Swine acute diarrhea syndrome coronavirus nucleocapsid protein antagonizes the IFN response through inhibiting TRIM25 oligomerization and functional activation of RIG-I/TRIM25.

Swine acute diarrhea syndrome coronavirus (SADS-CoV), an emerging Alpha-coronavirus, brings huge economic loss in swine industry. Interferons (IFNs) participate in a frontline antiviral defense mechanism triggering the activation of numerous downstream antiviral genes. Here, we demonstrated that TRIM25 overexpression significantly inhibited SADS-CoV replication, whereas TRIM25 deficiency markedly increased viral yield. We found that SADS-CoV N protein suppressed interferon-beta (IFN-ß) production induced by Sendai virus (SeV) or poly(I:C). Moreover, we determined that SADS-CoV N protein interacted with RIG-I N-terminal two caspase activation and recruitment domains (2CARDs) and TRIM25 coiled-coil dimerization (CCD) domain. The interaction of SADS-CoV N protein with RIG-I and TRIM25 caused TRIM25 multimerization inhibition, the RIG-I-TRIM25 interaction disruption, and consequent the IRF3 and TBK1 phosphorylation impediment. Overexpression of SADS-CoV N protein facilitated the replication of VSV-GFP by suppressing IFN-ß production. Our results demonstrate that SADS-CoV N suppresses the host IFN response, thus highlighting the significant involvement of TRIM25 in regulating antiviral immune defenses.

Analysis of Chicken IFITM3 Gene Expression and Its Effect on Avian Reovirus Replication.

Interferon-inducible transmembrane protein 3 (IFITM3) is an antiviral factor that plays an important role in the host innate immune response against viruses. Previous studies have shown that IFITM3 is upregulated in various tissues and organs after avian reovirus (ARV) infection, which suggests that IFITM3 may be involved in the antiviral response after ARV infection. In this study, the chicken IFITM3 gene was cloned and analyzed bioinformatically. Then, the role of chicken IFITM3 in ARV infection was further explored. The results showed that the molecular weight of the chicken IFITM3 protein was approximately 13 kDa. This protein was found to be localized mainly in the cytoplasm, and its protein structure contained the CD225 domain. The homology analysis and phylogenetic tree analysis showed that the IFITM3 genes of different species exhibited great variation during genetic evolution, and chicken IFITM3 shared the highest homology with that of Anas platyrhynchos and displayed relatively low homology with those of birds such as Anser cygnoides and Serinus canaria. An analysis of the distribution of chicken IFITM3 in tissues and organs revealed that the IFITM3 gene was expressed at its highest level in the intestine and in large quantities in immune organs, such as the bursa of Fabricius, thymus and spleen. Further studies showed that the overexpression of IFITM3 in chicken embryo fibroblasts (DF-1) could inhibit the replication of ARV, whereas the inhibition of IFITM3 expression in DF-1 cells promoted ARV replication. In addition, chicken IFITM3 may exert negative feedback regulatory effects on the expression of TBK1, IFN-γ and IRF1 during ARV infection, and it is speculated that IFITM3 may participate in the innate immune response after ARV infection by negatively regulating the expression of TBK1, IFN-γ and IRF1. The results of this study further enrich the understanding of the role and function of chicken IFITM3 in ARV infection and provide a theoretical basis for an in-depth understanding of the antiviral mechanism of host resistance to ARV infection.

Interferons in Viral Infections.

Interferons (IFNs) are cytokines that inhibit viral replication in host cells by triggering innate immune responses through the transcriptional induction of various IFN-stimulated genes (ISGs) [...].

The IFIH1-A946T risk variant promotes diabetes in a sex-dependent manner.

Type 1 diabetes (T1D) is an autoimmune disease in which pancreatic islet ß-cells are attacked by the immune system, resulting in insulin deficiency and hyperglycemia. One of the top non-synonymous single-nucleotide polymorphisms (SNP) associated with T1D is in the interferon-induced helicase C domain-containing protein 1 (IFIH1), which encodes an anti-viral cytosolic RNA sensor. This SNP results in an alanine to threonine substitution at amino acid 946 (IFIH1A946T) and confers an increased risk for several autoimmune diseases, including T1D. We hypothesized that the IFIH1A946T risk variant, (IFIH1R) would promote T1D pathogenesis by stimulating type I interferon (IFN I) signaling leading to immune cell alterations. To test this, we developed Ifih1R knock-in mice on the non-obese diabetic (NOD) mouse background, a spontaneous T1D model. Our results revealed a modest increase in diabetes incidence and insulitis in Ifih1R compared to non-risk Ifih1 (Ifih1NR) mice and a significant acceleration of diabetes onset in Ifih1R females. Ifih1R mice exhibited a significantly enhanced interferon stimulated gene (ISG) signature compared to Ifih1NR, indicative of increased IFN I signaling. Ifih1R mice exhibited an increased frequency of plasma cells as well as tissue-dependent changes in the frequency and activation of CD8+ T cells. Our results indicate that IFIH1R may contribute to T1D pathogenesis by altering the frequency and activation of immune cells. These findings advance our knowledge on the connection between the rs1990760 variant and T1D. Further, these data are the first to demonstrate effects of Ifih1R in NOD mice, which will be important to consider for the development of therapeutics for T1D.

Interferon-induced protein with tetratricopeptide repeats 5 of black fruit bat (Pteropus alecto) displays a broad inhibition of RNA viruses.

Bats are natural host reservoirs and have adapted a unique innate immune system that permits them to host many viruses without exhibiting symptoms. Notably, bat interferon stimulated genes (ISGs) have been shown to play antiviral roles. Interferon induced protein with tetratricopeptide repeats 5 (IFIT5) is a well-characterised ISG in humans with antiviral activities against negative-sense RNA viruses via inhibiting viral transcription. Here, we aim to investigate if Pteropus alecto (pa) IFIT5 (paIFIT5) possess the ability to inhibit negative-sense RNA viruses. Initially, gene syntenic and comparative structural analyses of multiple animals highlighted a high level of similarity between Pteropus alecto and human IFIT5 proteins. Our results showed that paIFIT5 was significantly inducible by viral and dsRNA stimulation. Transient overexpression of paIFIT5 inhibited the replication of vesicular stomatitis virus (VSV). Using minireplicon and transcription reporter assays, we demonstrated the ability of paIFIT5 specifically to inhibit H17N10 polymerase activity. Mechanistically, we noticed that the antiviral potential of paIFIT5 against negative sense RNA viruses was retributed to its interaction with 5'ppp containing RNA. Taken together, these findings highlight the genetic and functional conservation of IFIT5 among mammals.

Development of a Cytosolic DNA Sensor Agonist Using GALA Peptide-Conjugated DNA and Long Single-Stranded DNA.

Cytosolic DNA sensors (CDSs) recognize DNA molecules that are abnormally located in the cytosol, thus leading to the activation of the stimulator of interferon genes (STING) and the induction of type 1 interferon. In turn, type 1 interferon evokes defensive reactions against viral infections and activates the immune system; therefore, the use of agonists of CDSs as cancer therapeutics and vaccine adjuvants is expected. Double-stranded DNA molecules with dozens to thousands of bases derived from bacteria and viruses are agonists of CDSs. However, DNA is a water-soluble molecule with a high molecular weight, resulting in poor cellular uptake and endosomal escape. In contrast, long single-stranded DNA (lssDNA) obtained by rolling circle amplification is efficiently taken up and localized to endosomes. Here we constructed a CDS-targeting lssDNA via the facilitation of its intracellular transport from endosomes to the cytosol. An endosome-disrupting GALA peptide was used to deliver the lssDNA to the cytosol. A peptide-oligonucleotide conjugate (POC) was successfully obtained via the conjugation of the GALA peptide with an oligonucleotide complementary to the lssDNA. By hybridization of the POC to the complementary lssDNA (POC/lssDNA), the CDS-STING pathway in dendritic cells was efficiently stimulated. GALA peptide-conjugated DNA seems to be a helpful tool for the delivery of DNA to the cytosol.

Identification of biomimetic nanoplatform-mediated delivery of si-ISG15 for treatment of triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is recognized as the most malicious form of breast cancer and exhibits an alarming tendency for recurrence, a heightened propensity for metastasis, and an overwhelmingly grim prognosis. Therefore, effective therapy approaches for TNBC are urgently required. In this study, the interferon-stimulated gene 15 (ISG15) expression level was analyzed by bioinformatics and verified by Western blot analysis. The effects of ISG15 on the proliferation and metastasis of TNBC cells were assessed using MTT, Colony formation, EdU, Transwell, and Flow cytometry assays. We also developed a cancer cell-biomimetic nanoparticle delivery system and evaluated its therapeutic efficacy in vivo. In this study, we reported that ISG15 was upregulated in TNBC, and its high expression level correlated with an increased risk of tumorigenesis. Through in vitro and in vivo studies, we discovered that ISG15 knockdown drastically suppressed cell proliferation, invasion, and migration and induced apoptosis in TNBC cells. Our findings revealed that ISG15 was a candidate therapeutic target in TNBC because of its key role in malignant growth and invasion. Moreover, co-immunoprecipitation showed that ISG15 exerted oncogenic functions through its interaction with ATP binding cassette subfamily E member 1 and activated the Janus kinase/signal transducers and activators of the transcription signaling pathway. Furthermore, we created a nanoparticle-based siRNA camouflaged using a cancer cell membrane vesicle delivery system (the CM@NP complex) and confirmed its therapeutic effects in vivo. Our findings confirmed that ISG15 may play a pivotal oncogenic role in the development of TNBC and that CM@siRNA-NP complexes are an effective delivery system and a novel biological strategy for treating TNBC.

Lamivudine, Doravirine, and Cabotegravir Downregulate the Expression of Human Endogenous Retroviruses (HERVs), Inhibit Cell Growth, and Reduce Invasive Capability in Melanoma Cell Lines.

This study explores the impact of antiretroviral administration on the expression of human endogenous retroviruses (HERVs), cell growth, and invasive capability of human melanoma cell lines in culture. We investigated three antiretrovirals-lamivudine, doravirine, and cabotegravir-in A375, FO-1, and SK-Mel-28, BRAF-mutated, and in MeWo, P53-mutated, melanoma cell lines. The findings indicate a general capability of these drugs to downregulate the expression of HERV-K Pol and Env genes and hinder cell viability, mobility, and colony formation capacity of melanoma cells. The antiretroviral drugs also demonstrate selectivity against malignant cells, sparing normal human epithelial melanocytes. The study reveals that the integrase inhibitor cabotegravir is particularly effective in inhibiting cell growth and invasion across different cell lines in comparison with lamivudine and doravirine, which are inhibitors of the viral reverse transcriptase enzyme. The investigation further delves into the molecular mechanisms underlying the observed effects, highlighting the potential induction of ferroptosis, apoptosis, and alterations in cell cycle regulatory proteins. Our findings showed cytostatic effects principally revealed in A375, and SK-Mel-28 cell lines through a downregulation of retinoblastoma protein phosphorylation and/or cyclin D1 expression. Signs of ferroptosis were detected in both A375 cells and FO-1 cells by a decrease in glutathione peroxidase 4 and ferritin expression, as well as by an increase in transferrin protein levels. Apoptosis was also detected in FO-1 and SK-Mel-28, but only with cabotegravir treatment. Moreover, we explored the expression and activity of the stimulator of interferon genes (STING) protein and its correlation with programmed death-ligand 1 (PD-L1) expression. Both the STING activity and PD-L1 expression were decreased, suggesting that the antiretroviral treatments may counteract the detrimental effects of PD-L1 expression activation through the STING/interferon pathway triggered by HERV-K. Finally, this study underscores the potential therapeutic significance of cabotegravir in melanoma treatment. The findings also raise the prospect of using antiretroviral drugs to downregulate PD-L1 expression, potentially enhancing the therapeutic responses of immune checkpoint inhibitors.

Altered peripheral blood leukocyte subpopulations, function, and gene expression in children with Down syndrome: implications for respiratory tract infection.

OBJECTIVES: We tested the hypothesis that aberrant expression of Hsa21-encoded interferon genes in peripheral blood immune cells would correlate to immune cell dysfunction in children with Down syndrome (DS). STUDY DESIGN: We performed flow cytometry to quantify peripheral blood leukocyte subtypes and measured their ability to migrate and phagocytose. In matched samples, we measured gene expression levels for constituents of interferon signaling pathways. We screened 49 children, of which 29 were individuals with DS. RESULTS: We show that the percentages of two peripheral blood myeloid cell subtypes (alternatively-activated macrophages and low-density granulocytes) in children with DS differed significantly from typical children, children with DS circulate a very different pattern of cytokines vs. typical individuals, and higher expression levels of type III interferon receptor Interleukin-10Rb in individuals with DS correlated with reduced migratory and phagocytic capacity of macrophages. CONCLUSIONS: Increased susceptibility to severe and chronic infection in children with DS may result from inappropriate numbers and subtypes of immune cells that are phenotypically and functionally altered due to trisomy 21 associated interferonopathy.

IFNL1 rs30461 polymorphism as a risk factor for COVID-19 severity: A cross-sectional study.

INTRODUCTION: The molecular basis of the progression of some COVID-19 patients to worse outcomes is not entirely known. Interferons-lambda-1/interleukin-29 (IFN-λ1/IL-29) is a member of the type III IFNs with a strong antiviral activity. Given the scant data on the potential role of IFN-λ1/IL-29 in COVID-19, we investigated the association of IFN-λ1/IL-29 serum level and the IFNL1 single-nucleotide polymorphism (SNP) (rs30461) with severe course of COVID-19. MATERIAL AND METHODS: This cross-sectional study included 400 COVID-19 patients, in which 262 mild COVID-19 patients and 138 severe COVID-19 patients were recruited and compared. The IFN-λ1/IL-29 serum levels were assessed in both the mild and severe COVID-19 groups. All participants were genotyped for the IFNL1 SNP (rs30461) by allelic discrimination RT-PCR using specific Taqman probes and primers. The associations between IFNL1 variants and risk of severe COVID-19 were examined via the logistic regression analysis. RESULTS: The serum IFN-λ1/IL-29 levels showed no statistically significant difference between mild and severe COVID-19 patients (P = 0.993). The genotype and allele frequencies of IFNL1 SNP (rs30461) were significantly different between the mild and severe groups, in which the minor G allele carried a highly significant risk of severe COVID-19 compared with the wild A allele [OR (95 %CI): 2.1 (1.5-2.9), P ≤ 0.001]. In multivariate analysis, the A/G and G/G genotypes of IFNL1 SNP (rs30461) were independent predictors of COVID-19 severity (P < 0.05). CONCLUSION: The study concluded that the IFNL1 SNP (rs30461) may constitute an independent risk factor for COVID-19 severity.

Differential Interferon Signaling Regulation and Oxidative Stress Responses in the Cerebral Cortex and Cerebellum Could Account for the Spatiotemporal Pattern of Neurodegeneration in Niemann-Pick Disease Type C.

Niemann-Pick disease type C (NPC) is a fatal neurodegenerative condition caused by genetic mutations of the NPC1 or NPC2 genes that encode the NPC1 and NPC2 proteins, respectively, which are believed to be responsible for cholesterol efflux from late-endosomes/lysosomes. The pathogenic mechanisms that lead to neurodegeneration in NPC are not well understood. There are, however, well-defined spatiotemporal patterns of neurodegeneration that may provide insight into the pathogenic process. For example, the cerebellum is severely affected from early disease stages, compared with cerebral regions, which remain relatively spared until later stages. Using a genome-wide transcriptome analysis, we have recently identified an aberrant pattern of interferon activation in the cerebella of pre-symptomatic Npc1-/- mice. Here, we carried out a comparative transcriptomic analysis of cerebral cortices and cerebella of pre-symptomatic Npc1-/- mice and age-matched controls to identify differences that may help explain the pathological progression within the NPC brain. We report lower cerebral expression of genes within interferon signaling pathways, and significant differences in the regulation of oxidative stress, compared with the cerebellum. Our findings suggest that a delayed onset of interferon signaling, possibly linked to lower oxidative stress, may account for the slower onset of cerebral cortical pathology in the disease.

Alternative translation contributes to the generation of a cytoplasmic subpopulation of the Junín virus nucleoprotein that inhibits caspase activation and innate immunity.

The highly pathogenic arenavirus, Junín virus (JUNV), expresses three truncated alternative isoforms of its nucleoprotein (NP), i.e., NP53kD, NP47kD, and NP40kD. While both NP47kD and NP40kD have been previously shown to be products of caspase cleavage, here, we show that expression of the third isoform NP53kD is due to alternative in-frame translation from M80. Based on this information, we were able to generate recombinant JUNVs lacking each of these isoforms. Infection with these mutants revealed that, while all three isoforms contribute to the efficient control of caspase activation, NP40kD plays the predominant role. In contrast to full-length NP (i.e., NP65kD), which is localized to inclusion bodies, where viral RNA synthesis takes place, the loss of portions of the N-terminal coiled-coil region in these isoforms leads to a diffuse cytoplasmic distribution and a loss of function in viral RNA synthesis. Nonetheless, NP53kD, NP47kD, and NP40kD all retain robust interferon antagonistic and 3'-5' exonuclease activities. We suggest that the altered localization of these NP isoforms allows them to be more efficiently targeted by activated caspases for cleavage as decoy substrates, and to be better positioned to degrade viral double-stranded (ds)RNA species that accumulate in the cytoplasm during virus infection and/or interact with cytosolic RNA sensors, thereby limiting dsRNA-mediated innate immune responses. Taken together, this work provides insight into the mechanism by which JUNV leverages apoptosis during infection to generate biologically distinct pools of NP and contributes to our understanding of the expression and biological relevance of alternative protein isoforms during virus infection.IMPORTANCEA limited coding capacity means that RNA viruses need strategies to diversify their proteome. The nucleoprotein (NP) of the highly pathogenic arenavirus Junín virus (JUNV) produces three N-terminally truncated isoforms: two (NP47kD and NP40kD) are known to be produced by caspase cleavage, while, here, we show that NP53kD is produced by alternative translation initiation. Recombinant JUNVs lacking individual NP isoforms revealed that all three isoforms contribute to inhibiting caspase activation during infection, but cleavage to generate NP40kD makes the biggest contribution. Importantly, all three isoforms retain their ability to digest double-stranded (ds)RNA and inhibit interferon promoter activation but have a diffuse cytoplasmic distribution. Given the cytoplasmic localization of both aberrant viral dsRNAs, as well as dsRNA sensors and many other cellular components of innate immune activation pathways, we suggest that the generation of NP isoforms not only contributes to evasion of apoptosis but also robust control of the antiviral response.

Genomic and biological variation in bat IFNs: An antiviral treatment approach.

Bat-borne viruses have attracted considerable research, especially in relation to the Covid-19 pandemic. Although bats can carry multiple zoonotic viruses that are lethal to many mammalian species, they appear to be asymptomatic to viral infection despite the high viral loads contained in their bodies. There are several differences between bats and other mammals. One of the major differences between bats and other mammals is the bats' ability to fly, which is believed to have induced evolutionary changes. It may have also favoured them as suitable hosts for viruses. This is related to their tolerance to viral infection. Innate immunity is the first line of defence against viral infection, but bats have metamorphosed the type of responses induced by innate immunity factors such as interferons. The expression patterns of interferons differ, as do those of interferon-related genes such as interferon regulatory factors and interferon-stimulated genes that contribute to the antiviral response of infected cells. In addition, the signalling pathways related to viral infection and immune responses have been subject to evolutionary changes, including mutations compared to their homologues in other mammals and gene selection. This article discusses the differences in the interferon-mediated antiviral response in bats compared to that of other mammals and how these differences are correlated to viral tolerance in bats. The effect of bat interferons related genes on human antiviral response against bat-borne viruses is also discussed.

Three-Dimensional Chromosomal Landscape Revealing miR-146a Dysfunctional Enhancer in Lupus and Establishing a CRISPR-Mediated Approach to Inhibit the Interferon Pathway.

OBJECTIVE: The diminished expression of microRNA-146a (miR-146a) in systemic lupus erythematosus (SLE) contributes to the aberrant activation of the interferon pathway. Despite its significance, the underlying mechanism driving this reduced expression remains elusive. Considering the integral role of enhancers in steering gene expression, our study seeks to pinpoint the SLE-affected enhancers responsible for modulating miR-146a expression. Additionally, we aim to elucidate the mechanisms by which these enhancers influence the contribution of miR-146a to the activation of the interferon pathway. METHODS: Circular chromosome conformation capture sequencing and epigenomic profiles were applied to identify candidate enhancers of miR-146a. CRISPR activation was performed to screen functional enhancers. Differential analysis of chromatin accessibility was used to identify SLE-dysregulated enhancers, and the mechanism underlying enhancer dysfunction was investigated by analyzing transcription factor binding. The therapeutic value of a lupus-related enhancer was further evaluated by targeting it in the peripheral blood mononuclear cells (PBMCs) of patients with SLE through a CRISPR activation approach. RESULTS: We identified shared and cell-specific enhancers of miR-146a in distinct immune cells. An enhancer 32.5 kb downstream of miR-146a possesses less accessibility in SLE, and its chromatin openness was negatively correlated with SLE disease activity. Moreover, CCAAT/enhancer binding protein α, a down-regulated transcription factor in patients with SLE, binds to the 32.5-kb enhancer and induces the epigenomic change of this locus. Furthermore, CRISPR-based activation of this enhancer in SLE PBMCs could inhibit the activity of interferon pathway. CONCLUSION: Our work defines a promising target for SLE intervention. We adopted integrative approaches to define cell-specific and functional enhancers of the SLE critical gene and investigated the mechanism underlying its dysregulation mediated by a lupus-related enhancer.

Gender-dependent multiple cross-phenotype association of interferon lambda genetic variants with peripheral blood profiles in healthy individuals.

BACKGROUND: Type III interferons (IFN), also called as lambda IFNs (IFN-λs), are antiviral and immunomodulatory cytokines that are evolutionarily important in humans. Given their central roles in innate immunity, they could be influencing other aspects of human biology. This study aimed to examine the association of genetic variants that control the expression and/or activity of IFN-λ3 and IFN-λ4 with multiple phenotypes in blood profiles of healthy individuals. METHODS: In a cohort of about 550 self-declared healthy individuals, after applying several exclusion criteria to determine their health status, we measured 30 blood parameters, including cellular, biochemical, and metabolic profiles. We genotyped them at rs12979860 and rs28416813 using competitive allele-specific PCR assays and tested their association with the blood profiles under dominant and recessive models for the minor allele. IFN-λ4 variants rs368234815 and rs117648444 were also genotyped or inferred. RESULTS: We saw no association in the combined cohort under either of the models for any of the phenotypes. When we stratified the cohort based on gender, we saw a significant association only in males with monocyte (p = 1 × 10-3 ) and SGOT (p = 7 × 10-3 ) levels under the dominant model and with uric acid levels (p = 0.01) under the recessive model. When we tested the IFN-λ4 activity modifying variant within groupings based on absence or presence of one or two copies of IFN-λ4 and on different activity levels of IFN-λ4, we found significant (p < 0.05) association with several phenotypes like monocyte, triglyceride, VLDL, ALP, and uric acid levels, only in males. All the above significant associations did not show any confounding when we tested for the same with up to ten different demographic and lifestyle variables. CONCLUSIONS: These results show that lambda interferons can have pleiotropic effects. However, gender seems to be an effect modifier, with males being more sensitive than females to the effect.

Single-cell resolution of longitudinal blood transcriptome profiles in rheumatoid arthritis, systemic lupus erythematosus and healthy control pregnancies.

OBJECTIVES: Comparative longitudinal analyses of cellular composition and peripheral blood gene expression in Rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and healthy pregnancies. METHODS: In total, 335 whole blood samples from 84 RA, SLE and healthy controls before pregnancy, at each trimester, 6 weeks, 6 months and 12 months post partum were analysed. We combined bulk and single cell RNA analyses for cell-type estimation, validated by flow cytometry, before combining this in a cell-type adjusted analysis for an improved resolution of unrecognised gene expression changes associated with RA and SLE pregnancies. RESULTS: Patients were well regulated throughout pregnancy, and few had pregnancy complications. In SLE, the interferon signature was augmented during pregnancy, and the pregnancy signature was continued post partum. An altered cell type composition strongly influences the profile. In the pregnancy signature, transcripts involved in galactosylation potentially altering the effector functions of autoantibodies became more evident. Several genes in the adjusted RA signature are expressed in mucosal associated invariant T cells. CONCLUSION: We found distinct RA, SLE and pregnancy signatures, and no expression patterns could be attributed to medication or disease activity. Our results support the need for close postpartum follow-up of patients with SLE. Gene expression patterns in RA were closer to healthy controls than to SLE, and primarily became evident after cell-type adjustment. Adjusting for cell abundance unravelled gene expression signatures less associated with variation in cell-composition and highlighted genes with expression profiles associated with changes in specialised cell populations.

Prokaryotic expression of soluble IFN-λ1 recombinant protein with cold-shock system.

Interferon (IFN)-λ1, a member of type III IFN, possesses unique antiviral, anti-tumor, and immune modulation properties. IFN-λ alone or combined with other drugs is considered an essential therapeutic regimen in the clinic. Obtaining high-quality, biologically-active recombinant human IFN-λ1 (rhIFN-λ1) is of great practical significance. In this study, pCold-II-IFN-λ1 expression plasmid was correctly constructed, the rhIFN-λ1 was expressed in BL21(DE3) E.coli and reached the highest level under the optimal condition of 15 °C culture temperature and at 1 µg/L IPTG induction for 12 h. The soluble rhIFN-λ1 was purified by Ni-NTA affinity chromatography. The purified rhIFN-λ1 can effectively activate the JAK1-STAT1 signaling pathway and induce the expression of a large number of interferon-stimulated genes (ISG) including ISG15, ISG54, ISG56, TRAIL, OAS1, MX1, IRF7 and IRF9. In addition, rhIFN-λ1 can effectively inhibit the growth/proliferation of cervical cancer HeLa cells in a dose-dependent pattern. Collectively, the soluble rhIFN-λ1 was successfully expressed in BL21(DE3) E.coli with the cold-shock system, and the purified rhIFN-λ1 demonstrated excellent biological activity. This study lays a solid basis for acquiring high-quality rhIFN-λ1 and its clinical application.

SARS-CoV-2 RNA stabilizes host mRNAs to elicit immunopathogenesis.

SARS-CoV-2 RNA interacts with host factors to suppress interferon responses and simultaneously induces cytokine release to drive the development of severe coronavirus disease 2019 (COVID-19). However, how SARS-CoV-2 hijacks host RNAs to elicit such imbalanced immune responses remains elusive. Here, we analyzed SARS-CoV-2 RNA in situ structures and interactions in infected cells and patient lung samples using RIC-seq. We discovered that SARS-CoV-2 RNA forms 2,095 potential duplexes with the 3' UTRs of 205 host mRNAs to increase their stability by recruiting RNA-binding protein YBX3 in A549 cells. Disrupting the SARS-CoV-2-to-host RNA duplex or knocking down YBX3 decreased host mRNA stability and reduced viral replication. Among SARS-CoV-2-stabilized host targets, NFKBIZ was crucial for promoting cytokine production and reducing interferon responses, probably contributing to cytokine storm induction. Our study uncovers the crucial roles of RNA-RNA interactions in the immunopathogenesis of RNA viruses such as SARS-CoV-2 and provides valuable host targets for drug development.

Disrupted RNA editing in beta cells mimics early-stage type 1 diabetes.

A major hypothesis for the etiology of type 1 diabetes (T1D) postulates initiation by viral infection, leading to double-stranded RNA (dsRNA)-mediated interferon response and inflammation; however, a causal virus has not been identified. Here, we use a mouse model, corroborated with human islet data, to demonstrate that endogenous dsRNA in beta cells can lead to a diabetogenic immune response, thus identifying a virus-independent mechanism for T1D initiation. We found that disruption of the RNA editing enzyme adenosine deaminases acting on RNA (ADAR) in beta cells triggers a massive interferon response, islet inflammation, and beta cell failure and destruction, with features bearing striking similarity to early-stage human T1D. Glycolysis via calcium enhances the interferon response, suggesting an actionable vicious cycle of inflammation and increased beta cell workload.

Function analysis of fish PACT gene in response to virus infection.

PACT (interferon-inducible double-stranded RNA-dependent protein kinase activator A) is a cellular protein which can activate PKR in dsRNA-independent manner. However, the role of PACT in fish virus infection remains largely unknown. In this study, a PACT homologue from grouper (Epinephelus coioides)(EcPACT) was cloned and characterized. The open reading frame of EcPACT has a full length of 924 bp and encodes a protein of 307 amino acids with a predicted molecular weight of 33.29 kDa. Similar to mammals, EcPACT contains three dsRBD domains. EcPACT shares 99.67 % homology with E. lanceolatus. Real-time fluorescence quantitative PCR results showed that EcPACT mRNA was widely expressed in all tissues and abundantly expressed in brain, blood, head kidney and kidney. In addition, SGIV and RGNNV infection significantly upregulated the transcript levels of EcPACT. Subcellular localization analysis showed that EcPACT was mainly distributed in the nucleus. Overexpression of EcPACT inhibited the replication of SGIV and RGNNV in vitro and positively regulated the expression of interferon (IFN) and pro-inflammatory factors. The results provide a better understanding of the relationship between PACT and viral infection in fish.