The productive infection of influenza A virus (IAV) depends on host factors. However, the involvement of long non-coding RNAs (lncRNAs) in IAV infection remains largely uninvestigated. In this work, we have discovered a human lncRNA, named lncRNA-PAAN (PA-associated noncoding RNA) that enhances IAV replication. The level of lncRNA-PAAN increases upon infection of IAV, but not other viruses, nor interferon treatment, suggesting specific up-regulation of lncRNA-PAAN expression by IAV. Silencing lncRNA-PAAN significantly decreases IAV replication through impairing the activity of viral RNA-dependent RNA polymerase (RdRp). This function of lncRNA-PAAN is a result of its association with viral PA protein, a key component of IAV RNA polymerase complex. Consequently, depletion of lncRNA-PAAN prevents the formation of functional RdRp. Together, these results suggest that lncRNA-PAAN promotes the assembly of viral RNA polymerase, thus warranting efficient viral RNA synthesis. Elucidating the functions of lncRNAs in IAV infection is expected to advance our understanding of IAV pathogenesis and open new avenues to the development of novel anti-IAV therapeutics.

Enterovirus-A71 (EV-A71) is the second most common causative agent after coxsackievirus A16 of hand, foot, and mouth disease. The capsids of EV-A71 consist of 60 copies of each of the four viral structural proteins (VP1–VP4). VP1 is highly exposed and surface accessible, playing a central role in virus particle assembly, attachment, and entry. To gain insight into the role of highly conserved residues at positions 75, 78, and 88 in the capsid protein VP1 in these processes, an alanine-scanning analysis was performed using an infectious cDNA clone of EV-A71. Our study revealed that the substitutions of VP1-T75A, VP1-T78A, and VP1-G88A could affect the assembly of the virus capsid proteins, resulting in the production of abnormal virions with reduced infectivity. Specifically, the substitution of VP1-T75A affected the maturation cleavage of the VP0 precursor, leading to deficiencies in binding to receptor scavenger receptor class B2 (SCARB2), viral attachment, internalization, and even uncoating. For the mutants of T78A and G88A, a significant reduction in virion-associated genomic RNA was observed, suggesting that more noninfectious empty particles were produced during viral assembly. Interestingly, the VP1-T75A variant showed weak replication in cell cultures but demonstrated increased virulence in BALB/c neonatal mice, which might be due to the difference in viral receptors among mammalian species. Taken together, our data revealed the important role of the highly conserved residues T75, T78, and G88 in VP1 protein in the infectivity of EV-A71. Characterizing these novel determinants of EV-A71 virulence would contribute to rationally developing effective treatments and broadly protective vaccine candidates. IMPORTANCE: EV-A71 causes hand, foot, and mouth disease in children. In this study, we discovered three highly conserved residues at positions 75, 78, and 88 of the capsid protein VP1 as the potential virulence determinants of EV-A71, which can influence viral replication by regulating the assembly of EV-A71. Mechanistic studies revealed that VP1-T75A could affect the maturation cleavage of the VP0 precursor, resulting in deficiencies in binding to the receptor SCARB2, viral attachment, internalization, and even uncoating. For the mutants of T78A and G88A, more noninfectious empty particles were produced during viral assembly. The discovery of these novel determinants of EV-A71 virulence will promote the study of the pathogenesis of enteroviruses.

The effective antiviral agents that treat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are urgently needed around the world. The 3C-like protease (3CL^pro) of SARS-CoV-2 plays a pivotal role in virus replication; it also has become an important therapeutic target for the infection of SARS-CoV-2. In this work, we have identified Darunavir derivatives that inhibit the 3CL^pro through a high-throughput screening method based on a fluorescence resonance energy transfer (FRET) assay in vitro. We found that the compounds 29# and 50# containing polyphenol and caffeine derivatives as the P2 ligand, respectively, exhibited favorable anti-3CL^pro potency with EC50 values of 6.3 μM and 3.5 μM and were shown to bind to SARS-CoV-2 3CL^pro in vitro. Moreover, we analyzed the binding mode of the DRV in the 3CL^pro through molecular docking. Importantly, 29# and 50# exhibited a similar activity against the protease in Omicron variants. The inhibitory effect of compounds 29# and 50# on the SARS-CoV-2 3CL^pro warrants that they are worth being the template to design functionally improved inhibitors for the treatment of COVID-19.

Enterovirus A71 (EV-A71) causes hand-foot-mouth disease, which may progress to severe neurological and systemic complications in young children and infants. To deliver viral genomic RNA into the target cell for replication, the mature EV-A71 capsid particle undergoes drastic structural changes to become an expanded A particle, which is marked by the movement of the N-terminal region of viral protein VP1 from the interior to the capsid surface and the dislodgement of the pocket factor. It is not completely understood which amino acids support these key activities of VP1 during EV-A71 entry. To answer this question, we investigated the functions of amino acids that are highly conserved in VP1 across all genotypes of EV-A71 by performing reverse genetic mutagenesis and functional experiments. Among the 21 highly conserved amino acids, 20 affect the replication of EV-A71. Notably, 7 of the 21 highly conserved amino acids are located in the 71-amino-acid N-terminal region, and mutation of any of these seven amino acids is lethal to EV-A71. Further mechanistic studies showed that the most detrimental defect occurred at the step of viral capsid uncoating and viral RNA release during virus entry, which is supported by the results of structural analyses showing the disruption of complex hydrogen bonding and hydrophobic networking due to mutating these amino acids. Our data have thus unraveled the genetic determinants that govern the key function of the N-terminal region of VP1 during EV-A71 entry and viral RNA release. IMPORTANCE: Hand, foot, and mouth disease (HFMD) annually affects millions of children worldwide. EV-A71 is a major cause of HFMD in the Asia-Pacific region. Notably, EV-A71 infection can lead to severe neurological syndromes and even death in young children; however, no specific vaccines or drugs are available. Among picornavirus capsid proteins, VP1 is the most external, surface-accessible, and immunodominant protein. However, it is not completely understood which amino acids support these key activities of VP1 during EV-A71 entry. Here, we report that 7 of the 21 highly conserved amino acids within the 71-amino-acid N-terminal region of VP1, and mutation of any of these 7 amino acids is lethal to EV-A71 by disrupting capsid uncoating and RNA release during entry. Overall, our findings highlight that the highly conserved N-terminal region residues of VP1 play a crucial role in viral infectivity and may contribute to the development of broad-spectrum anti-EV-A71 vaccines and drugs.

Rising drug resistance undermines current anti-influenza virus therapies. Although targeting the hemagglutinin (HA)–sialic acid receptor interaction is a promising strategy, progress is impeded by the lack of subtype-independent screening models. Herein, we established a fluorescence-based cell high-throughput model using fluorescein isothiocyanate-conjugated Sambucus Nigra Lectin (FITC-SNA) as a stable HA surrogate and α-2,6-sialyltransferase (ST6GAL1)-overexpressing MDCK cells to mimic the HA–receptor interface. This platform was designed to serve as an efficient primary screening tool to rapidly filter large compound libraries for potential binders to the receptor-binding interface. Screening 10,000 compounds identified Obatoclax Mesylate and Ethylparaben as primary hits. Both exhibited broad-spectrum HA inhibition activity, validating the model’s capability to identify compounds interfering with viral attachment. Further cellular antiviral assays revealed cytotoxicity for both compounds, resulting in low selectivity indexes (SIs), indicating that while these molecules effectively target the interaction site, they require substantial structural optimization for therapeutic use. Molecular docking confirmed their binding to type A H1N1, H3N2, and B/Victoria HA proteins, while ADMET predictions highlighted specific structural optimization needs to mitigate toxicity. In conclusion, this subtype-independent, highly specific high-throughput screening (HTS) model provides an efficient and reliable platform for early-stage influenza drug discovery and lead compound development.

Rising drug resistance underscores the urgent need for novel anti-influenza strategies. Targeting the hemagglutinin (HA)–sialic acid receptor interaction offers a promising broad-spectrum approach, yet progress is impeded by the lack of screening models that report the receptor-recognition step in a subtype-independent manner. Herein, we established a robust cell-based ELISA platform for high-throughput screening (HTS) that operates on the subtype-independent receptor-recognition step. The model utilizes ST6GAL1-overexpressing MDCK cells to enrich human-type α2,6-sialic acid receptors and employs horseradish peroxidase (HRP)-conjugated Sambucus nigra lectin (SNA) as a functional, though not structural, surrogate for the receptor-recognition step, yielding a high Z’ factor of 0.78. Given that SNA is a lectin rather than HA, the assay cannot by itself distinguish HA-specific blockade from membrane-mediated or virucidal effects. Orthogonal validation is therefore required for every hit. Screening a library of 2600 compounds identified two hits, Hypericin and protoporphyrin IX (PPIX), which effectively blocked SNA–receptor recognition with IC₅₀ values of 18.39 ± 6.23 μM and 9.77 ± 3.05 μM, respectively. An orthogonal flow cytometry assay with FITC-conjugated SNA reproduced this inhibition on intact cells in a dose-dependent manner (IC₅₀ = 17.68 ± 9.54 μM for Hypericin and 25.31 ± 19.47 μM for PPIX), confirming that the inhibition is not an artifact of the colorimetric readout. Hemagglutination inhibition (HA) assays with distinct pre-incubation strategies indicated that both compounds act on the viral particle rather than on the host receptor. Surface plasmon resonance (SPR) and molecular docking studies further indicated direct, dose-dependent but low-affinity binding to HA proteins. Notably, cytopathic effect (CPE) assays demonstrated their potent antiviral efficacy across H1N1 and H3N2 subtypes, with PPIX exhibiting exceptionally potent activity against H3N2 (EC₅₀ = 0.50 ± 0.17 μM). Taken together, these data identify Hypericin and PPIX as viral-side attachment inhibitors with weak HA binding and multimodal antiviral activity. Because neither a virucidal nor a fusion-blocking control was included, the relative contribution of HA engagement to their overall antiviral effect remains to be established. In conclusion, this BSL-1-compatible ELISA platform, which probes the subtype-independent receptor-recognition step, provides an efficient tool for discovering broad-spectrum influenza entry inhibitors, and the identified hits offer novel pharmacological scaffolds for further structural optimization.