Search Results (269)

RNA viruses, such as SARS-CoV-2 and influenza, pose a persistent threat to global public health. Their high mutation rates undermine the effectiveness of conventional direct-acting antivirals (DAAs) and facilitate drug resistance. As obligate intracellular parasites, RNA viruses rely extensively on host cellular machinery and metabolic pathways throughout their life cycle. This dependency has prompted a strategic shift in antiviral research—from targeting the mutable virus to targeting relatively conserved host dependency factors (HDFs). In this review, we systematically analyze how RNA viruses exploit HDFs at each stage of infection: utilizing host receptors for entry; remodeling endomembrane systems to establish replication organelles; hijacking transcriptional, translational, and metabolic systems for genome replication and protein synthesis; and co-opting trafficking and budding machinery for assembly and egress. By comparing strategies across diverse RNA viruses, we highlight the broad-spectrum potential of HDF-targeting approaches, which offer a higher genetic barrier to resistance, providing a rational framework for developing host-targeting antiviral therapies. Full article

ADP-ribosylation factor (Arf) proteins are small GTPases that regulate intracellular membrane trafficking and actin remodeling through tightly controlled cycles of GTP binding and hydrolysis. Arf1, a central coordinator of Golgi and endosomal transport, and Arf6, which regulates plasma membranes and endosomal dynamics, have both been implicated in late stages of the HIV-1 life cycle. However, the mechanisms by which these GTPases support viral assembly and release remain incompletely defined. Here, we provide direct evidence that both Arf1 and Arf6 are required for efficient trafficking of the HIV-1 Gag polyprotein, assembly, and virion production. Perturbation of Arf1 function using either GTP-locked (Q71L) or GDP-locked (T31N) mutants significantly reduced virus release, impaired Gag association with membrane compartments, and prevented its accumulation at the plasma membrane. Manipulation of Arf1 cycling through the GTPase-activating protein AGAP1 further demonstrated that dynamic transitions between GTP- and GDP-bound states are essential for productive Gag trafficking. Similarly, expression of a constitutively active Arf6 mutant (Q67L) misrouted Gag to intracellular membranes and markedly suppressed virion release. Importantly, disruption of Arf1 or Arf6 activity did not affect the expression, surface levels, or intracellular distribution of the host restriction factor BST-2. Together, these findings identify Arf1- and Arf6-mediated trafficking pathways as critical host determinants of HIV-1 assembly and release and establish that their functions operate independently of BST-2 antagonism. Full article

The highly pathogenic avian influenza (HPAI) H5N1 virus poses a growing global health threat, particularly following its unprecedented spillover into dairy cattle and subsequent transmission to more than 1000 dairy farms in 18 states. This study investigates the host cell responses to distinct H5N1 strains (bovine- and mink-derived H5N1) in the presence and absence of raw milk across diverse mammalian cell lines (MDCK, MDBK, A549, Vero, MV1). Our findings reveal that the bovine-derived H5N1 strain exhibits more robust replication than the mink-derived H5N1 and demonstrates intra-host viral evolution with emerging amino acid substitutions detectable by deep sequencing. Although raw milk supplementation did not directly enhance viral replication in vitro, it significantly modulated host gene expression, often dampening key antiviral interferon-stimulated gene (ISG) responses and disrupting essential host cellular processes like intracellular trafficking and sialic acid biosynthesis. These host gene modulations are cell-type- and strain-specific, suggesting a complex interplay that may theoretically influence virus–host dynamics, though the biological significance of these in vitro observations requires validation through infectious virus assays and in vivo studies. This hypothesis-generating work provides preliminary insights into H5N1-milk interactions, highlighting the need for further mechanistic investigation to assess potential implications for viral transmission in dairy environments. Full article

Interleukin-10 (IL-10), a potent anti-inflammatory cytokine, plays a vital role in regulating immune responses across various infectious and inflammatory conditions. While IL-10 is essential for preventing excessive tissue damage and maintaining immune homeostasis (e.g., respiratory syncytial virus), its elevated levels could result in immunosuppression during viral infections, enabling viruses to evade host defenses (e.g., foot-and-mouth disease virus). This review aims to elucidate the mechanisms through which IL-10 mediates immunosuppression in viral infections and to explore the implications of these mechanisms for therapeutic intervention. The key scientific concepts outlined in this review include the mechanisms of IL-10 production and its varied impacts on the immune response during viral infections. Specifically, we discuss the multifaceted inhibitory effects of IL-10 on innate and adaptive immunity, including its implications for antigen presentation, T cells activation, pro-inflammatory cytokine production, immune cell differentiation, trafficking, apoptosis, and co-inhibitory expression related to T cells exhaustion. Finally, we discuss the therapeutic potential of targeting IL-10, such as monoclonal antibodies and small molecule inhibitors, and their potential to restore effective immune responses. By summarizing current knowledge on IL-10’s role in viral infections, this review offers a thorough insight into its immunosuppressive mechanisms and their therapeutic potential, paving the way for innovative treatment strategies in viral diseases. Full article

Extracellular vesicles (EVs) are rapidly gaining recognition as critical mediators of inter-cellular communication during viral infections. To contribute to fill the gap in knowledge regarding the role of EVs in adenovirus infection, we used human adenovirus type 4 of species Mastadenovirus exoticum (HAdV-E4), a prevalent respiratory and ocular pathogen, and characterized the cargo and biological properties of EVs released by HAdV-E4-infected A549 lung epithelial cells at a pre-lytic stage of infection. Using immunocapture-based isolation and multi-omics approaches, we found that infection profoundly alters the EV uploaded proteome and small non-coding RNA repertoire. Mass spectrometry identified 268 proteins unique to EVs purified from infected cells (AdV-EVs), with enrichment in pathways supporting vesicle trafficking and viral protein translation, and importantly also a few virus-encoded proteins. A small RNA transcriptome analysis showed differential uploading in AdV-EVs of various small non-coding RNAs, including snoRNAs, as well as the presence of virus associated RNAs I and II. Notably, AdV-EVs contained viral genomic DNA and could initiate productive infection upon delivery to naïve cells in the absence of detectable viral particles. Our data suggest that EVs released during the HAdV-E4 infection may serve as vehicles for non-lytic viral dissemination and highlight their possible role in intra-host dissemination Full article

The dengue virus (DENV) exploits host cell exosome pathways to disseminate and evade immunity. However, the host factors enabling this process remain poorly defined. Here, we demonstrate that DENV infection robustly induces expression of the short isoform of Reticulon 3 (RTN3S) in hepatic (Huh7) and monocytic cells, and that RTN3S is a critical driver of infectious exosome biogenesis. RTN3S physically associates with double-stranded viral RNA and the DENV non-structural protein 3 (NS3) in infected cells, indicating its integration into the viral replication complex. Loss of RTN3 markedly reduced exosome production and the exosomal export of viral RNA and proteins, demonstrating that RTN3S is required for efficient exosome-mediated viral release. Conversely, overexpression of full-length RTN3S dramatically increased the release of infectious virus-containing exosomes; truncation of the RTN3S C-terminal domain abolished this enhancement, confirming the essential role of the C-terminus in RTN3S’s pro-viral exosomal function. In DENV-infected monocytes, we observed a shift toward a CD16-positive intermediate phenotype, accompanied by the upregulation of genes involved in vesicle biogenesis and stress response. These infected monocytes also secreted higher levels of inflammatory cytokines. Similarly, monocytes from Dengue patients exhibited high RTN3 expression, which correlated with an expansion of intermediate (CD16⁺) subsets and enriched expression of vesicle trafficking machinery genes. These findings reveal a previously unrecognized mechanism by which DENV hijacks RTN3S to promote the formation of infectious exosomes, thereby facilitating viral dissemination and immune evasion. RTN3S thus represents a novel element of the Dengue pathogenesis and a potential target for host-directed antiviral strategies. Full article

Chimeric antigen receptor (CAR) T cell therapy has revolutionized the treatment of certain hematologic malignancies, yet its success in solid tumors has been limited by antigen heterogeneity, an immunosuppressive tumor microenvironment, and barriers to cell trafficking and persistence. To expand the reach of cellular immunotherapy, multiple immune cell types—γδ T cells, invariant NKT cells, virus-specific T cells, natural killer (ΝΚ) cells, and myeloid effectors such as macrophages and dendritic cells—are now being explored as alternative or complementary CAR platforms. Each lineage brings unique advantages, such as the innate cytotoxicity and safety profile of CAR NK cells, the tissue infiltration and microenvironment-modulating capacity of CAR macrophages, or the MHC-independent recognition offered by γδ T cells. Recent advances in pharmacological strategies, synthetic biology, and artificial intelligence provide additional opportunities to overcome barriers and optimize CAR design and manufacturing scale-up. Here, we review the state of the art in engineering diverse immune cells for solid tumor therapy, highlight safety considerations across autologous, allogeneic, and in vivo CAR cell therapy approaches, and provide our perspective on which platforms might best address current unmet clinical needs. Collectively, these developments lay the foundation for next-generation strategies to achieve durable immunotherapy responses in solid tumors. Full article

The rabies virus (RABV) phosphoprotein (P protein) has multiple functions, including acting as the essential non-catalytic cofactor of the viral polymerase (L protein) for genome replication and transcription; the principal viral antagonist of the interferon (IFN)-mediated innate immune response; and the chaperone for the viral nucleoprotein (N protein). Although P protein is known to undergo phosphorylation by cellular kinases, the location and functions of the phosphorylation sites remains poorly defined. Here, we report the identification by mass-spectrometry (MS) of residues of P protein that are modified by phosphorylation in mammalian cells, including several novel sites. Analysis of P protein with phospho-mimetic and phospho-inhibitory mutations of three novel residues/clusters that were commonly identified by MS (Ser48, Ser183/187, Ser217/219/220) indicate that phosphorylation at each of these sites does not have a major influence on nuclear trafficking or antagonistic functions toward IFN signalling pathways. However, phosphorylation of Ser48 in the N-terminus of P protein impaired function in transcription/replication and in the formation of replication structures that contain complexes of P and N proteins, suggestive of altered interactions of these proteins. The crystal structure of P protein containing the S48E phospho-mimetic mutation indicates that Ser48 phosphorylation facilitates the binding of residues 41–52 of P protein into the RNA-binding groove of non-RNA-bound N protein (N⁰), primarily through the formation of a salt bridge with Arg434 of N protein. These data indicate that Ser48 modification regulates the cycling of P-N⁰ chaperone complexes that deliver N protein to RNA to enable transcription/replication, such that enhanced interaction due to S48E phospho-mimetic mutation reduces N protein delivery to the RNA, inhibiting subsequent transcription/replication processes. These data are, to our knowledge, the first to implicate phosphorylation of RABV P protein in conserved replication functions of the P gene. Full article

Nucleocytoplasmic trafficking is a highly regulated process that allows the cell to control the partitioning of proteins and nucleic acids between the cytosolic and nuclear compartments. The Ebola virus minor matrix protein VP24 (eVP24) hijacks this process by binding to a region on the NPI-1 subfamily of karyopherin alpha (KPNA) nuclear importers. This region overlaps with the activated transcription factor STAT1 binding site on KPNAs, preventing STAT1 nuclear localization and activation of antiviral gene transcription. However, the molecular interactions of eVP24-KPNA5 binding that lead to the nuclear localization of eVP24 remain poorly characterized. Here, we show that trafficking of eVP24 into the nucleus by KPNA5 requires simultaneous binding of cargo. We also describe the conformational dynamics of KPNA5 and interactions with eVP24 and cargo nuclear localization sequences (NLS) using biophysical approaches. Our results reveal that eVP24 binding to KPNA5 does not impact cargo NLS binding to KPNA5, indicating that simultaneous binding of both cellular cargo and eVP24 to KPNA5 is likely required for nuclear trafficking. Together, these results provide a biophysical basis for how Ebola virus VP24 protein gains access to the nucleus during Ebola virus infection. Full article

Ebola virus (EBOV) causes severe hemorrhagic fevers in humans, and effective countermeasures remain limited. The EBOV-encoded major matrix protein VP40 is essential for viral assembly, budding, and particle release, making it a promising target for antiviral drug development. However, no approved drugs currently target the viral particle formation process. In this study, we established a simple and highly sensitive screening system to evaluate VP40-mediated virus-like particle (VLP) formation under biosafety level −2 conditions. The system uses the HiBiT luminescence-based reporter fused to VP40, allowing for the detection of VP40 release. Our results demonstrate that the HiBiT sequence fused at the N-terminus [HiBiT-VP40 (N)] retains VP40′s ability to form VLPs, supporting its use as a functional reporter. Furthermore, we validated the system by assessing the role of Rab11-dependent trafficking in VP40-mediated budding and by evaluating the effect of nocodazole, a microtubule depolymerizer, on VLP release. This novel screening system provides a convenient and reliable platform for screening potential inhibitors targeting the late stages of EBOV infection, including viral particle formation and release. Additionally, its potential adaptability to other filoviruses suggests wide applicability in the discovery and development of additional novel therapeutic agents. Full article

SNX11, a sorting nexin protein localized on the endosomal membrane, is an important protein closely related to protein sorting and endosomal trafficking. Previously, through a genome-wide CRISPR screening, we identified SNX11 as a critical protein for the entry of Dabie bandavirus. SNX11 deletion significantly inhibits the replication of Dabie bandavirus. We further discovered that the loss of SNX11 alters endosomal pH, potentially affecting the release process of Dabie bandavirus from endosomes to the cytoplasm. However, the mechanism by which SNX11 modulates endosomal pH and whether SNX11 deletion similarly inhibits other viruses remain to be elucidated. This study reveals that SNX11 can interact with the V1 subunit of the endosomal proton pump V-ATPase, affecting the expression level of this subunit on the endosomal membrane and thereby disrupting the assembly of V-ATPase. Additionally, we found that SNX11 deletion significantly inhibits the replication of dengue virus, hantavirus, and influenza virus. These findings suggest that SNX11 may be a key protein in the process of viral infection and could serve as a broad-spectrum antiviral target. Full article

Henipaviruses are emerging zoonotic viruses that have caused deadly outbreaks in humans and livestock across several regions of the world. The fusion (F) protein of henipaviruses plays a critical role in viral entry into host cells and represents a key determinant of viral pathogenicity. This review provides a comprehensive analysis of current knowledge regarding trafficking, activation, as well as the role in particle assembly, of henipavirus F proteins. We discuss the unique characteristics of henipavirus F proteins compared to other paramyxovirus fusion proteins, with particular emphasis on their distinctive trafficking and activation mechanisms. Attention is also given to novel henipaviruses that have been detected in hosts other than bats, namely rodents and shrews. These viruses are sufficiently different that the International Committee on Taxonomy of Viruses has proposed a new genus for them, the Parahenipaviruses. We discuss how variations in F protein characteristics between Henipaviruses, Parahenipaviruses, and yet-unclassified henipa-like viruses might influence their trafficking and activation. Understanding these molecular mechanisms is crucial for developing effective therapeutic strategies against henipavirus infections and for predicting the emergence of novel henipavirus strains with pandemic potential. Full article

The ability of a virus to be propagated within a host cell is dependent on a multitude of dynamic virus–host interactions. Live-cell imaging is an invaluable approach in the study of virus replication cycles and virus–host interactions as it can allow for the direct visualisation of key events and interactions in real time. These details can provide unique insights into many aspects of viral infections including the cellular pathways that are exploited by viruses, the evasion of host immune defences, and viral pathogenesis. This review summarises the live-cell fluorescence imaging approaches that have been developed and applied to study Flaviviridae virus family members that are responsible for significant public health burdens and outbreaks which, in many instances, are increasing in frequency and severity. We discuss how these approaches have expanded our understanding of fundamental stages of viral replication cycles by enabling the direct visualisation of the localisation, trafficking, and interactions of virus particles, proteins, and genomes at distinct stages. The strategies that can be employed to enhance the biological relevance of live-cell fluorescence imaging acquisitions are discussed, along with how live-cell imaging approaches can be further developed to increase resolution, enable multi-colour imaging, and support the long-term visualisation of multiple stages of a viral replication cycle. Full article

Hendra virus (HeV) is a highly pathogenic member of the Henipavirus genus (family Paramyxoviridae, order Mononegavirales), for which all basic replication processes are located in the cytoplasm. The HeV matrix (M) protein plays essential roles in viral assembly and budding at the plasma membrane, but also undergoes dynamic nuclear and nucleolar trafficking, accumulating in nucleoli early in infection, before relocalising to the plasma membrane. We previously showed that M targets sub-nucleolar compartments—the fibrillar centre (FC) and dense fibrillar component (DFC)—to modulate rRNA biogenesis by mimicking a process occurring during a nucleolar DNA-damage response (DDR). Here, we show that M protein sub-nucleolar localisation is regulated by ubiquitination, which controls its redistribution between the FC-DFC and granular component (GC). The mutagenesis of a conserved lysine (K258) reported to undergo ubiquitination, combined with the pharmacological modulation of ubiquitination, indicated that a positive charge at K258 is required for M localisation to the FC-DFC, while ubiquitination regulates subsequent egress from the FC-DFC to the GC. M proteins from multiple Henipaviruses exhibited similar ubiquitin-dependent sub-nucleolar trafficking, indicating a conserved mechanism. These findings reveal a novel mechanism regulating viral protein transport between phase-separated sub-nucleolar compartments and highlight ubiquitination as a key modulator of intra-nucleolar trafficking. Full article

Measles, hepatitis C, and COVID-19 are significant human diseases caused by RNA viruses. While vaccines exist to prevent infections, there are a small number of currently available therapeutic agents that can effectively treat these diseases after infection occurs. This study explores a new therapeutic strategy using a small molecule designated polyprenyl immunostimulant (PI) to increase innate immune responses and combat viral infections. Using a multi-disciplinary approach, this study quantifies the effects of PI in mice and THP-1 cells using flow cytometry to identify immune phenotypic markers and mass spectroscopy to monitor the metabolomic profiles of immune cells perturbed by PI treatment. The metabolomic studies identified that sphinganine and ceramide, which are precursors of sphingosine-1-phosphate (S1P), were the common metabolites upregulated in THP-1 and mice blood. Sphingosine-1-phosphate can mediate the trafficking of T cells, whereas ceramide can signal the activation and proliferation of T cells, thereby modulating the mammalian host’s immunity. To demonstrate proof-of-principle, a case study was conducted to examine the benefit of administering PI to improve the outcomes of a feline co-infected with two distinct RNA viruses—feline leukemia virus and feline infectious peritonitis virus. Both viruses produce deadly symptoms that closely resemble RNA viruses that infect humans. The results identify quantifiable cellular and metabolic markers arising from PI treatment that can be used to establish a platform measuring the efficacy of PI in modulating the innate immune system. Full article