Antiviral Mechanism Research Articles

Structural basis for the concerted antiphage activity in the SIR2-HerA system.

Recently, a novel two-gene bacterial defense system against phages, encoding a SIR2 NADase and a HerA ATPase/helicase, has been identified. However, the molecular mechanism of the bacterial SIR2-HerA immune system remains unclear. Here, we determine the cryo-EM structures of SIR2, HerA and their complex from Paenibacillus sp. 453MF in different functional states. The SIR2 proteins oligomerize into a dodecameric ring-shaped structure consisting of two layers of interlocked hexamers, in which each subunit exhibits an auto-inhibited conformation. Distinct from the canonical AAA+proteins, HerA hexamer alone in this antiphage system adopts a split spiral arrangement, which is stabilized by a unique C-terminal extension. SIR2 and HerA proteins assemble into a ∼1.1 MDa torch-shaped complex to fight against phage infection. Importantly, disruption of the interactions between SIR2 and HerA largely abolishes the antiphage activity. Interestingly, binding alters the oligomer state of SIR2, switching from a dodecamer to a tetradecamer state. The formation of the SIR2-HerA binary complex activates NADase and nuclease activities in SIR2 and ATPase and helicase activities in HerA. Together, our study not only provides a structural basis for the functional communications between SIR2 and HerA proteins, but also unravels a novel concerted antiviral mechanism through NAD+ degradation, ATP hydrolysis, and DNA cleavage.

Effect of cyclosporin A on respiratory viral replication in fully differentiated exvivo human airway epithelia.

Cyclosporin A (CsA), an immunosuppressive drug used in transplant recipients, inhibits graft rejection by binding to cyclophilins and competitively inhibiting calcineurin. While concerns about respiratory infections in immunosuppressed patients exist, contradictory data emerged during the COVID-19 pandemic, prompting investigations into CsA's impact on viral infections. This study explores CsA's antiviral effects on SARS-CoV-2 Omicron BA.1, Delta variants, and human parainfluenza virus 3 (HPIV3) using an exvivo model of human airway epithelium (HAE). CsA exhibited a dose-dependent antiviral effect against the SARS-CoV-2 Delta variant, reducing viral load over 10 days. However, no significant impact was observed against SARS-CoV-2 Omicron or HPIV3, indicating a virus-specific effect. At high concentrations, CsA was associated with an increase of IL-8 and a decrease of IFNλ expression in infected and noninfected HAE. This study highlights the complexity of CsA's antiviral mechanisms, more likely involving intricate inflammatory pathways and interactions with specific viral proteins. The research provides novel insights into CsA's effects on respiratory viruses, emphasizing the need for understanding drug-virus interactions in optimizing therapeutic approaches for transplant recipients and advancing knowledge on immunosuppressive treatments' implications on respiratory viral infections. Limitations include the model's inability to assess T lymphocyte activation, suggesting the necessity for further comprehensive studies to decipher the intricate dynamics of immunosuppressive treatments on respiratory viral infections.

Synthesis, structural modification, and biological activity of a novel bisindole alkaloid iheyamine A

Diseases caused by plant viruses and pathogens pose a serious threat to crop yield and quality. Traditional pesticides have gradually developed drug resistance and brought certain environmental safety issues during long-term overuse. There is an urgent need to discover new candidate compounds to address these issues. In this study, we achieved the efficient synthesis of iheyamine A and its derivatives, and discovered their excellent antiviral activities against tobacco mosaic virus (TMV). Most compounds displayed higher antiviral activities against TMV than commercial ribavirin at 500 μg/mL, with compounds 3a (Inactive effect IC50: 162 µg/mL), 3d (Inactive effect IC50: 249 µg/mL), 6p (Inactive effect IC50: 254 µg/mL), and 7a (Inactive effect IC50: 234 µg/mL) exhibiting better antiviral activities than ningnanmycin at 500 μg/mL (Inactive effect IC50: 269 µg/mL). Meanwhile, the structure–activity relationships of this type of compounds were systematically studied. We chose 3a for further antiviral mechanism research and found that it can directly act on viral coat protein (CP). The interaction of 3a and CP was further verified via molecular docking. These compounds also showed broad-spectrum fungicidal activities against 8 plant pathogenic fungi, especially for P. piricola. This study provides a reference for the role of iheyamine alkaloids in combating plant pathogenic diseases.

Impact of a Functional Dairy Powder and Its Primary Component on the Growth of Pathogenic and Probiotic Gut Bacteria and Human Coronavirus 229E.

Milk boasts an array of potent bioactive compounds, such as lactoferrin (Lf), immunoglobulins, and functional proteins, all delivering substantial therapeutic benefits. In this study, Immune Powder (a functional dairy formulation) and its primary component called Fractionated Milk Protein (FMP) containing Lf, zinc, and immunoglobulins and formulated by Ausnutria Pty Ltd. were evaluated for their potential broad-spectrum pharmacological activity. In particular, this study investigated the antibacterial (against pathogenic Escherichia coli), prebiotic (promoting Lactobacillus delbrueckii growth), anti-inflammatory (inhibition of NO production in RAW264.7 macrophages), and antiviral (against human coronavirus 229E) effects of the samples. In addition, the impact of simulated gastric digestion on the efficacy of the samples was explored. LCMS-based proteomics was implemented to unveil cellular and molecular mechanisms underlying antiviral activity. The Immune Powder demonstrated antibacterial activity against E. coli (up to 99.74 ± 11.47% inhibition), coupled with prebiotic action (10.84 ± 2.2 viability fold-change), albeit these activities diminished post-digestion (p < 0.01). The Immune Powder effectively mitigated NO production in lipopolysaccharide-stimulated RAW264.7 macrophages, with declining efficacy post-digestion (p < 0.0001). The Immune Powder showed similar antiviral activity before and after digestion (p > 0.05) with up to 3-fold improvement. Likewise, FMP exhibited antibacterial potency pre-digestion at high concentrations (95.56 ± 1.23% inhibition at 125 mg/mL) and post-digestion at lower doses (61.82 ± 5.58% inhibition at 3906.25 µg/mL). FMP also showed enhanced prebiotic activity post-digestion (p < 0.0001), NO inhibition pre-digestion, and significant antiviral activity. The proteomics study suggested that the formulation and its primary component shared similar antiviral mechanisms by inhibiting scavenger receptor binding and extracellular matrix interaction.

TRIM103 activates the RLRs pathway to enhance antiviral response by targeting VP5 and VP7

Grass carp (Ctenopharyngodon idella), crucial to global inland aquaculture with a production of 5.8 million tones in 2020, faces significant challenges from hemorrhagic disease caused by grass carp reovirus (GCRV). Rapid mutations compromise current vaccines, underscoring the need for a deeper understanding of antiviral mechanisms to enhance molecular marker-assisted selection. This study investigates the role of Tripartite Motif (TRIM) family in the innate immune response of grass carp, focusing on TRIM103 from Ctenopharyngodon Idella (CiTRIM103), a member of the TRIM-B30.2 family, which includes proteins with the B30.2 domain at the N-terminus, known for antiviral properties in teleosts. CiTRIM103 bind to the outer coat proteins VP5 and VP7 of GCRV. This binding is theorized to strengthen the function of the RIG-I-like Receptor (RLR) signaling pathway, crucial for antiviral responses. Demonstrations using overexpression and RNA interference (RNAi) techniques have shown that CiTRIM103 effectively inhibits GCRV replication. Moreover, molecular docking and pulldown assays suggest potential binding interactions of CiTRIM103's B30.2 domain with GCRV outer coat proteins VP5 and VP7. These interactions impede viral replication, enhance RLR receptor expression, and activate key transcription factors to induce type I interferons (IFNs). These findings elucidate the antiviral mechanisms of CiTRIM103, provide a foundation for future Molecular genetic breeding in grass carp.

Model informed dose regimen optimizing in development of leritrelvir for the treatment of mild or moderate COVID-19.

Leritrelvir (RAY1216) acts as a main protease inhibitor that hinders the cleavage of viral precursor proteins, thereby inhibiting virus replication of SARS-CoV-2). This antiviral mechanism has shown significant efficacy against the novel coronavirus. Preclinical studies have demonstrated the potent antiviral activity and favorable safety profile of this compound. This study aims to develop a pharmacokinetic model for leritrelvir, with and without ritonavir as a pharmacokinetic enhancer and to evaluate the necessity of co-administration with ritonavir and to investigate different dosage regimens. The model establishment was based on plasma concentration data from a phase I trial involving 72 subjects in single-ascending dose (SAD), multiple-ascending dose (MAD), and a food effect cohort. Analysis was conducted using a nonlinear mixed-effects model, and clinical trial simulations were carried out. The findings of this study demonstrate a favorable safety profile for leritrelvir. With simulation suggests that a 400mg thrice-daily (TID) regimen may be optimal to maintain the trough concentrations (Ctrough) above levels required for inhibiiting viral replication. While ritonavir was found to enhance exposure, it was deemed unnecessary. Gender and food consumption were identified as significant covariates affecting pharmacokinetic parameters, however, no dose adjustments were deemed necessary. This findings supported by subsequent phase II and phase III trials validated the appropriateness of a 400mg TID regimen for the administration of leritrelvir.

Effects of the oxoaporphine alkaloid hernandonine on dengue virus. Evidence for its mechanisms of action

BackgroundDengue, caused by the dengue virus (Orthoflavivirus dengue, encompassing DENV types 1-4), is a member of the Flaviviridae family. The symptoms of dengue range from subclinical or mild manifestations to potentially fatal complications. The management of severe dengue is exceptionally challenging due to the absence of effective antiviral medications. In this context, natural products, whether in the form of pure compounds or standardized plant extracts, have emerged as a promising source for the development of novel antiviral therapeutics. Hernandonine, an oxoaporphine alkaloid found in Hernandia nymphaeifolia (C. Presl) Kubitzki. serves both as a metabolite and an inhibitor of human immunodeficiency virus type 1 (HIV-1) integrase. PurposeThis study investigated the ability of hernandonine to inhibit DENV infection and explored its potential mechanisms. Study designTo assess the in vitro anti-DENV activity, cells or induced pluripotent stem cell (iPSC)-derived cerebral organoids were exposed to hernandonine before or after infection with DENV. Along with hernandonine, the endocytosis modulators, genistein, wortmannin, Methyl-β-cyclodextrin (MβCD) and lovastatin, were used in the assays. MethodsThe DENV infectivity and virion production in cells or cerebral organoids treated with compounds were determined. Various methods, including cell and cerebral organoids imaging, protein and gene detection were conducted to explore their antiviral mechanisms. ResultsThe results revealed notable antiviral properties of hernandonine, particularly in inhibiting DENV during the early stages of infection. Mechanistic analysis demonstrated that, akin to genistein, wortmannin, methyl-β-cyclodextrin (MβCD), and lovastatin, hernandonine exerted an influence on cholesterol-rich lipid rafts. It also restrained the pseudopodial movement ability of cells, potentially through the downregulation of cytoskeleton and endocytosis regulatory genes or protein expression. Moreover, hernandonine's virucidal activity was demonstrated. Hernandonine's inhibition of DENV infection was further validated in a disease-relevant iPSC-derived cerebral organoids model, a novel DENV-2 infection system worthy of further application. ConclusionThis study evidenced the potential of hernandonine as a novel candidate in the fight against DENV infection.

Exploring antiviral effect and mechanism of Jinye Baidu granules（JYBD）against influenza A virus through network pharmacology and in vitro and invivo experiments

Ethnopharmacological relevanceJinye Baidu granules (JYBD) have been used to treat acute respiratory tract infections and demonstrated clinical efficacy for the treatment of emerging or epidemic respiratory viruses such as SARS-CoV-2 and influenza virus. Aim of the studyThis study is to investigate the antiviral effect of JYBD against influenza A viruses (IAV) in vitro and in vivo and elucidate its underlying mechanism. Materials and methodsUltra-high-performance liquid chromatography connected with Orbitrap mass spectrometer (UHPLC-Orbitrap MS) was employed to describe the chemical profile of JYBD. The potential pathways and targets involved in JYBD against IAV infection were predicted by network pharmacology. The efficacy and mechanism of JYBD were validated through both in vivo and in vitro experiments. Moreover, combination therapy with JYBD and the classic anti-influenza drugs was also investigated. ResultsA total of 126 compounds were identified by UHPLC-Orbitrap MS, of which 9 compounds were unambiguously confirmed with reference standards. JYBD could significantly inhibit the replication of multiple strains of IAV, especially oseltamivir-resistant strains. The results of qRT-PCR and WB demonstrated that JYBD could inhibit the excessive induction of pro-inflammatory cytokines induced by IAV infection and regulate inflammatory response through inhibiting JAK/STAT, NF-κB and MAPK pathways. Moreover, both JYBD monotherapy or in combination with oseltamivir could alleviate IAV-induced severe lung injury in mice. ConclusionsJYBD could inhibit IAV replication and mitigate virus-induced excessive inflammatory response. Combinations of JYBD and neuraminidase inhibitors conferred synergistic suppression of IAV both in vitro and in vivo. It might provide a scientific basis for clinical applications of JYBD against influenza virus infected diseases.

Import of extracellular 2′-3′cGAMP by the folate transporter, SLC19A1, establishes an antiviral response that limits herpes simplex virus-1

Recently it was discovered that extracellular 2′-3′cGAMP can activate the STING pathway in a cGAS-independent fashion by being transported across the cell membrane via the folate transporter, SLC19A1, the first identified extracellular antiporter of this critical signaling molecule in cancer cells. We hypothesized that this non-canonical activation of STING pathway would function to establish an antiviral state similar to that seen with the paracrine antiviral activities of interferon. Herein, we report that treatment of the monocytic cell line, THP-1 cells and SH-SY5Y neuronal cell line with exogenous 2′-3′cGAMP induces interferon production and establishes an antiviral state that limits herpes simplex virus-1 (HSV-1), a ubiquitous virus with high seropositivity in the human population. Using either pharmaceutical inhibition or genetic knockout of SLC19A1 blocks the 2′-3′cGAMP-induced inhibition of viral replication. Our data indicate SLC19A1 functions as a newly identified antiviral mediator for extracellular 2′-3′cGAMP. This work presents novel and important findings about an antiviral mechanism which information could aid in the development of better antiviral drugs in the future.

Antiviral Activity of Selective Estrogen Receptor Modulators against Severe Fever with Thrombocytopenia Syndrome Virus In Vitro and In Vivo.

Severe fever with thrombocytopenia syndrome virus (SFTSV), also known as the Dabie Banda virus, is an emerging tick-borne Bunyavirus that causes severe fever with thrombocytopenia syndrome (SFTS). Currently, symptomatic treatment and antiviral therapy with ribavirin and favipiravir are used in clinical management. However, their therapeutical efficacy is hardly satisfactory in patients with high viral load. In this study, we explored the antiviral effects of selective estrogen receptor modulators (SERMs) on SFTSV infection and the antiviral mechanisms of a representative SERM, bazedoxifene acetate (BZA). Our data show that SERMs potently inhibited SFTSV-induced cytopathic effect (CPE), the proliferation of infectious viral particles, and viral RNA replication and that BZA effectively protected mice from lethal viral challenge. The mode of action analysis reveals that BZA exerts antiviral effects during the post-entry stage of SFTSV infection. The transcriptome analysis reveals that GRASLND and CYP1A1 were upregulated, while TMEM45B and TXNIP were downregulated. Our findings suggest that SERMs have the potential to be used in the treatment of SFTSV infection.

MicroRNA-296-5p Expression in COVID-19 Patients and its Relationship with Inflammatory Cytokines

Objectives: The cytokine storm, triggered by the activation of certain pro-inflammatory genes in the second phase of COVID-19, is associated with severe acute respiratory disorder. Evidence suggests that microRNAs (miR) and cytokine levels can play pivotal roles in host cell antiviral defense mechanisms. This study aimed to investigate the changes in IFN-γ inducible protein 10 (IP-10) and miR-296-5p expression, as well as their relationship with some inflammatory cytokines and biochemical variables in COVID-19 patients. Methods: This retrospective single-center study was conducted on 30 COVID-19 patients and 30 controls. The expression of miR-296-5p and IP-10 genes in peripheral blood mononuclear cells (PBMCs) was evaluated using real-time PCR. Serum levels of IL-6, TNF-α, and CRP were measured by ELISA. Results: Higher expression levels of miR-296-5p and IP-10 genes were observed in COVID-19 patients compared to controls (P=0.001). Furthermore, IP-10, IL-6, and TNF-α levels were significantly higher (PP&lt;0.01) in COVID-19 patients than in controls. The results also showed positive correlations between miR-296-5p expression and serum levels of IL-6, CRP, and TNF-α in patients with COVID-19. ROC curve analysis of miR[1]296-5p in COVID-19 patients showed an area under the curve of 0.830, 95% CI (0.658-0.815), P&lt;0.001, with an optimal cut-off point of 0.32965. Conclusion: Our results suggest a regulatory role for miR-296-5p in cytokine secretion in COVID-19. The results indicate that the expression of miR-296-5p and IP-10 genes in PBMCs might serve as convenient novel biomarkers for the prognosis of COVID-19.

Unveiling the Antiviral Potential of Minocycline: Modulation of Nuclear Export of Viral Ribonuclear Proteins during Influenza Virus Infection.

Influenza A virus (IAV) poses a global threat worldwide causing pandemics, epidemics, and seasonal outbreaks. Annual modification of vaccines is costly due to continual shifts in circulating genotypes, leading to inadequate coverage in low- and middle-income countries like India. Additionally, IAVs are evolving resistance to approved antivirals, necessitating a search for alternative treatments. In this study, the antiviral role of the FDA-approved antibiotic minocycline against IAV strains was evaluated in vitro and in vivo by quantifying viral gene expression by qRT-PCR, viral protein levels by Western blotting, and viral titers. Our findings demonstrate that minocycline at a non-toxic dose effectively inhibits IAV replication, regardless of viral strain or cell line. Its antiviral mechanism operates independently of interferon signaling by targeting the MEK/ERK signaling pathway, which is crucial for the export of viral ribonucleoproteins (vRNPs). Minocycline prevents the assembly and release of infectious viral particles by causing the accumulation of vRNPs within the nucleus. Moreover, minocycline also inhibits IAV-induced late-stage apoptosis, further suppressing viral propagation. The antiviral activity of minocycline against IAVs could offer a promising solution amidst the challenges posed by influenza and the limitations of current treatments.

Transient Expression Vector Construction, Subcellular Localisation, and Evaluation of Antiviral Potential of Flagellin BP8-2.

This study used the DNA of Bacillus amyloliquefaciens Ba168 as a template to amplify the flagellin BP8-2 gene and ligate it into the fusion expression vector pCAMBIA1300-35S-EGFP after digestion for the construction of the expression vector pCAMBIA1300-EGFP-BP8-2. Next, using Nicotiana benthamiana as receptor material, transient expression was carried out under the mediation of Agrobacterium tumefaciens C58C1. Finally, the transient expression and subcellular localisation of flagellin BP8-2 protein were analysed using the imaging of co-transformed GFP under laser confocal microscopy. The results showed that flagellin BP8-2 was localised in the cell membrane and nucleus, and the RT-PCR results showed that the BP8-2 gene could be stably expressed in tobacco leaf cells. Furthermore, there was stronger antiviral activity against tobacco mosaic virus (TMV) infection in Nicotiana glutinosa than in BP8-2 and ningnanmycin, with an inhibitory effect of 75.91%, protective effect of 77.45%, and curative effect of 68.15%. TMV movement and coat protein expression were suppressed, and there was a high expression of PR-1a, PAL, and NPR1 in BP8-2-treated tobacco leaf. These results suggest that flagellin BP8-2 inhibits TMV by inducing resistance. Moreover, BP8-2 has low toxicity and is easily biodegradable and eco-friendly. These results further enrich our understanding of the antiviral mechanisms of proteins and provide alternatives for controlling viral diseases in agriculture.

Multi-omics analysis of antiviral interactions of Elizabethkingia anophelis and Zika virus

The microbial communities residing in the mosquito midgut play a key role in determining the outcome of mosquito pathogen infection. Elizabethkingia anophelis, originally isolated from the midgut of Anopheles gambiae possess a broad-spectrum antiviral phenotype, yet a gap in knowledge regarding the mechanistic basis of its interaction with viruses exists. The current study aims to identify pathways and genetic factors linked to E. anophelis antiviral activity. The understanding of E. anophelis antiviral mechanism could lead to novel transmission barrier tools to prevent arboviral outbreaks. We utilized a non-targeted multi-omics approach, analyzing extracellular lipids, proteins, metabolites of culture supernatants coinfected with ZIKV and E. anophelis. We observed a significant decrease in arginine and phenylalanine levels, metabolites that are essential for viral replication and progression of viral infection. This study provides insights into the molecular basis of E. anophelis antiviral phenotype. The findings lay a foundation for in-depth mechanistic studies.

Acidic polysaccharide from the edible insect Protaetia brevitarsis seulensis activates antiviral immunity to suppress norovirus infection

Edible insects are gaining attention as potential nutraceutical sources with immunomodulatory properties. This study reports purification and structural characterization of polysaccharides from Protaetia brevitarsis seulensis larvae (PBSL) with antiviral activity against murine norovirus. Four polysaccharide fractions purified from PBSL water extracts exhibited varying molecular weights (458.5–627.3 kDa) and monosaccharide compositions, including glucose (42.4–99.2 %), galactose (5.9–13.9 %), rhamnose (0.7–18.7 %), arabinose (3.8–5.4 %), and glucuronic acid (0–15.3 %). The immunomodulatory activity, assessed by interferon-β (IFN-β) production, positively correlated with higher galactose, mannose, rhamnose, and uronic acid contents. Among the fractions, PBS-P, eluted with 0.5 M NaCl, demonstrated superior in vitro antiviral activity with IFN-β production exceeding 8-fold compared to other fractions and 82-fold higher than PBSL water extract, confirming it as the main antiviral active component. Structural analysis revealed PBS-P backbone consisted of α-(1 → 4)-D-Glcp, α-(1 → 4,6)-D-Glcp, α-(1 → 4)-D-GlcpA, α-(1 → 3)-D-Galp and α-(1 → 4)-D-Manp residues, and branched chains of α-D-Glcp-(1→, and α-L-Arap-(1 → 2)-α-L-Rhap-(1 → residues. PBS-P suppressed norovirus replication by stimulating IFN-β, TNF-α, and activating NF-κB, STAT1/2, and TBK1-IRF3 pathways, and its oral administration reduced viral loads in infected mice intestines. This study provides the first report on the detailed structural feature of polysaccharide from an edible insect and its antiviral mechanism, highlighting its potential as a new antiviral agent.

Design, synthesis, and biological activities of arecoline derivatives containing 1,3,4-oxadiazole structure

Pesticides play an important role in the development of agriculture, as they can prevent and control crop diseases and pests, improve crop yield and quality. However, the abuse and improper use of pesticides can lead to negative impacts such as environmental pollution and pest resistance issues. There is an urgent need to develop green, safe, and efficient pesticides. In this work, natural product arecoline was selected as parent structure, a series of arecoline derivatives were designed, synthesized, and systematically investigated antiviral activities against tobacco mosaic virus (TMV). These compounds were found to have good to excellent anti-TMV activities for the first time. The antiviral activities of 4a, 4 h, 4 l, 4p, 6a, 6c, and 6f are higher than that of ningnanmycin. Compounds 4 h (EC50 value 146 µg/mL) and 4p (EC50 value 161 µg/mL) with simple structures and excellent activities emerged as new antiviral candidates. We chose 4 h to further investigate the antiviral mechanism, which revealed that it can cause virus fragmentation by acting on the viral coat protein (CP). We further validated this result through molecular docking. These compounds also displayed broad-spectrum fungicidal activities against 8 plant pathogenic fungi. This work lays the theoretical foundation for the application of arecoline derivatives in the agricultural field.

TRIMming down Flavivirus Infections.

Flaviviruses comprise a large number of arthropod-borne viruses, some of which are associated with life-threatening diseases. Flavivirus infections are rising worldwide, mainly due to the proliferation and geographical expansion of their vectors. The main human pathogens are mosquito-borne flaviviruses, including dengue virus, Zika virus, and West Nile virus, but tick-borne flaviviruses are also emerging. As with any viral infection, the body's first line of defense against flavivirus infections is the innate immune defense, of which type I interferon is the armed wing. This cytokine exerts its antiviral activity by triggering the synthesis of hundreds of interferon-induced genes (ISGs), whose products can prevent infection. Among the ISGs that inhibit flavivirus replication, certain tripartite motif (TRIM) proteins have been identified. Although involved in other biological processes, TRIMs constitute a large family of antiviral proteins active on a wide range of viruses. Furthermore, whereas some TRIM proteins directly block viral replication, others are positive regulators of the IFN response. Therefore, viruses have developed strategies to evade or counteract TRIM proteins, and some even hijack certain TRIM proteins to their advantage. In this review, we summarize the current state of knowledge on the interactions between flaviviruses and TRIM proteins, covering both direct and indirect antiviral mechanisms.

Positive selection analyses identify a single WWE domain residue that shapes ZAP into a more potent restriction factor against alphaviruses.

The host interferon pathway upregulates intrinsic restriction factors in response to viral infection. Many of them block a diverse range of viruses, suggesting that their antiviral functions might have been shaped by multiple viral families during evolution. Host-virus conflicts have led to the rapid adaptation of host and viral proteins at their interaction hotspots. Hence, we can use evolutionary genetic analyses to elucidate antiviral mechanisms and domain functions of restriction factors. Zinc finger antiviral protein (ZAP) is a restriction factor against RNA viruses such as alphaviruses, in addition to other RNA, retro-, and DNA viruses, yet its precise antiviral mechanism is not fully characterized. Previously, an analysis of 13 primate ZAP orthologs identified three positively selected residues in the poly(ADP-ribose) polymerase-like domain. However, selective pressure from ancient alphaviruses and others likely drove ZAP adaptation in a wider representation of mammals. We performed positive selection analyses in 261 mammalian ZAP using more robust methods with complementary strengths and identified seven positively selected sites in all domains of the protein. We generated ZAP inducible cell lines in which the positively selected residues of ZAP are mutated and tested their effects on alphavirus replication and known ZAP activities. Interestingly, the mutant in the second WWE domain of ZAP (N658A) is dramatically better than wild-type ZAP at blocking replication of Sindbis virus and other ZAP-sensitive alphaviruses due to enhanced viral translation inhibition. The N658A mutant is adjacent to the previously reported poly(ADP-ribose) (PAR) binding pocket, but surprisingly has reduced binding to PAR. In summary, the second WWE domain is critical for engineering a more potent ZAP and fluctuations in PAR binding modulate ZAP antiviral activity. Our study has the potential to unravel the role of ADP-ribosylation in the host innate immune defense and viral evolutionary strategies that antagonize this post-translational modification.

Antiviral activity of vitamin D derivatives against severe fever with thrombocytopenia syndrome virus in vitro and in vivo

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a tick-borne virus that causes the severe fever thrombocytopenia syndrome, which manifests as fever and haemorrhage, accompanied by severe neurological complications. To date, no specific antiviral drugs have been approved for this indication. Herein, we investigated whether vitamin D derivatives inhibit SFTSV both in vitro and in vivo. An in vitro study demonstrated that vitamin D derivatives significantly suppressed viral RNA replication, plaque formation, and protein expression in a dose-dependent manner. Subsequently, in vivo studies revealed that doxercalciferol and alfacalcidol were associated with increased survival and reduced viral RNA load in the blood. Time-of-addition assay suggested that vitamin D derivatives primarily acted during the post-entry phase of SFTSV infection. However, cytopathic effect protective activity was not observed in RIG-I immunodeficient cell line Huh7.5, and the administration of vitamin D derivatives did not improve the survival rates or reduce the blood viral loads in adult A129 mice. Further transcriptome exploration into the antiviral mechanism revealed that alfacalcidol stimulates host innate immunity to exert antiviral effects. To expand the application of vitamin D derivatives, in vitro and in vivo drug combination assays were performed, which highlighted the synergistic effects of vitamin D derivatives and T-705 on SFTSV. The combination of alfacalcidol and T-705 significantly enhanced the therapeutic effects in mice. This study highlights the potential of vitamin D derivatives against SFTSV and suggests that they may have synergistic effects with other compounds used in the treatment of SFTSV infection.

Bacterial homologs of innate eukaryotic antiviral defenses with anti-phage activity highlight shared evolutionary roots of viral defenses

Prokaryotes have evolved a multitude of defense systems to protect against phage predation. Some of these resemble eukaryotic genes involved in antiviral responses. Here, we set out to systematically project the current knowledge of eukaryotic-like antiviral defense systems onto prokaryotic genomes, using Pseudomonas aeruginosa as a model organism. Searching for phage defense systems related to innate antiviral genes from vertebrates and plants, we uncovered over 450 candidates. We validated six of these phage defense systems, including factors preventing viral attachment, R-loop-acting enzymes, the inflammasome, ubiquitin pathway, and pathogen recognition signaling. Collectively, these defense systems support the concept of deep evolutionary links and shared antiviral mechanisms between prokaryotes and eukaryotes.