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Herpes simplex virus type 1 (HSV-1) poses a persistent public health challenge, particularly due to the emergence of drug-resistant strains and the limited efficacy of current monotherapies. Through an unbiased multi-omic approach, we identify a previously lesser-known viral strategy in which HSV-1 hijacks cyclin-dependent kinase (CDK) signaling to disrupt host cell cycle and translational control, specifically via the eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1). Targeted knockdown of CDKs confirmed their critical role in mediating 4EBP1 dephosphorylation during infection. Mechanistic evaluation of BX795, a previously known modulator of the 4EBP1 pathway, revealed an alternative route of translational repression mediated through CDKs. To further support this conclusion, we demonstrated that a distinct small-molecule CDK inhibitor, GW8510, exhibits potent antiviral activity against HSV-1 and functions as a true mechanistic analog of BX795. Together, these findings uncover a previously unrecognized CDK-4EBP1 regulatory axis exploited by HSV-1 and identify GW8510 as a promising candidate for host-directed antiviral intervention.IMPORTANCEHerpes simplex virus type 1 remains a major clinical burden, and resistance to existing therapies underscores the need for alternative strategies. This study reveals a mechanism by which HSV-1 regulates host cell cycle and translation control through cyclin-dependent kinase signaling and the 4E-binding protein 1 pathway. By revealing that pharmacological inhibition of this pathway suppresses viral replication, we identify a host-directed therapeutic approach that circumvents challenges associated with viral resistance to the current drugs. The demonstration of potent antiviral activity by GW8510, a small-molecule cyclin-dependent kinase inhibitor, establishes a promising foundation for translational development and highlights the potential of targeting host regulatory networks to combat viral infection.

Multiple genes are involved in the pathogenicity of influenza A virus. Our previous study reported two naturally occurring amino acid mutations in the polymerase acidic (PA) protein as crucial determinants of the virulence of Eurasian avian-like H1N1 (EA H1N1) influenza viruses. PA-X, an accessory protein encoded by the PA gene, is thought to play a role in viral pathogenicity and regulation of host immune response, but its specific function remains unclear. In this study, we found that two genetically similar EA H1N1 influenza viruses, A/swine/Liaoning/FX38/2017 (FX38) and A/swine/Liaoning/SY72/2018 (SY72), induced significantly different suppression levels of host protein synthesis. The difference in host shutoff activity induced by PA-X protein was the key factor affecting the inhibition of host gene expression. Loss of PA-X expression significantly reduced its host shutoff activity, thereby enhancing host antiviral immune response. PA-X deficiency had no apparent effect on polymerase activity or replication capacity. We pinpointed a single residue 122V involved in the ability of PA-X to inhibit host gene expression and thereby modulate the host antiviral response. Notably, PA-X 122V was highly conserved among multiple subtypes of influenza A viruses and vital for maintaining the inhibitory effects on the host protein synthesis. Together, these findings demonstrate that the PA-X protein plays a major role in the suppression of host protein synthesis during influenza virus infection and elucidate the molecular mechanism by which the amino acid residue 122V in PA-X facilitates its suppression effects on host innate immune responses.

Importance: PA gene, encoding PA protein and several accessory proteins including PA-X, PA-N155, and PA-N182, is a key factor determining the pathogenicity of influenza A virus. In this study, we found that PA-X is crucial for suppression of host protein synthesis during viral infection. Loss of PA-X expression significantly reduced its host shutoff activity, thereby enhancing host antiviral immune responses. Furthermore, we pinpointed a crucial amino acid, 122V, involved in the host shutoff activity of PA-X and found that 122V is highly conserved among multiple subtypes of influenza A viruses. These findings deepen our understanding of the mechanisms by which PA-X modulates viral pathogenesis and the host immune response.

The MarR-family regulator MhqR of Staphylococcus aureus (SaMhqR) was previously characterized as a quinone-sensing repressor of the mhqRED operon. Here, we solved the crystal structures of apo-SaMhqR and the 2-methylbenzoquinone (MBQ)-bound SaMhqR complex. AlphaFold3 modeling was used to predict the structure of SaMhqR in complex with its operator DNA. In the DNA-bound SaMhqR state, S65 and S66 of an allosteric α3-α4 loop adopted a helically wound conformation to elongate helix α4 for optimal DNA binding. Key residues for MBQ interaction were identified as F11, F39, E43, and H111, forming the MBQ-binding pocket. MBQ binding prevented the formation of the extended helix α4 in the allosteric loop, leading to steric clashes with the DNA. Molecular dynamics (MD) simulations revealed an increased intrinsic dynamics within the allosteric loop and the β1/β2-wing regions after MBQ binding to prevent DNA binding. Using mutational analyses, we validated that F11, F39, and H111 are required for quinone sensing in vivo, whereas S65 and S66 of the allosteric loop and D88, K89, V91, and Y92 of the β1/β2-wing are essential for DNA binding in vitro and in vivo. In conclusion, our structure-guided modeling and mutational analyses identified a quinone-binding pocket in SaMhqR and the mechanism of SaMhqR inactivation, which involves local structural rearrangements of an allosteric loop and high intrinsic dynamics to prevent DNA interactions. Our results provide novel insights into the redox mechanism of the conserved SaMhqR repressor, which functions as an important determinant of quinone and antimicrobial resistance in S. aureus.IMPORTANCEStaphylococcus aureus is a major human pathogen that can cause life-threatening infections in humans. However, treatment options are limited due to the prevalence of antimicrobial-resistant isolates in the hospital and the community. The MarR-type repressor SaMhqR was described to control resistance toward quinones and quinone-like antimicrobials. However, the redox-regulatory mechanism of SaMhqR by quinones was unknown. In this work, we explored the DNA-binding and quinone-sensing mechanism of SaMhqR and identified a quinone-binding pocket and an allosteric loop, which facilitates DNA binding activity via a helical wound conformation and adapts an unstructured coiled conformation upon quinone binding to inhibit DNA binding. A similar mechanism has been recently discovered for the regulation of uric acid resistance by UrtR family repressors (W. S. Song, D. U. Ki , H. Y. Cho, O. H. Kwon, H. Cho, S. I. Yoon, Nucleic Acids Res 52:13192-13205, 2024, https://doi.org/10.1093/nar/gkae922). Our results contribute to a better understanding of antimicrobial resistance regulation, which may be exploited for future drug design to combat multidrug-resistant S. aureus.

Candida albicans causes severe mucosal and systemic infections, with hypha formation playing a key role in its virulence. Hyphal invasion via endocytosis is mediated predominantly through interactions between Als3p and the epidermal growth factor receptor (EGFR). Subsequent EGFR activation by candidalysin, a hyphal-secreted cytolytic peptide toxin encoded by the ECE1 gene, induces receptor signaling and immune responses. While EGFR ubiquitination critically regulates receptor trafficking and signaling, its involvement during C. albicans infection has remained unexplored. Here, we demonstrate that C. albicans induces EGFR ubiquitination, leading to altered trafficking and lysosomal degradation in an ECE1- and ALS3-dependent manner. This correlates with changes in EGFR ligand expression, adaptor recruitment, and protein ubiquitination in oral epithelial cells. In a mouse model of oropharyngeal candidiasis, wild-type C. albicans and ece1Δ/Δ and als3Δ/Δ mutant strains were found to differentially regulate Egfr expression, ubiquitin pathway-associated genes, and protein ubiquitination. Furthermore, conditional EGFR knockout was protective during infection. Together, our findings reveal that C. albicans infection modulates the host ubiquitin system, including direct effects on EGFR, highlighting a novel aspect of host-fungal interactions.IMPORTANCECandida albicans is a common fungal pathogen that causes both mucosal infections, such as thrush, and life-threatening systemic diseases. A key step in infection is the fungus invading epithelial tissues and activating the host epidermal growth factor receptor (EGFR). We discovered that C. albicans alters how EGFR is regulated by inducing its ubiquitination, a modification that leads to receptor degradation. This process depends on two major fungal virulence factors: the adhesin Als3p and Ece1p, the polypeptide that contains the candidalysin toxin. The fungus also broadly increases protein ubiquitination in oral epithelial cells. In a mouse model of oral infection, loss of EGFR in epithelial tissues reduced disease severity, suggesting that the receptor helps the fungus establish infection. These findings reveal a previously unrecognized strategy by which C. albicans manipulates protein ubiquitination and regulation in epithelial cells, offering new insights into fungal pathogenesis and potential therapeutic approaches that target host pathways.

The virus factories (VFs) of infectious bursal disease virus (IBDV) are biomolecular condensates formed through liquid-liquid phase separation (LLPS). A major component of the IBDV VF is the nonstructural protein VP3, but the molecular basis underlying VF formation remains poorly understood. Here, we demonstrate that VP3 was necessary but not sufficient for phase-separated biomolecular condensates to form. Using live-cell imaging of cells transfected with fluorescent reporter-tagged proteins, our data suggested that the minimal components required to form these structures were VP3, the viral polymerase (VP1), and viral RNA (vRNA). Furthermore, using protein modeling and molecular dynamics simulations, we determined that the 36 amino acid carboxy (C)-terminus of VP3 forms a highly dynamic intrinsically disordered region (IDR). When this was removed, puncta were significantly less numerous (P < 0.0001), smaller (P < 0.0001), and more irregular in shape than puncta formed in the presence of wt VP3, demonstrating that the VP3 C-terminal IDR promoted their formation. Moreover, by fluorescence recovery after photobleaching, the VP3ΔC puncta had a significantly reduced mobile fraction (0.29) as compared to full-length VP3 puncta (0.70) (P < 0.001), demonstrating that the VP3 C-terminal IDR modulated their physical properties. In summary, our data reveal that VP3 forms part of a higher-order complex with VP1, and likely vRNA, to drive LLPS and the formation of IBDV VFs, and that the VP3 C-terminus encodes an IDR that is essential for modulating the physical properties of the resultant structures.

Importance: Liquid-liquid phase separation (LLPS) is a phenomenon of growing interest in cell biology. It is a part of the replication cycles of diverse viruses, but our understanding of the molecular basis that underpins the mechanism of phase separation is incomplete. We previously demonstrated that the virus factories of the birnavirus IBDV, a major agricultural pathogen, are biomolecular condensates formed through LLPS. In this study, we discovered that VP3 was necessary but not sufficient for condensates to form, and the minimal components of these structures were VP3, VP1, and likely vRNA. We also discovered that the C-terminal 36 amino acid region of IBDV VP3 encoded a highly dynamic intrinsically disordered region that promoted the formation of the cytoplasmic puncta and modulated their physical properties. This work contributes to a more detailed understanding of birnavirus replication at the molecular level and to the study of LLPS as a phenomenon.

The atmosphere harbors a diverse and dynamic reservoir of microorganisms, yet their distribution in the atmosphere and response to environmental variation remains a subject of ongoing investigation. In this study, we compared airborne bacterial and fungal communities at subalpine forest (NWT) and steppe grassland (CPER) sites, over diel, vertical, and seasonal gradients. Air samples were collected at three heights over 4 months at NWT with concurrent sampling at CPER during two of those months. Fungal communities exhibited greater site-specific variability and sensitivity to environmental factors than bacterial communities, particularly at NWT, where vertical stratification and diel cycles significantly structured microbial diversity. In comparison, bacterial communities were temporally dynamic but showed weaker responses to local environmental conditions and minimal site-level differences. This may indicate broader dispersal and a ubiquitous set of bacterial taxa. Environmental drivers, such as atmospheric moisture and air pressure, strongly influenced microbial beta-diversity at NWT, while air temperature and wind speed impacted diversity at CPER, again highlighting ecosystem-specific responses. Despite compositional differences, a subset of shared bacterial and fungal ASVs was consistently detected across sites, with most shared ASVs detected at greater heights at NWT. This, along with wind patterns moving eastward from NWT toward CPER, indicates potential atmospheric transport between sites, with taxa dispersal being filtered by height. These results underscore the role of ecosystem structure, meteorological conditions, and air mass movement in shaping the aerobiome and suggest that airborne microbial communities are shaped by both local emission and long-range atmospheric transport processes.IMPORTANCEUnderstanding the drivers of airborne microbial community structure is essential for predicting microbial dispersal, ecosystem connectivity, and responses to environmental change. This study reveals that atmospheric fungal and bacterial communities are shaped by distinct ecological and environmental factors, with fungi exhibiting stronger site-specific responses and vertical stratification than bacteria. The contrasting patterns between subalpine forest and grassland ecosystems underscore how local conditions influence microbial diversity and transport potential. Importantly, the detection of shared taxa, especially at greater sampling heights, suggests that atmospheric transport may connect distant ecosystems and that certain taxa are ubiquitous. These findings highlight the complexity of the aerobiome and its sensitivity to spatial and temporal dynamics, providing new insights into microbial distribution and the role of the atmosphere in microbial exchange across landscapes.