New nonacyclic duclauxin derivatives with potent anti-influenza activities from Antarctic fungus Penicillium sp. CPCC 401065.

In the context of de novo structure based drug design, the rise of in silico tools shows a promising strategy to search new molecules to address several diseases. In this work, a program with a Grammatical Evolution approach, lead to the design of 4 new molecules that can inhibit the SARS-CoV-2 RNA dependent RNA polymerase. Those were build by an exploration of a chemical space, defined previously by a pharmacophore model, with several bioisosteric fragments arrange by the grammatical evolution code into 1D molecular strings, which were then converted into 3D molecules and evaluated against the desire target using automated docking calculations. The novel drug candidates demonstrate the capacity to manifest ligand efficiencies at the biological target that are analogous to those exhibited by Remdesivir. The molecules in question also demonstrate pharmacokinetic profiles that are analogous. Two of these molecules have been observed to maintain stable interaction properties with the RNA-dependent RNA polymerase over the course of 200 nanoseconds of molecular dynamics simulation. These molecules primarily interact with the active site through weak hydrogen bonds. The software we developed for this contribution is available in the following URL: https://github.com/masotelof/GEMolecularDesign.

AlphaFold-driven structure-guided identification of NS5 RdRp-targeting antiviral leads against Kyasanur Forest Disease Virus.

Southern rice black-streaked dwarf virus (SRBSDV) is a destructive pathogen of rice that is transmitted by the white-backed planthopper (WBPH, Sogatella furcifera). Identifying infectious reservoirs within the SRBSDV cycle is critical for developing effective disease management strategies. This research identifies Chinese sprangletop (Leptochloa chinensis), a noxious weed commonly found in rice ecosystems, as a previously unrecognized natural host of SRBSDV. SRBSDV infection was detected inL. chinensissamples collected from rice paddies exhibiting SRBSDV symptoms. Transcriptomic analyses, observation of SRBSDV virions, and typical profiles of SRBSDV-derived small interfering RNAs provided evidence of active, low-level, and asymptomatic viral infection. Genomic comparisons revealed minor genetic divergence in the viral RNA-dependent RNA polymerase (RdRP) gene, suggesting host-specific adaptation without compromising transmissibility. Further investigations using transmission experiments demonstrated that WBPHs microinjected with SRBSDV obtained from L. chinensis efficiently transmitted the virus to rice seedlings at a rate of 35.7%. This study emphasizes the necessity of integrating weed management into SRBSDV control strategies to disrupt viral reservoirs and mitigate outbreaks.

Norovirus is a major enteric pathogen with pandemic potential and disproportionately high mortality in low-income countries, particularly among young children. Despite its global health burden, no approved vaccine or specific antiviral therapy is currently available. In this study, we targeted two key viral proteins, viral protein 1 (VP1) and RNA-dependent RNA polymerase (RdRp). The workflow included protein modeling, structural stability assessment, molecular docking, molecular dynamics (MD) simulations, non-covalent interaction (NCI) analysis, protein contact atlas evaluation, and pharmacokinetic (ADME-Tox) profiling. Molecular docking results indicated strong binding affinities of selected phytochemicals-Zingiberol, Cardeonolide, Boeravinone B, β-Elemene, and Fisetin-toward both VP1 and RdRp, with binding energies ranging from - 7.8 to - 9.4kcal/mol. MD simulations further demonstrated the structural stability of protein-ligand complexes, with stable RMSD values (~ 0.3nm for RdRp and 0.3-0.5nm for VP1) and only minor transient fluctuations observed in VP1. RMSF analysis revealed localized flexibility, while radius of gyration, hydrogen bonding patterns, and solvent-accessible surface area collectively confirmed overall conformational stability throughout the simulation period. Complementary protein contact atlas and NCI analyses supported the persistence and robustness of protein-ligand interactions, showing comparable or improved stability relative to reference antivirals ribavirin and nitazoxanide. Additionally, all selected compounds exhibited favorable drug-likeness and acceptable ADME-Tox properties. Collectively, these findings suggest that the identified phytochemicals may serve as promising antiviral candidates against norovirus, although further validation through in vitro and in vivo studies is required.

Hepatitis C virus (HCV) manipulates host cellular pathways to create favourable conditions that support its replication and persistence. Identifying virus-host interactions helps to prevent disease progression and supports the development of host-directed antiviral therapies (HDTs) with a reduced risk of drug resistance due to viral mutations. In this study, a proteomic approach using in vitro pulldown assay and mass spectrometry identified α- and β-tubulin as novel cellular proteins physically associated with the viral RNA-dependent RNA polymerase (NS5B). This association was validated in hepatic (Huh7) and non-hepatic (HEK293T) cells. Further analysis confirmed that the interaction between NS5B and α/β-tubulin is an indirect interaction mediated by unidentified protein(s). Domain mapping analysis using NS5B-deletion mutants localized the tubulin-interacting region of NS5B to its N-terminal domain. Nocodazole, a known inhibitor of α- and β-tubulin polymerization, significantly reduced the association between NS5B and α-tubulin in vitro and in vivo settings, but had no notable impact on the NS5B/β-tubulin interaction. Additionally, nocodazole treatment markedly inhibited RNA replication of HCV subgenomic replicon in Huh7 cells in a dose-dependent manner. These results suggest an association between tubulin proteins and HCV RNA replication, potentially involving the interaction with NS5B. In addition, NS5B expression was associated with increased cell proliferation, indicating a possible link between NS5B/tubulin interaction, microtubule dynamics, and cellular transformation. Further studies are required to determine whether these associations reflect direct functional roles in HCV RNA replication, trafficking, virus assembly and/or cellular transformation.

In this study, a novel double-stranded RNA (dsRNA) mycovirus, tentatively designated Fusarium solani partitivirus 4 (FsPV4), was isolated from the Fusarium solani strain GF7, a phytopathogenic fungus responsible for tobacco root rot. The genome of FsPV4 consists of two dsRNA segments, referred to as dsRNA1 (2312bp in length) and dsRNA2 (2213bp in length). The dsRNA 1 and dsRNA 2 were predicted to encode an RNA-dependent RNA polymerase (RdRp) and a coat protein (CP), respectively. Sequence analysis revealed that the RdRp of FsPV4 showed significant sequence similarity to the RdRps of partitiviruses, with Aplosporella javeedii partitivirus 1 being the best match (identity: 60.76%). Phylogenetic analysis of the RdRp showed that FsPV4 clustered robustly within the genus Betapartitivirus of the family Partitiviridae. This represents the first report of a betapartitivirus infecting F. solani, providing a potential candidate for the biological control of F. solani-mediated plant diseases.

Surveillance of tick-associated viruses may contribute to our understanding of viral diversity and evolution. Here, we identified a novel victorivirus in a sample derived from a human-biting tick, Amblyomma testudinarium, in Japan. The viral sequence was identified by metatranscriptomic sequencing of total RNA extracted from Vero cells treated with tick homogenate. The viral sequence was 4621 bp in length and contained two major open reading frames predicted to encode a putative coat protein (CP) and a putative RNA-dependent RNA polymerase (RdRp), respectively. The two open reading frames overlapped at the tetranucleotide sequence AUGA. The C-terminal region of putative CP was enriched in alanine, glycine, and proline residues. All these features are similar to those commonly observed in victoriviruses. Phylogenetic analyses based on the amino acid sequences of the putative CP and RdRp showed that the virus belongs to the genus Victorivirus of the family Pseudototiviridae. We therefore designated this putative virus as Amblyomma testudinarium-associated victorivirus 1 (ATaVV1). These findings expand our current knowledge of hidden viral diversity in tick-associated samples.

NAE inhibitor MLN4924 effectively suppresses Coxsackievirus B3 replication.

Mycoviruses are ubiquitous among fungal species. However, their diversity and distribution across fungal taxa remain incompletely characterized. Here, we report the identification and molecular analysis of a previously undescribed mycovirus from Rhizoctonia solani AG-4 HG III strain Rs292. This virus was provisionally designated Rhizoctonia solani partitivirus Rs292 (RsPV-Rs292). Its viral genome comprised two sections of double-stranded RNA (dsRNA), measuring 1,924bp (dsRNA1) and 1,929bp (dsRNA2), with dsRNA1 consisting of a single open reading frame (ORF1) potentially encoding an RNA-dependent RNA polymerase (RdRp) of 609 residues with conserved catalytic motifs characteristic of partitiviruses. The smaller segment, dsRNA2, contained ORF2, putatively encoding a capsid protein (CP) of 574 residues. BLASTP searches indicated marked sequence similarities of both RdRp and CP with members of the family Partitiviridae. pairwise sequence comparisons of RdRp and CP, RdRp amino acid sequence-based phylogenetic analysis placed RsPV-Rs292 within the genus Alphapartitivirus, supporting its classification as a novel species in this group.

Viral infections continue to pose a significant health problem to all parts of the world because of a high rate of mutation and the ability to resist traditional single-target antivirals. Multi-target inhibition of core viral proteins (main protease, RNA-dependent RNA polymerase, and spike glycoprotein) with plant metabolites as the target of drug discovery is a promising alternative. This review presented a systematic review of the antiviral potential and mechanism of plantderived compounds published from 2020 to 2025. The systematic search of the literature was performed with the help of PubMed, Scopus, Web of Science, Science Direct, and EMBASE in accordance with PRISMA 2020. Peer-reviewed articles that reported precise molecular targets, quantifiable antiviral activity (IC50, EC50 or %inhibition), and mechanistic information were included. Qualitative synthesis of data was done because of heterogeneity in methodology. Sixty studies were eligible. Flavonoids (quercetin, kaempferol) had an inhibitory effect on viral protease and polymerase with an IC50 value of 2-12 μM. The terpenoids (glycyrrhizin, betulinic acid) inhibited spike-ACE2 binding and viral fusion with up to 80% infectivity in vitro. The polymerases of hepatitis and influenza viruses were inhibited by polyphenols (EGCG, resveratrol). The bioavailability increased 150-200% with nano formulations, which enhanced therapeutic potency. Metabolites of plants have direct antiviral and host immunomodulatory properties. Nevertheless, a lack of clinical validation and inconsistency in the standardization of phytochemicals are critical issues. Plant-based metabolites are highly promising, multi-target antiviral leads. Nanotechnology, together with standardized pharmacological assessment and enhanced regulatory validation, has the potential to hasten clinical translation of plant metabolites into safe, broad-spectrum antiviral therapies.

Increasing intrinsic protein disorder improves CD8+ immunogenicity for a SARS CoV2 vaccine candidate antigen.

A novel oral vaccine strategy utilizing a plasmid with CAG promoter-driven alphavirus RdRp for Co-expression of NA epitope and adjuvant in attenuated Salmonella boosts immune responses against H9N2 in mice.

Kiwifruit (Actinidia spp.) has long been appreciated for its desirable flavor and high nutritional value. In this study, leaf samples with chlorotic mottling symptoms were collected from kiwifruit plants in Hanzhong, Shaanxi Province of China. Using high-throughput sequencing, a novel totivirus tentatively designated Actinidia totivirus 1 (AcToV1) was identified. Its complete genome sequence was determined using RT-PCR and RACE techniques. The genome of AcToV1 is 5,060 nucleotides in length and contains two open reading frames (ORFs), encoding a coat protein (CP) of 793 amino acids and an RNA-dependent RNA polymerase (RdRp) of 841 amino acids. It also features a typical slippery heptanucleotide motif, a conserved element characteristic of the genus Totivirus. AcToV1 shares the highest genome-wide nucleotide sequence identity of 57.6% with Panax notoginseng virus A (PnVA; GenBank accession NO. KT388111). Its CP and RdRp also exhibit the highest amino acid sequence identities to PnVA, at 44.0% and 56.7%, respectively. Phylogenetic analysis further supported that AcToV1 clusters closely with PnVA and Hubei toti-like virus 2 (HTLV2). Therefore, AcToV1 be considered a new species of the genus Totivirus.

Tick-borne encephalitis virus (TBEV) causes severe neurological disease. However, whether host restricts its replication and the underlying mechanisms remain incompletely delineated. Here, we identify the E3 ubiquitin ligase RNF138 as an intrinsic restriction factor targeting the viral RNA-dependent RNA polymerase (RdRp) NS5. Mechanistically, RNF138 directly interacts with the RdRp domain of NS5 through its ubiquitin-interacting motif, catalyzes K48-linked polyubiquitination and induces degradation of TBEV NS5. Ubiquitin-remnant profiling and mutational analyses identify K372, K462, and K470 within NS5 as the ubiquitination sites, and mutating these residues leads to NS5 resistant to RNF138-induced degradation. Functionally, RNF138 suppresses the replication of TBEV as well as Zika virus, a representative mosquito-borne flavivirus, through recognizing and degrading NS5 proteins. Ectopic RNF138 reduces viral RNA levels in the brain and peripheral tissues of TBEV-infected mice, mitigates neuroinflammatory responses, and improves survival. Notably, the antiviral activity is conserved among several mammalian RNF138 orthologs but absent in the arthropod homologs, highlighting a host-specific antiviral adaptation. Together, these findings reveal RNF138-mediated ubiquitination and degradation of NS5 as a mechanism of intrinsic defense against TBEV and provide a framework for exploring targeted destabilization of conserved flaviviral replicases.

A universal RdRp-targeted primer set for broad, non-specific detection of Quinvirinae and other Betaflexiviridae infecting stone fruits.

Orthobunyaviruses, comprising more than 130 species, constitute a significant threat to human and animal health, yet no effective broad-spectrum treatments or vaccines are currently available. Here, we initially identified an evolutionarily constrained lysine residue, highly conserved across all 22 Orthobunyavirus serogroups, within the α30 helix of the RNA-dependent RNA polymerase (RdRp). Functional interrogation of this site by charge-reversal substitution (K-to-E) substantially impaired polymerase activity and led to marked attenuation of Ebinur Lake virus (EBIV), as evidenced by impaired replication, attenuated cytopathogenicity, and reduced virulence in mice. The similar attenuation observed in Bunyamwera virus (BUNV), the prototype virus of the genus, and Oya virus (OYAV), a member of the Simbu serogroup, demonstrates that this residue fulfills a conserved functional role. Biochemical and cellular analyses further revealed a graded, charge-dependent mutational effect, indicating that attenuation arises predominantly from disruption of the essential interaction between RdRp and the 3' terminus of viral genomic RNA (3' vRNA). Structural modeling and mutagenesis further suggest that the K-to-E substitution creates a new hydrogen bond that may also contribute to viral attenuation. Together, these findings establish the conserved lysine as a critical regulator of RdRp function and offer a rational strategy for broad-spectrum attenuation of orthobunyaviruses.

Curcumin-based nanomaterials for dual-target inhibition of SARS-CoV-2 omicron main protease and HMPV RNA polymerase: A computational study.

Crimean-Congo Haemorrhagic Fever Virus (CCHFV) is found across Africa, Asia, and the Middle East where it can cause Haemorrhagic outbreaks with high case fatality rates. Central to the viral life cycle is the viral L-protein, a crucial and multifunctional protein which both transcribes and replicates the viral genome. Here, we present the cryoEM structures of an RNA free and a 5' promoter bound complex, describing the core catalytic RNA-dependent RNA polymerase (RdRp). We observe an RdRp that is substantially larger than related L-proteins and contains domain insertions unique to the nairovirus family. The 5' RNA promoter is found in a tight RNA hairpin stabilised by a single base pair, with 5' binding triggering the closure of protein over the RNA. Functional analysis of the endonuclease and RdRp activities reveals an enzyme which is capable of both activities and demonstrate RdRp inhibition by known antiviral nucleosides. These data advance our understanding of the molecular mechanisms behind genome replication and transcription, that will help inform future antiviral development.

Nucleotide analogues (NAs) have been successfully used for the treatment of various RNA virus infections by selectively targeting the viral RNA-dependent RNA polymerase (RdRp) for incorporation into the viral genome. However two major families of human-infecting RNA viruses, Coronaviridae (CoV) and Arenaviridae, encode exonuclease domains that may recognize and remove incorporated NAs, thus providing natural resistance against some of these drugs. Both polymerization and excision reactions are mechanistically centered on the nucleotide α-phosphate, enabling the potential for sequential inhibition of both RNA synthesis and repair. Here, we provide structural evidence of inversion of configuration at the phosphorus center during polymerization, demonstrating that the SARS-CoV-2 RdRp proceeds through an SN2 mechanism. A 2.39 Å resolution cryo-EM structure of a ternary replication complex bound to RNA and an α-thio-modified NTP shows that incorporation of the preferred SP isomer at the 3' end of the RNA yields a phosphorothioate linkage in the RP configuration. This RP-phosphorothioate RNA product shows reduced cleavage by both the SARS-CoV-2 and three arenavirus RNA exonucleases, revealing a stereochemical preference opposite to that of structurally related DNA exonucleases. This observation contradicts the prevailing assumption that sulfur substitution at the metal-coordinating oxygen universally blocks catalysis. Instead, RNA exonuclease stereoselectivity appears to be shaped not only by metal-sulfur interactions but also by the geometry of nucleophile activation. These findings provide mechanistic insights into phosphoryl transfer in viral polymerases and exonucleases and highlight opportunities to counteract intrinsic nuclease-mediated resistance against antiviral nucleotide analogues.