing the pathways and processes leading to translational arrest of plant viruses. This model can serve as a framework to formulate hypotheses on how TA halts viral replication, inspiring new leads for the development of antiviral resistance in crops.

TLR3 recognition of viral double-stranded RNA in human dental pulp cells is important for the innate immunity

Dental caries or trauma can expose human dental pulp cells (DPCs) to various oral microorganisms, which play an important role in the development of an innate immune response. In the present study, we examined the expression of Toll-like receptors (TLRs) for sensing microbe-associated molecular patterns in human DPCs. Interestingly, real-time PCR analysis demonstrated that TLR3 is the most highly expressed among 10 different TLRs in human DPCs. Poly(I:C), a representative TLR3 ligand mimicking viral double-stranded RNA, potently induced IL-8 expression in a time- and dose-dependent manner. Concordantly, poly(I:C) treatment substantially increased the expression of pro-inflammatory cytokines and chemokines such as IL-6, CCL2, and CXCL10. Human DPCs transfected with TLR3 siRNA exhibited decreased IL-8 production compared with non-targeting siRNA-transfected cells, suggesting that the expression of poly(I:C)-induced inflammatory cytokines is dependent on TLR3. IL-8 secretion induced by poly(I:C) was down-regulated by MAP kinase inhibitors, indicating that the MAP kinase pathway contributes to IL-8 production. Furthermore, C/EBPβ and NF-κB were essential transcriptional factors for poly(I:C)-induced IL-8 expression, as demonstrated by the transient transfection and reporter gene assay. Since lipoproteins are known as major immunostimulatory components of bacteria, human DPCs were treated with poly(I:C) together with Pam2CSK4, a synthetic lipopeptide mimicking bacterial lipoproteins. Pam2CSK4 and poly(I:C) co-treatment synergistically increased IL-8 production in comparison to Pam2CSK4 or poly(I:C) alone, implying that co-infection of viruses and bacteria can synergistically induce inflammatory responses in the dental pulp. Taken together, these results suggest that human DPCs potentially sense and respond to viral double-stranded RNAs, leading to effective induction of innate immune responses.

Mechanisms of length-dependent recognition of viral double-stranded RNA by RIG-I

Retinoic acid-inducible gene I (RIG-I) is the most front-line cytoplasmic viral RNA sensor and induces antiviral immune responses. RIG-I recognizes short double-stranded (dsRNA) (< 500 bp), but not long dsRNA (> 500 bp) to trigger antiviral signaling. Since RIG-I is capable of binding with dsRNA irrespective of size, length-dependent RIG-I signaling remains elusive. Here, we demonstrated that RIG-I bound to long dsRNA with slow kinetics. Remarkably, RIG-I/short dsRNA complex efficiently dissociated in an ATP hydrolysis-dependent manner, whereas RIG-I/long dsRNA was stable and did not dissociate. Our study suggests that the dissociation of RIG-I from RIG-I/dsRNA complex could be a step for efficient antiviral signaling. Dissociated RIG-I exhibited homo-oligomerization, acquiring ability to physically associate with MAVS, and biological activity upon introduction into living cells. We herein discuss common and unique mechanisms of viral dsRNA recognition by RIG-I and MDA5.

Infection Defects of RNA and DNA Viruses Induced by Antiviral RNA Interference.

Immune recognition of viral genome-derived double-stranded RNA (dsRNA) molecules and their subsequent processing into small interfering RNAs (siRNAs) in plants, invertebrates, and mammals trigger specific antiviral immunity known as antiviral RNA interference (RNAi). Immune sensing of viral dsRNA is sequence-independent, and most regions of viral RNAs are targeted by virus-derived siRNAs which extensively overlap in sequence. Thus, the high mutation rates of viruses do not drive immune escape from antiviral RNAi, in contrast to other mechanisms involving specific virus recognition by host immune proteins such as antibodies and resistance (R) proteins in mammals and plants, respectively. Instead, viruses actively suppress antiviral RNAi at various key steps with a group of proteins known as viral suppressors of RNAi (VSRs). Some VSRs are so effective in virus counter-defense that potent inhibition of virus infection by antiviral RNAi is undetectable unless the cognate VSR is rendered nonexpressing or nonfunctional. Since viral proteins are often multifunctional, resistance phenotypes of antiviral RNAi are accurately defined by those infection defects of VSR-deletion mutant viruses that are efficiently rescued by host deficiency in antiviral RNAi. Here, we review and discuss in vivo infection defects of VSR-deficient RNA and DNA viruses resulting from the actions of host antiviral RNAi in model systems.

Is the Association of the Rare rs35667974 IFIH1 Gene Polymorphism With Autoimmune Diseases a Case of RNA Epigenetics?

Interferon induced with helicase C domain-containing protein 1 (IFIH1) gene encodes a cytoplasmic RNA helicase otherwise known as melanoma differentiation-associated 5 (MDA5), a RIG-1-like RNA helicase that recognizes viral RNA and is involved in innate immunity through recognition of viral RNA. Upon binding to double-stranded (ds) RNA, MDA5 forms a filamentous assembly along the length of dsRNA and utilizes molecular signatures to discriminate self, versus non-self on the basis of dsRNA length and methylation. Its missense variant rs35667974 is protective for type 1 diabetes, psoriasis, and psoriatic arthritis, but is also found to be associated with an increased risk for ankylosing spondylitis, Crohn’s disease, and ulcerative colitis. To gain insight into the complex role of this variant we performed a structural analysis of MDA5 in complex with dsRNA using molecular dynamics simulations. Our data suggest that while the Ile923Val mutation of the rs35667974 variant does not affect binding to native dsRNA significantly, it displays a destabilizing effect in the presence of 2′-O uridine methylation. Thus, the presence of 2′-O-methylation at the dsRNA introduces a sensing signature that leads to selective reduction of the overall MDA catalytic activity. This study represents an evaluation of the role of the shared rs35667974 variant of autoimmune locus IFIH1, reported to lead to selectively reduced catalytic activity of the modified MDA5 phenotype and, as a consequence, reduced negative feedback on cytokine and chemokine signaling and selectively protection against autoimmunity.

LGP2 Promotes Type I Interferon Production To Inhibit PRRSV Infection via Enhancing MDA5-Mediated Signaling.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important pathogens in the global pig industry, which modulates the host's innate antiviral immunity to achieve immune evasion. RIG-I-like receptors (RLRs) sense viral RNA and activate the interferon signaling pathway. LGP2, a member of the RLR family, plays an important role in regulating innate immunity. However, the role of LGP2 in virus infection is controversial. Whether LGP2 has a role during infection with PRRSV remains unclear. Here, we found that LGP2 overexpression restrained the replication of PRRSV, while LGP2 silencing facilitated PRRSV replication. LGP2 was prone to interact with MDA5 and enhanced viral RNA enrichment and recognition by MDA5, thus promoting the activation of RIG-I/IRF3 and NF-κB signaling pathways and reinforcing the expression of proinflammatory cytokines and type I interferon during PRRSV infection. Meanwhile, there was a decreased protein expression of LGP2 upon PRRSV infection in vitro. PRRSV Nsp1 and Nsp2 interacted with LGP2 and promoted K63-linked ubiquitination of LGP2, ultimately leading to the degradation of LGP2. These novel findings indicate that LGP2 plays a role in regulating PRRSV replication through synergistic interaction with MDA5. Moreover, targeting LGP2 is responsible for PRRSV immune evasion. Our work describes a novel mechanism of virus-host interaction and provides the basis for preventing and controlling PRRSV. IMPORTANCE LGP2, a member of retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), shows higher-affinity binding to RNA and work synergism with RIG-I or MDA5. However, LGP2 has divergent responses to different viruses, which remains controversial in antiviral immune responses. Here, we present the detailed process of LGP2 in positively regulating the anti-PRRSV response. Upon PRRSV infection, LGP2 was prone to bind to MDA5 and enhanced MDA5 signaling, manifesting the enrichment of viral RNA on MDA5 and the activation of downstream IRF3 and NF-κB, which results in increased proinflammatory cytokines and type I interferon expression, ultimately inhibiting PRRSV at the early stage of infection. Moreover, PRRSV Nsp1 and Nsp2 interacted with LGP2 via ubiquitin-proteasome pathways, thus blocking LGP2-mediated immune response. This research helps us understand the host recognition and innate antiviral response to PRRSV infection by neglected pattern recognition receptors, which sheds light on the detailed mechanism of virus-host interaction.

RNA interference, an emerging component of antiviral immunity in mammals.

Antiviral RNA interference (RNAi) is an immune pathway that can, in certain conditions, protect mammalian cells against RNA viruses. It depends on the recognition and dicing of viral double-stranded RNA by a protein of the Dicer family, which leads to the production of viral small interfering RNAs (vsiRNAs) that sequence-specifically guide the degradation of cognate viral RNA. If the first line of defence against viruses relies on type-I and type-III interferons (IFN) in mammals, certain cell types such as stem cells, that are hyporesponsive for IFN, instead use antiviral RNAi via the expression of a specific antiviral Dicer. In certain conditions, antiviral RNAi can also contribute to the protection of differentiated cells. Indeed, abundant vsiRNAs are detected in infected cells and efficiently guide the degradation of viral RNA, especially in cells infected with viruses disabled for viral suppressors of RNAi (VSRs), which are virally encoded blockers of antiviral RNAi. The existence and importance of antiviral RNAi in differentiated cells has however been debated in the field, because data document mutual inhibition between IFN and antiviral RNAi. Recent developments include the engineering of a small molecule inhibitor of VSR to probe antiviral RNAi in vivo, as well as the detection of vsiRNAs inside extracellular vesicles in the serum of infected mice. It suggests that using more complex, in vivo models could allow to unravel the contribution of antiviral RNAi to immunity at the host level.

TLR3 Recognition of Viral Double-Stranded RNA in Human Dental Pulp Cells is Important for the Innate Immunity

TLR3 Recognition of Viral Double-Stranded RNA in Human Dental Pulp Cells is Important for the Innate Immunity

IRF1 Is Required for MDA5 (IFIH1) Induction by IFN-α, LPS, and poly(I:C) in Murine Macrophages

Melanoma differentiation-associated protein 5 (MDA5) induces type I interferons (IFNs) after the recognition of viral RNA. In addition, gain-of-function mutations in the interferon induced with helicase C domain 1 (IFIH1) gene, which encodes MDA5, lead to type I interferonopathies. Here, we show that Mda5 is highly expressed in murine macrophages and is regulated by pro-inflammatory stimuli such as the cytokines IFN-α and IFN-γ, the TLR ligand LPS, and a mimic of dsRNA, poly(I:C). Mda5 induction is mediated through the production of reactive oxygen species. The induction by IFN-α or LPS occurs at the transcriptional level since the Mda5 mRNA half-life before and after induction is very stable. Interestingly, STAT1 is required for Mda5 induction by IFN-α, LPS, or poly(I:C). The time course of induction of at least 3 h and the need for protein synthesis indicate that Mda5 requires an intermediate protein for transcription. In transient transfection experiments, we found that a 105-bp fragment of this gene, between −1153 and −1258 bp relative to the transcription start site, is required for transcription. In this specific region, we observed a sequence containing an IRF-binding motif, which, when mutated, abolishes the induction of Mda5. This sequence is strongly conserved in the IFIH1 promoters of eutherian mammals and in other distant species. Kinetic experiments, chromatin immunoprecipitation assays, and gene-silencing experiments revealed that IRF1 is required for induction of Mda5 expression.

RNA interference is the basis of human antiviral defense

Comparative analysis of antiviral protective mechanisms in protozoa and RNA interference of multicellular organisms has revealed their similarity, also providing a clue to understanding the adaptive immunity. In this article, we present the latest evidence on the importance of RNA-guided gene regulation in human antiviral defense. The role of neutralizing antibodies and interferon system in viral invasion is considered. The new concept has been introduced, i.e., antiviral protection of any living organism is based on the intracellular RNA-guided mechanisms. Simple and effective defense against viruses is that spacer segment of the viral DNA is inserted into the cellular chromosomes. Upon re-infection, the RNA transcript of the spacer directs nuclease enzymes against the foreign genome. This is a really adaptive immune defense that any cell potentially possesses. In humans, the interferon system provides an additional tool for early suppression of viral infections which shifts the cells to the alert regimen, thus preventing further spread of infection. The main task of the human central immune system is to maintain integrity and combat foreign organisms. Accordingly, a suitable index of acquired antiviral immunity should be a presence of specific spacer markers in DNA samples from reconvalescent persons, rather than detection of neutralizing antibodies, B and T memory cells.This article is addressed primarily to general medical community, and its practical conclusions are as follows:1. Presence or absence of specific antibodies to SARS-CoV-2 is not a prognostic sign of the disease. Detection of specific antibodies in blood simply reflects the fact that the person has contacted with the viral agent. Absence of antibodies does not mean a lack of such contact, and the persons with high titers of specific antibodies are not protected from re-infection with SARS-CoV-2.2. PCR testing: The PCR results may remain “false positive” in those subjects who have had COVID-19, if the genetic material is taken from the site of initial virus contraction (mainly, nasopharynx). In our opinion, negative PCR tests for COVID-19 in blood plasma and urine will be a more correct index for the absence of the disease, even with positive PCR tests from the nasopharyngeal samples.3. It is necessary to draw attention of general practitioners to potential usage of retinol in prevention and treatment of COVID-19, given the importance of RLR receptors in recognition of viral RNAs and positive experience of vitamin A administration in measles, another dangerous viral disease.

Zika virus causes placental pyroptosis and associated adverse fetal outcomes by activating GSDME.

Zika virus (ZIKV) can be transmitted from mother to fetus during pregnancy, causing adverse fetal outcomes. Several studies have indicated that ZIKV can damage the fetal brain directly; however, whether the ZIKV-induced maternal placental injury contributes to adverse fetal outcomes is sparsely defined. Here, we demonstrated that ZIKV causes the pyroptosis of placental cells by activating the executor gasdermin E (GSDME) in vitro and in vivo. Mechanistically, TNF-α release is induced upon the recognition of viral genomic RNA by RIG-I, followed by activation of caspase-8 and caspase-3 to ultimately escalate the GSDME cleavage. Further analyses revealed that the ablation of GSDME or treatment with TNF-α receptor antagonist in ZIKV-infected pregnant mice attenuates placental pyroptosis, which consequently confers protection against adverse fetal outcomes. In conclusion, our study unveils a novel mechanism of ZIKV-induced adverse fetal outcomes via causing placental cell pyroptosis, which provides new clues for developing therapies for ZIKV-associated diseases.