Salmonella enterica serovar Typhimurium (S. Typhimurium) is a Gram-negative enterobacteria of public health concern. The lipopolysaccharide (LPS) leaflet of the Gram-negative outer membrane (OM) has a defensive role and modulates the interactions between the bacterial cell and the microenvironment. Due to the increasing number of antimicrobial-resistant bacteria, interest in alternative treatments, such as phage therapy, has grown. Thus, understanding the mechanisms of phage–host interaction become increasingly important. The bacteriophages Lederbergvirus P22 (P22) and Nonanavirus nv9NA (9NA) infect S. Typhimurium after binding and depolymerizing the O-antigen portion of LPS¹. In vitro studies have shown that a complete LPS structure (lipid A, core, and O-antigen) is essential for P22 and 9NA’s DNA release^2,3. However, the dynamics of the early steps that precede infection, in which phages locate, recognize, and cleave the LPS O-antigen before DNA entry, remain largely unresolved. To address this, we developed LPS-enriched supported lipid bilayers (LPS-SLBs) as model membranes that mimic the S. Typhimurium OM, showcasing their usability as versatile biomimetic platforms for tracking microbial virus interactions. In addition, we investigated phage–surface interactions by tracking single 9NA particles on distinct LPS-SLB surfaces using total internal reflection fluorescence (TIRF) microscopy. This allowed us to monitor phage diffusion on the model membrane surface and characterize the dynamic events leading to the start of the infection. Our results provide mechanistic insight into how the OM composition of Gram-negative hosts guides the efficiency of bacteriophage infection.

References

1. Broeker, N. K. & Barbirz, S. Mol. Microbiol. 105, 353–357 (2017).
2. Andres, D. et al. Mol. Microbiol. 83, 1244–1253 (2012).
3. Stephan, M. S. et al. Preprint at https://doi.org/10.1101/2024.08.19.608551 (2024).

Introduction: Zika fever is a disease caused by Orthoflavivirus zikaense (Zika virus, ZIKV), mainly transmitted by mosquitoes of the Aedes genus. The symptoms are classified as dengue-like, however, more severe cases can present neurological disorders, such as Guillain-Barré syndrome and microcephaly in newborns of infected pregnant women. ZIKV belongs to the family Flaviviridae that is characterized by viruses with a positive sense single stranded RNA. However, no vaccines or antiviral drugs are currently available against ZIKV, making the search for compounds with antiviral activity essential. Objective: Evaluate the antiviral activity of a Schiff base derived from amantadine coordinated to cobalt(II) on ZIKV replication. Methods: Cell viability and antiviral assays were conducted to determine the 50% effective concentration (EC₅₀), 50% cytotoxic concentration (CC₅₀), and selectivity index (SI = CC₅₀/EC₅₀). Immunofluorescence assays were used to measure infection levels. Vero E6 cells were infected with the ZIKV_PE243 strain at a multiplicity of infection (MOI) of 0.01. Results: The cobalt–Schiff base complex (Co-atdSali) reduced ZIKV infection by more than 90%, while the starting Co(II) salt alone and the free Schiff base (atdSali) used in the synthesis of Co-atdSali reduced infection by 44% and 8%, respectively. The complex also showed strong inhibition of ZIKV replication in the dose–response assay, with a selectivity index (SI) of 14.7, compared with 3.9 for Co(II) salt and 1.6 for atdSali. Conclusion: The Co-atdSali complex exhibits enhanced antiviral activity compared to its free ligands, indicating the potential of metal coordination as a promising approach for developing new antiviral drug candidates against ZIKV.

Following the emergence of SARS-CoV-2 in humans, the scientific community has ramped up its efforts to better understand the biology, ecology and spillover risk of SARS-CoV-2-related viruses (SC2r-CoVs), primarily circulating in horseshoe bats. Here, we present a holistic approach to going from field sampling to experimental, phylogenetic, and ecological characterization of novel SC2r-CoVs. We sampled acuminate horseshoe bats from an artificial cave in Thailand and identified two SC2r-CoV lineages co-circulating in the same population. Our sampling identifies the first co-infection of an ACE2- and a non-ACE2-using SC2r-CoV, raising important implications about the interactions between related coronaviruses that utilize different entry receptors. Despite the new ACE2-using virus’s ability to bind to human ACE2, it exhibits reduced fusogenicity and replication in vitro, and lower pathogenicity and transmissibility in hamsters compared to SARS-CoV-2, highlighting the latter’s key biological differences to its bat-infecting relatives. Finally, we implemented a state-of-the-art phylogeographic approach to trace the movement of the bat SC2r-CoVs presented here across their evolutionary history. We show that some of their genomic segments recently moved from Laos to Thailand, reinforcing the notion that SC2r-CoVs continuously travel across their host ranges in Southeast Asia. The multi-faceted approach we employ here provides comprehensive insights into the biology and ecology of the viruses closest to SARS-CoV-2 and paves the way for effectively assessing the spillover potential of animal viruses.

Nucleproteins are essential for multiple parts of the virus life cycle, including genome compaction for post-entry shielding of the viral genome from host pattern recognition receptors and for packaging during the assembly of new virions. Due to their vital role in infection, nucleoprotein sequences are also usually well conserved within viral families. Such factors make nucleoproteins an ideal antiviral target. We therefore sought to selectively target and degrade the nucleoproteins of different viruses through various synthetic restriction approaches as a proof-of-principal for an antiviral strategy. One of these approaches utilises TRIM21, an intracellular antibody receptor known to neutralise adenovirus (AdV) by clustering around antibody-coated AdV capsids and targeting the virus for degradation. We fused the RING E3 ligase domain of TRIM21 either to the nucleoprotein directly or to nanobodies that bind the nucleoprotein. We tested the ability of these constructs to restrict model RNA viruses including influenza A virus (IAV) and Hazara virus (HAZV) through the expression of these synthetic restriction factors in target cells and via electroporation-dependent neutralisation assays. RING-nanobodies, which bind IAV nucleoprotein, were able to potently restrict infection. Similarly, the expression of HAZV nucleoprotein fused directly to RING was also able to restrict subsequent HAZV infection. The current work aims to understand the underlying molecular mechanisms behind how these synthetic restriction factors function to prevent infection and whether they can be adapted to restrict other RNA viruses. Overall, the targeted degradation of viral nucleoproteins could serve as a novel therapeutic approach against a multitude of pathogenic viruses.

SARS-CoV-2 initiates infection through its spike (S) glycoprotein, which binds the angiotensin-converting enzyme 2 (ACE2) receptor on host cells. The S protein comprises two subunits: S1, containing the receptor-binding domain (RBD) that recognizes ACE2, and S2, which mediates membrane fusion. Following ACE2 binding, host proteases such as TMPRSS2 or cathepsins cleave the S protein, exposing the S2 fusion peptide and enabling viral–host membrane fusion. Consequently, SARS-CoV-2 enters cells either via a TMPRSS2-dependent cell-surface route or an endocytic route requiring cathepsin activation in acidic endosomes.

Recent genome-wide CRISPR screens identified Niemann-Pick C1 (NPC1) as an essential host factor for SARS-CoV-2 infection. Our compound screen revealed Tubeimosides as potent inhibitors of viral entry that specifically target NPC1. NPC1 is a late-endosomal membrane protein that, with NPC2, transports cholesterol but also serves as an intracellular receptor for filoviruses through its luminal domain C (NPC1-C). We found that the SARS-CoV-2 RBD similarly binds NPC1-C and that NPC1 is required for viral entry.

NPC1 knockout cells showed reduced viral entry, particularly in TMPRSS2-negative cells, confirming NPC1’s function in the endocytic, not the cholesterol-transport, pathway. NPC1 loss did not affect SARS-CoV-2–mediated cell–cell or virion–virion fusion, indicating its specific involvement in endosomal fusion. The Omicron variant, which favors endocytic entry, exhibited heightened dependence on NPC1. Live-cell imaging showed spike–NPC1 complexes localized to late endosomes/lysosomes, while biochemical assays revealed enhanced spike–NPC1 binding and diminished spike–ACE2 binding under acidic conditions. Artificial exposure of NPC1-C on the cell surface permitted spike interaction and fusion, further amplified at low pH.

Together, these findings demonstrate that NPC1-C directly mediates membrane fusion in late endosomes. SARS-CoV-2 thus undergoes a receptor switch—from ACE2 to NPC1—during endocytic entry.

The Chikungunya virus (CHIKV) is a positive-sense RNA alphavirus that circulates in nature by alternating replication between mammalian hosts and mosquito vectors, primarily Aedes aegypti and Aedes albopictus. Remarkably, while CHIKV infection causes significant disease in mammals, mosquitoes remain asymptomatic, reflecting the presence of powerful disease tolerance mechanisms within the vector. Previous studies have revealed that a key mechanism underlying this tolerance is the synthesis of viral DNA (vDNA) using the CHIKV RNA genome as a template. This conversion is mediated by endogenous retrotransposon-encoded reverse transcriptases (RTs) within the mosquito. Pharmacological inhibition of this process using classical reverse transcriptase inhibitors such as zidovudine (AZT) abrogates vDNA synthesis and impairs disease tolerance, leading to increased viral susceptibility. However, the molecular pathways governing vDNA biogenesis, as well as the distinct impacts of different classes of reverse transcriptase inhibitors in mosquitoes, remain poorly characterized.

To address these gaps, we investigated the mechanisms of vDNA synthesis both in vivo and in vitro. We mapped the reverse-transcribed regions of the CHIKV genome and characterized the kinetics of vDNA synthesis following infection. In parallel, we evaluated the comparative efficacy of five RT inhibitors—AZT, d4T, 3TC, nevirapine, and tenofovir—representing nucleoside analogs, nucleotide analogs, and allosteric inhibitors, in blocking vDNA synthesis in both mosquitoes and mosquito-derived cells. For each compound, we assessed cellular toxicity, viral loads, cell viability, and cytopathic effects, alongside quantification of vDNA inhibition. Finally, we performed transcriptomic analysis to dissect the broader impact of RT inhibition on mosquito antiviral immune mechanisms, cellular metabolism, and stress responses during CHIKV infection.

Our findings elucidate core molecular features of vDNA synthesis and demonstrate distinct inhibitor effects, advancing understanding of mosquito tolerance to viral disease and offering potential avenues for vector-targeted control strategies.

Dengue virus (DENV) is one of the most prevalent arboviruses worldwide, and the lack of specific antiviral treatments highlights the need to develop new therapeutic strategies based on natural compounds. This study evaluated the antiviral potential of Carica papaya leaf extracts against DENV replication in cell culture, considering the presence of bioactive metabolites previously reported in the literature, including flavonoids, alkaloids, terpenoids, and phenolic compounds. Extracts were obtained through maceration and ultrasound-assisted extraction using different solvent systems. The extraction yields varied significantly depending on the solvent employed, with the hexane extract showing the highest recovery, while methanol and ethanol also allowed an efficient extraction of bioactive fractions. Cytotoxicity assays in BHK-21 cells after 24, 48, and 72 hours of incubation revealed mean cytotoxic concentrations ≥1 mg/mL for all extracts, indicating low toxicity in cell culture. Antiviral assays performed through viral plaque quantification demonstrated that all extracts inhibited DENV replication, with the hexane extracts exhibiting the lowest mean inhibitory concentration values, indicating a stronger antiviral effect at lower concentrations. Overall, these findings suggest that Carica papaya leaf extracts, particularly those obtained with nonpolar solvents, contain metabolites with promising antiviral properties that have not been previously described in the literature, as most studies have focused on extracts obtained with polar solvents. The identification and characterization of these compounds represent an essential step toward the development of plant-derived antiviral agents targeting dengue virus.

HIV/SIV infection leads to eventual diminished proliferation and loss of functions of CD8+ T cells. Understanding the mechanisms involved in CD8+ T-cell dysfunction and how to reinvigorate it is critical in the strategy to cure HIV. It is known that N-glycosylation modulates T-cell functions in cancer and viral infections by increasing surface retention of exhaustion markers and by preventing antigen recognition at the immune synapse. Galectin-3 (Gal-3), a predominant lectin with a high affinity for glycoprotein, forms a lattice after binding with N-glycan, restricts the clustering of T cell receptors and co-receptors in the immune synapse, and impairs T-cell functions. Herein, we speculate that N-glycosylation modulated by Gal-3 and other involved pathways may negatively impact CD8 + T-cell function in HIV/SIV infection. In this study, eight Chinese rhesus macaques (cRM) were infected with SIVmac239M and treated with antiretroviral therapy daily for over 6 months. CD8+ T cells were isolated from PBMCs at pre-infection and SIV+ on ART time points. Expression of Gal-3 was evaluated by quantitative real-time PCR. N-glycan branching on the CD8+ T cells was analyzed by flow cytometry. Swainsonine, which blocks N-glycosylation by inhibiting a-mannosidase, was used to treat dysfunctional CD8+ T cells purified from PBMCs of ART-suppressed SIV-infected cRM ex vivo. CD8+ T-cell proliferation and cytokine production after swainsonine treatment were evaluated by flow cytometry. We found that, compared to pre-infection, increased Gal-3 in PBMCs and N-glycan branching on CD8+ T cells were observed in ART-suppressed cRM. N-glycosylation inhibited the proliferation of CD8+ T cells. The intervention on N-glycosylation by swainsonine successfully restored the proliferation and increased the cytokine production of CD8+ T cells isolated from ART-suppressed SIV-infected cRM. In conclusion, N-glycosylation contributes to CD8+ T-cell dysfunction during HIV/SIV infection. Therapeutic strategies targeting N-glycosylation may therefore restore this critical antiviral response and enhance viral control.

Kaposi’s sarcoma-associated herpesvirus (KSHV) is an oncogenic gammaherpesvirus with a biphasic life cycle. KSHV infection of oral epithelial cells supports lytic replication and transmission into B cells where viral genome heterochromatinization drives KSHV into latency. Epigenetic factors that counteract heterochromatin could thus be targets to block lytic infection. Here, we evaluated the role and regulation of host BRCA1 associated protein (BAP1) in KSHV’s life cycle. BAP1 deubiquitinates H2AK119Ub, a repressive mark abundant on latent KSHV genomes, but its role during infection is unknown. BAP1 can also function as a scaffold to recruit histone methyltransferase MLL3 complexed with histone demethylase UTX and acetyltransferase CBP to support host gene transcription via removal of H3K27me3 and H3K27Ac deposition, respectively. We hypothesized that BAP1 might support KSHV’s lytic cycle as an enzyme or a scaffold leading to KSHV euchromatinization. We found that siRNA-mediated depletion of BAP1 in telomerase-immortalized gingival keratinocytes (TIGKs) reduced viral gene expression and KSHV copy number at 24 hours post-infection (hpi). Inhibiting BAP1 enzymatic activity did not affect H2AK119Ub levels at lytic promoters nor lytic gene expression, while BAP1 depletion resulted in increased H3K27me3 and decreased H3K27Ac marks at lytic promoters. MLL3 depletion in TIGKs inhibited lytic infection, in support of a functional BAP1/MLL3 axis. Strikingly, immunofluorescence revealed that BAP1 highly accumulates during de novo KSHV infection in the nucleus of TIGKs expressing lytic proteins, but not in adjacent infected cells that lack lytic proteins. Conversely, depletion of BAP1 did not impair lytic infection by Herpes simplex virus (HSV-1) in TIGKs, indicating that the requirement for BAP1 is virus-specific. In summary, we propose that BAP1 may be co-opted specifically by KSHV and tethered to the KSHV genome during lytic infection to recruit MLL3 complexes to promote lytic infection.

Viral coinfections account for ~10-30% of all respiratory viral infections. They are particularly prevalent in children and are thought to play a critical role in influencing susceptibility, disease severity and transmission. In previous studies, we showed that coinfection of a human-derived respiratory cell line with laboratory-adapted strains of influenza A virus (IAV) and respiratory syncytial virus (RSV) generated hybrid viral particles (HVPs). These HVPs exhibited structural changes that affected both antigenicity and receptor tropism, suggesting an important role in virus fitness. To determine if HVPs could be formed under more physiologically relevant conditions, we infected A549s and differentiated human bronchial epithelial cells (hBECs) with a prototype strain of RSV A2 and a clinical isolate of IAV H3N2, as well as with clinical isolates of RSV and IAV H1N1. Using confocal microscopy, we observed that A549s coinfected by both virus combinations displayed filamentous structures resembling HVPs. In separate experiments, we confirmed the presence of coinfected cells in hBECs, suggesting opportunities for the formation of HVPs in the human respiratory epithelium. Further, IAV H1N1 replicated at higher levels in the presence of RSV in both systems, likely as a result of syncytia formation. Our findings suggest that HVPs can be formed during natural coinfections, with potential implications for virus pathogenesis due to increased IAV replication. Understanding the impact of viral coinfections and specifically how RSV might be responsible for the enhancement of infectivity of other respiratory viruses could help improve diagnosis, treatment, and infection control, especially in children, in whom coinfections are common.