Search Results (125)

Influenza viruses are a major global health threat because of their recurring seasonal burden and continuing pandemic potential. Central to the viral life cycle is the viral ribonucleoprotein complex (vRNP), the functional unit of the segmented genome, in which each negative-sense RNA segment is encapsidated by oligomeric nucleoprotein (NP) and bound at its termini by the polymerase complex (FluPol). Recent advances in structural biology have revealed high-resolution structures of FluPol in distinct conformations, NP-RNA helical assemblies, and intact vRNP architectures, providing a structural framework for understanding vRNP assembly, polymerase conformational switching, and RNA synthesis in the RNP context. By contrast, current models for vRNP trafficking and selective genome packaging still rely largely on virological, biochemical, and cell biological evidence, with only limited structural resolution. In this review, we synthesize current knowledge of vRNP assembly, transcription, replication, intracellular trafficking, and selective genome packaging, and discuss the major unresolved questions in each area as well as their implications for antiviral development. Full article

The Yezo virus (YEZV) is a recently discovered tick-borne orthonairovirus with pathogenic potential, causing acute febrile illness in humans. Viral nucleoproteins (N) play a key role in genome packaging, replication, and modulation of host immune responses, making their structural characterization essential for understanding viral pathogenesis and developing targeted countermeasures. However, the absence of structural data for YEZV proteins significantly hinders these efforts. This study presents the first solution structure of the YEZV N domain 1 (D1). A highly purified, soluble, tag-free recombinant YEZV N D1 was produced from the native sequence of the clinical YEZV isolate. The native-state conformation was resolved through an integrated approach combining size-exclusion chromatography coupled with small-angle X-ray scattering (SEC-SAXS), AlphaFold 3 structure prediction, and all-atom molecular dynamics simulations. The YEZV N D1 structure adopts a stable, predominantly α-helical globular fold that remains monomeric under near-physiological conditions. SEC-SAXS data show excellent agreement with computational models, revealing moderate conformational flexibility. The characterized recombinant YEZV N D1 and its first solution structure reported here providing essential insights into understanding of YEZV molecular architecture. These findings lay a foundation for rational serological assay development and structure-guided therapeutic design against this and other emerging orthonairoviruses. Full article

Paraspeckles are subnuclear ribonucleoprotein condensates that regulate host stress responses, including those triggered by viral infection. In vitro studies using non-neuronal cells have shown the involvement of specific paraspeckle components in facilitating the replication of certain viruses, including Herpes Simplex Virus 1 (HSV-1), but these processes have not been investigated in human neuronal cells, which represent a relevant target of the virus. We employed human neural precursor cells (NPCs), neurons, and brain organoids derived from hiPSCs to investigate the previously unexplored dynamics of paraspeckle components in HSV-1-infected human neuronal cells. Our results reveal cell-type-specific differences in the expression of paraspeckle genes in response to HSV-1 infection. Unlike other viruses, HSV-1 orchestrates a previously unreported redistribution of paraspeckle proteins, leading to their accumulation in viral replication compartments (VRCs). Importantly, the expression of the paraspeckle proteins NONO and SFPQ correlates with HSV-1 permissiveness in human neuronal cells and may be required to establish a nuclear environment favoring viral transcription/replication. This enhances our understanding of how stress-response pathways in cells can be exploited by viruses in a cell-type-specific manner. Full article

Approaches to delivering gene editing tools in the form of ribonucleoproteins may provide a safety advantage over the delivery of nucleic acids encoding ribonucleoproteins. Virus-based vectors are widely used as a delivery platform. However, the persistence of viral exogenous nucleic acids can cause increased genotoxicity. Virus-like particles (VLPs) do not contain an expression cassette and can act as a platform for the delivery of ready-made ribonucleoprotein complexes. The absence of nucleic acids in VLPs eliminates the risk of insertional mutagenesis compared to widely used lentiviruses or adeno-associated viruses. Therefore, we used VLPs to deliver the ribonucleoprotein complex MmCas12m–TadDE to disrupt the HIV-1 gag gene start codon. We detected VLP morphogenesis using electron microscopy. We confirmed the incorporation of MmCas12m–TadDE into VLPs. We achieved an editing efficiency of about 9% in some cases with minimal off-target effects, which confirms the prospect of using VLPs as a platform for delivering genomic editing tools. Full article

Background: Diabetic nephropathy (DN) is largely driven by transforming growth factor-β1 (TGF-β1)-mediated fibrosis. Clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) ribonucleoprotein (RNP) complexes offer precise gene disruption, yet effective non-viral delivery remains a challenge. This study developed cationic lipid-based hybrid nanostructured lipid carriers (NLCs) for intracellular delivery of TGFB1-targeting RNP as an early-stage platform for DN gene modulation. Methods: A single-guide RNA (sgRNA) targeting human TGFB1 was assembled with Cas9 protein (1:1 and 1:2 molar ratios). Hybrid NLCs comprising squalene, glyceryl trimyristate, and the cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) were formulated via optimized emulsification–sonication to achieve sub-100 nm particles. Physicochemical properties, including polydispersity index (PDI), were assessed via dynamic light scattering (DLS), while silencing efficacy in HEK293T cells was quantified using quantitative reverse transcription PCR (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA). Results: Optimized NLCs achieved hydrodynamic diameters of 65–99 nm (PDI < 0.5) with successful RNP complexation. The 1:2 Cas9:sgRNA formulation produced the strongest gene-editing response, reducing TGFB1 mRNA by 67% (p < 0.01) compared with 39% for the 1:1 ratio. This translated to a significant reduction in TGF-β1 protein (p < 0.05) within 24 h. Conclusions: DOTAP-based hybrid NLCs enable efficient delivery of CRISPR–Cas9 RNP and achieve significant suppression of TGFB1 expression at both transcriptional and protein levels. These findings establish a promising non-viral platform for upstream modulation of profibrotic signaling in DN and support further evaluation in kidney-derived cells and in vivo renal models. Full article

The SARS-CoV-2 nucleocapsid protein (Np) plays multifunctional roles in the viral life cycle. By interacting with host cellular proteins, Np regulates viral RNA transcription, replication, and immune evasion. It controls genome packaging and counteracts host RNA interference mediated antiviral responses through its RNA binding activity. Previous studies revealed a physical interaction between Np and DDX3X, a human DEAD-box RNA helicase that facilitates the replication of several viruses. This interaction enhances Np affinity for double-stranded RNA and inhibits DDX3X helicase activity. Since Np-RNA binding activity promotes ribonucleoprotein complex formation, targeting this interaction is a promising antiviral strategy. We generated truncated protein variants to define interaction regions between Np and DDX3X. Using AlphaFold modelling, we identified RecA2 as the key DDX3X domain involved in Np binding. Finally, to disrupt Np-RNA complex formation, we screened a small molecule library of putative binders of Np N-terminal region and identified two candidate inhibitors for further development. Full article

Background: Large-scale production of poultry viral vaccines increasingly requires robust suspension cell platforms. However, most avian cell lines, including DF-1, are strictly anchorage-dependent, limiting scalability. Aquaporin-1 (AQP1) regulates cell–cell adhesion and membrane dynamics, making it a potential target for engineering suspension growth. This study aimed to generate a stable DF-1 suspension cell line via AQP1 disruption and evaluate its potential for enhanced infectious bursal disease virus (IBDV) production. Methodology: DF-1 cells were engineered using a CRISPR/Cas9 ribonucleoprotein system to create a truncated AQP1 gene. DF-1/AQP1⁻ cells were assessed for morphology, tumorigenicity in nude mice, and genetic stability across 20 passages. Suspension growth, cell density, and viability were measured. Cells were infected with IBDV strain BJQ902, and viral titers were compared with wild-type DF-1 and monolayer DF-1/AQP1⁻ cells. Results: DF-1/AQP1⁻ cells maintained normal morphology, were non-tumorigenic, and retained stable AQP1 mutations. They grew as true suspension cultures without adaptation, reaching 4.0 × 10⁶ cells/mL with >95% viability. Suspension DF-1/AQP1⁻ cells cells produced significantly higher viral titers (9.0 log TCID₅₀/mL; 8.63 log EID₅₀/mL) than both monolayer DF-1/AQP1⁻ and wild-type DF-1 cells. Virus production time was shortened in suspension cultures. Conclusions: Targeted AQP1 disruption converts DF-1 cells into a stable, non-tumorigenic suspension cell line with markedly enhanced IBDV production, providing a scalable platform for next-generation avian vaccine manufacturing. Full article

Influenza A virus (IAV) poses a major threat to global public health, exerting immense pressure on human health and the economy. The IAV nucleoprotein (NP) is an ideal target for antiviral drug development. Through Mini-genome and Surface Plasmon Resonance assays, this study discovered and verified that mycophenolic acid methyl ester (MAE), a secondary metabolite produced by the marine algal-associated fungus Phaeosphaeria spartinae, can target the viral nucleoprotein to exert anti-IAV activity. Pull-down assays and immunofluorescence have revealed that MAE blocks the nuclear import of viral ribonucleoprotein complexes (vRNP) by interfering with the interaction between NP and IMP-α. It also affects the vRNP assembly process by regulating NP oligomerization and the interaction between NP and PB2. In addition, Sandwich ELISA and Electron Microscopy experiments showed that MAE can also inactivate viral particles to reduce the risk of infection. Comprehensive research results indicate that MAE exerts its effects by inhibiting the viral NP protein, which has laid an important foundation for the development of marine-derived NP-targeted drugs. Full article

The recent discovery of Orthonairovirus songlingense (SGLV) and Norwavirus beijiense (BJNV) in China has raised significant concern due to their potential to cause severe human disease. However, little is known about the structural features and function of their nucleoproteins, which play a key role in the viral life cycle. By combining small-angle X-ray scattering (SAXS) data and AlphaFold 3 simulations, we reconstructed the BJNV and SGLV nucleoprotein structures for the first time. The SGLV and BJNV nucleoproteins have structures that are broadly similar to those of Orthonairovirus haemorrhagiae (CCHFV) nucleoproteins despite low sequence similarity. Based on structural analysis, several residues located in the positively charged region of BJNV and SGLV nucleoproteins have been indicated to be important for viral RNA binding. A positively charged RNA-binding crevice runs along the interior of the SGLV and BJNV ribonucleoprotein complex (RNP), shielding the viral RNA. Despite the high structural similarity between SGLV and BJNV nucleoprotein monomers, their RNPs adopt distinct conformations. These findings provide important insights into the molecular mechanisms of viral genome packaging and replication in these emerging pathogens. Also, our work demonstrates that experimental SAXS data can validate and improve predicted AlphaFold 3 structures to reflect their solution structure and also provides the first low-resolution structures of the BJNV and SGLV nucleoproteins for the future development of POC tests, vaccines, and antiviral drugs. Full article

The virus-first theory presents a model in which viral lineages emerged before cells. This proposal aims to give the theory greater relevance by offering a plausible evolutionary framework that explains both (i) the origin of viruses from prebiotic chemistry and (ii) how viruses contributed to the emergence of cells. Here, we propose that viruses should be understood as a distinct class of ribonucleoprotein (RNP) systems, some of which evolved directly from the RNP-world. In our model, simple progenotes produced capsid-like particles through the evolution of a single gene encoding a self-assembling peptide. This allowed the formation of icosahedral shells around RNA genomes, as observed today in certain viral families whose capsids consist of ~60 identical subunits derived from a single gene product. These early capsids enabled mobility and protection, representing key intermediates toward biological complexity. Over time, some of those populations acquired additional peptides and evolved more elaborate architectures. Finally, the incorporation of lipid-binding domains in those capsid-like peptides allowed the formation of proteolipidic membranes akin to those found in modern cells. This model provides a gradualistic and logically coherent evolutionary path from the RNP-world to the emergence of cellular life, emphasizing the foundational role of viruses in early evolution. Full article

The pandemic of Coronavirus Disease 2019 has triggered a worldwide public health emergency. Its pathogen, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has developed multiple strategies for effectively evading the host immune defenses, including inhibition of interferon (IFN) signaling. Several viral proteins of SARS-CoV-2 are believed to interfere with IFN signaling. In this study, we found that the SARS-CoV-2 accessory protein ORF7a considerably impaired IFN-activated Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling via suppression of the nuclear translocation of IFN-stimulated gene factor 3 (ISGF3) and the activation of STAT2. ORF7a dampened STAT2 activation without altering the expression and phosphorylation of Janus kinases (JAKs). A co-immunoprecipitation (co-IP) assay was performed to gather ORF7a protein, but it failed to precipitate STAT2. Interestingly, mass spectrometry and immunoblotting analyses of the ORF7a co-IP product revealed that ORF7a interacted with an RNA-binding protein, heterogeneous nuclear ribonucleoprotein A2B1 (HNRNPA2B1), and HNRNPA2B1 was related to the inhibitory effect of ORF7a on STAT2 phosphorylation. Moreover, examination of ORF7a deletion constructs revealed that the C-terminal region of ORF7a (amino acids 96 to 122) is crucial for suppressing IFN-induced JAK/STAT signaling activation. In conclusion, we discovered that SARS-CoV-2 ORF7a antagonizes type I IFN-activated JAK/STAT signaling by interacting with HNRNPA2B1, and the C-terminal region of ORF7a is responsible for its inhibitory effect. Full article

Heterogeneous nuclear ribonucleoprotein H1 (hnRNPH1) is a multifunctional RNA-binding protein (RBP) that plays a central role in post-transcriptional regulation. Through its quasi-RNA recognition motifs and low-complexity domains, hnRNPH1 specifically binds guanine-rich RNA sequences, including G-quadruplex structures, to precisely modulate multiple aspects of RNA metabolism, such as alternative splicing, mRNA stability, translation, and subcellular localization. Accumulating evidence has implicated hnRNPH1 dysfunction in the pathogenesis of several human diseases. In cancer, hnRNPH1 often acts as a pro-tumorigenic factor, albeit in a context-dependent manner, influencing the alternative splicing of crucial oncogenes, mRNA stability, and tumor cell sensitivity to therapeutic agents. In the nervous system, hnRNPH1 is involved in neurodevelopment, neurodegenerative diseases, and drug addiction and plays an essential role in maintaining neuronal function and homeostasis. Furthermore, it exerts regulatory functions in reproductive system development and fertility and in non-neoplastic pathologies, including cardiovascular diseases, autoimmune disorders, and viral hepatitis. Given its pathophysiological significance, hnRNPH1 has emerged as a promising biomarker and therapeutic target. This review provides an overview of the structural basis and core molecular function of hnRNPH1. Its mechanisms of action and pathological significance in various diseases have also been detailed. Additionally, this review summarizes the current therapeutic strategies targeting hnRNPH1, discusses the associated challenges, outlines optimization approaches, and considers future research directions. Overall, this review aims to deepen our understanding of hnRNPH1 biology and inspire the development of novel diagnostic and therapeutic interventions. Full article

HIV-1 integrase (IN), an essential viral protein that catalyzes integration, also influences non-integration functions such as particle production and morphogenesis. The mechanism by which non-integration functions are mediated is not completely understood. Several factors influence these non-integration functions, including the ability of IN to bind to viral RNA. INI1 is an integrase-binding host factor that influences HIV-1 replication at multiple stages, including particle production and particle morphogenesis. IN mutants defective for binding to INI1 are also defective for particle morphogenesis, similar to RNA-binding-defective IN mutants. Studies have indicated that the highly conserved Repeat (Rpt) 1, the IN-binding domain of INI1, structurally mimics TAR RNA, and that Rpt1 and TAR RNA compete for binding to IN. Based on the RNA mimicry, we propose that INI1 may function as a “place-holder” for viral RNA to facilitate proper ribonucleoprotein complex formation required during the assembly and particle morphogenesis of the HIV-1 virus. These studies suggest that drugs that target IN/INI1 interaction may lead to dual inhibition of both IN/INI1 and IN/RNA interactions to curb HIV-1 replication. Full article

The transcription and replication of the genome of influenza A virus (IAV) take place in the nucleus of infected cells, which is catalyzed by the viral ribonucleoprotein (vRNP) complex. The nuclear import of the vRNP complex and its component proteins is essential for the efficient replication of IAV and is therefore prone to be targeted by host restriction factors. Herein, we found that host cellular protein keratin 6A (KRT6A) is a negative regulator of IAV replication because siRNA-mediated knockdown of KRT6A expression increased the growth titers of IAV, whereas exogenous overexpression of KRT6A reduced viral yields. The nuclear import of incoming vRNP complexes and newly synthesized nucleoprotein (NP) was significantly impaired when KRT6A was overexpressed. Further studies showed that KRT6A interacts with the four vRNP complex proteins—polymerase basic protein 1 (PB1), polymerase basic protein 2 (PB2), polymerase acidic protein (PA), and NP. Notably, the interaction between KRT6A and vRNP complex proteins had no effect on the nuclear import of PB2 or the PB1-PA heterodimer but impaired the interaction between NP and the nuclear import adaptor importin α3, thereby inhibiting the nuclear import of incoming vRNP complexes and newly synthesized NP. Moreover, KRT6A was further shown to suppress the assembly of the vRNP complex and consequently reduce viral polymerase activity. Together, our data uncover a novel role of KRT6A in counteracting the nuclear import and functions of the vRNP complex, thereby restricting the replication of IAV. Full article

Background/Objectives: N-Myristoyltransferase inhibitors (NMTi) represent a novel antiviral strategy against mammarenaviruses such as Lassa and Junin viruses. The Z matrix protein inhibits viral ribonucleoprotein (vRNP) activity in a dose-dependent manner. Here, we investigated whether Z-mediated vRNP inhibition depends on Z myristoylation or oligomerization. Methods: We used HEK293T cells transfected with wild-type (WT) or G2A-mutated Z constructs in LCMV minigenome (MG) assays. Cells were treated with the NMTi IMP-1088 and the proteasome inhibitor MG132. Z protein expression, vRNP activity, and VLP production were analyzed by immunofluorescence, western blotting, and colocalization analyses. Results: IMP-1088 treatment led to proteasome-mediated degradation of Z, reducing its inhibition of vRNP activity, which was restored by MG132. The non-myristoylated Z G2A mutant retained vRNP inhibitory activity but showed impaired oligomerization and budding capacity. These findings demonstrate that Z-mediated vRNP inhibition is independent of myristoylation and oligomerization. Conclusions: Z myristoylation and oligomerization are not required for its inhibitory vRNP activity. Targeting Z myristoylation with NMTi impairs virus assembly and budding without affecting Z-mediated inhibition of vRNP activity, supporting the development of NMTi as a promising broad-spectrum antiviral strategy against mammarenaviruses. Full article