Search Results (189)

Měnglà virus (MLAV) is a member of the genus Dianlovirus in the family Filoviridae, which also includes Ebola virus (EBOV) and Marburg virus (MARV). Whether MLAV poses a threat to human health is uncertain. However, the MLAV VP35 and VP40 proteins can impair IFNα/β gene expression and block IFNα/β-induced Jak-STAT signaling, respectively, suggesting the capacity to counteract human innate immune defenses. In this study, MLAV VP40 is demonstrated to impair the Sendai virus (SeV)-induced activation of the IFNβ promoter. Inhibition is independent of the MLAV VP40 PPPY late-domain motif that interacts with host proteins possessing WW-domains to promote viral budding. Similar IFNβ promoter inhibition was not detected for EBOV or MARV VP40. MLAV VP40 exhibited lesser capacity to inhibit TNFα activation of an NF-κB reporter gene. MLAV VP40 impaired IFNβ promoter activation by an over-expressed, constitutively active form of RIG-I and by the over-expressed IRF3 kinases TBK1 and IKKε. However, MLAV VP40 did not inhibit IFNβ promoter activation by constitutively active IRF3 5D. Consistent with these findings, MLAV VP40 inhibited SeV-induced IRF3 phosphorylation. Although IRF3 phosphorylation occurs in the cytoplasm, MLAV VP40 exhibits substantial nuclear localization, accumulating in foci in HeLa cell nuclei. In contrast, the VP40 of EBOV and MARV exhibited lower degrees of nuclear localization and did not accumulate in foci. MLAV VP40 interacts with importin alpha-1 (IMPα1), suggesting entry via the IMPα/IMPβ nuclear import pathway. Cumulatively, these data identify novel features that distinguish MLAV VP40 from its homologues in EBOV and MARV. Full article

We performed a diagnostic disease investigation on a wild smallmouth bass (Micropterus dolomieu) with skin ulcers that was collected from Lake Oahe, South Dakota, following reports from anglers of multiple fish with similar lesions. Gross and histologic lesions of ulcerative dermatitis, myositis, and lymphocytolysis within the spleen and kidneys were consistent with largemouth bass virus (LMBV) infection. LMBV was detected by conventional PCR in samples of a skin ulcer, and the complete genome sequence of the LMBV (99,184 bp) was determined from a virus isolate obtained from a homogenized skin sample. A maximum likelihood (ML) phylogenetic analysis based on the major capsid protein (MCP) gene alignment supported the LMBV isolate (LMBV-SD-2023) as a member of the species Ranavirus micropterus1, branching within the subclade of LMBV isolates recovered from North American largemouth (Micropterus salmoides) and smallmouth bass. This is the first detection of LMBV in wild smallmouth bass from South Dakota. The ultrastructure of the LMBV isolate exhibited the expected icosahedral shape of virions budding from cellular membranes. Viral nucleic acid in infected cells was visualized via in situ hybridization (ISH) within dermal granulomas, localized predominantly at the margin of epithelioid macrophages and central necrosis. Further sampling is needed to determine the geographic distribution, affected populations, and evolutionary relationship between isolates of LMBV. 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

Background/Objectives: Baculoviruses represent promising gene delivery vectors for mammalian systems, combining high safety profiles with substantial cargo capacity. While pseudotyping with vesicular stomatitis virus G-protein (VSV-G) enhances transduction efficiency, optimal expression strategies during the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) infection cycle remain unexplored. This study investigates how VSV-G expression timing affects pseudotype incorporation into budded virions (BVs) and subsequent transduction efficacy. Methods: Three recombinant AcMNPV constructs were generated, each expressing VSV-G under distinct baculoviral promoters (ie1, gp64, and p10) and GFP via a CMV promoter. VSV-G incorporation was verified by Western blot, while transduction efficiency was quantified in mammalian cell lines (fluorescence microscopy/flow cytometry) and rat hind limbs. Viral productivity was assessed through production kinetics and plaque assays. Results: All the pseudotyped viruses showed significantly enhanced transduction capacity versus controls, strongly correlating with VSV-G incorporation levels. The p10 promoter drove the highest VSV-G expression and transduction efficiency. Crucially, BV production yields and infectivity remained unaffected by VSV-G expression timing. The in vivo results mirrored the cell culture findings, with p10-driven constructs showing greater GFP expression at low doses (10⁴ virions). Conclusions: Strategic VSV-G expression via very late promoters (particularly p10) maximizes baculoviral transduction without compromising production yields. This study establishes a framework for optimizing pseudotyped BV systems, demonstrating that late-phase glycoprotein expression balances high mammalian transduction with preserved insect-cell productivity—a critical advancement for vaccine vector development. 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

Endocytosis is a specialized transport mechanism in which the cell membrane folds inward to enclose large molecules, fluids, or particles, forming vesicles that are transported within the cell. It plays a crucial role in nutrient uptake, immune responses, and cellular communication. However, many pathogens exploit the endocytic pathway to invade and survive within host cells, allowing them to evade the immune system and establish infection. Endocytosis can be classified as clathrin-mediated (CME) or clathrin-independent (CIE), based on the mechanism of vesicle formation. Unlike CME, which involves the formation of clathrin-coated vesicles that bud from the plasma membrane, CIE does not rely on clathrin-coated vesicles. Instead, other mechanisms facilitate membrane invagination and vesicle formation. CIE encompasses a variety of pathways, including caveolin-mediated, Arf6-dependent, and flotillin-dependent pathways. In this review, we discuss key features of CIE pathways, including cargo selection, vesicle formation, routes taken by internalized cargo, and the regulatory mechanisms governing CIE. Many viruses and bacteria hijack host cell CIE mechanisms to facilitate intracellular trafficking and persistence. We also revisit the exploitation of CIE by bacterial and viral pathogens, highlighting recent discoveries in entry mechanisms, intracellular fate, and host-pathogen interactions. Understanding how pathogens manipulate CIE in host cells can inform the development of novel antimicrobial and immunomodulatory interventions, offering new avenues for disease prevention and treatment. 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

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infections in infants, young children, and immunocompromised individuals. Currently, FDA-approved monoclonal antibody therapies are limited to infants and young children with severe RSV disease. As a result, there is an urgent need for comprehensive studies of RSV pathogenesis to support the development of new therapeutic strategies. Exchange proteins directly activated by cAMP (EPAC) have recently emerged as key regulators in various viral infections. Our previous work identified EPAC isoform 2 (EPAC2) as a critical factor in RSV replication and host innate immune responses. However, the molecular mechanisms underlying EPAC2’s role in RSV infection remain unclear. In this study, we investigated EPAC2-mediated RSV infection by identifying EPAC2-interacting proteins. Proteomics and immunoprecipitation analyses revealed that junction plakoglobin (JUP) interacts with EPAC2 in both mock- and RSV-infected cells, with this interaction notably enhanced during RSV infection. To determine JUP’s role in RSV infection, we compared viral replication in JUP-deficient and control cells. JUP downregulation significantly reduced the production of infectious RSV particles, likely by impairing viral budding and viral gene transcription. Moreover, our findings indicate that JUP is essential for an effective cellular immune response to RSV infection. Together, these results suggest that EPAC2 and JUP may cooperatively regulate RSV replication and dissemination. Full article

The structural integrity of viral envelopes is a critical determinant of infectivity for enveloped viruses, directly influencing vector stability, functional accuracy of surface-displayed epitopes, and preservation of native conformational states required for membrane protein studies. However, conventional purification methods often disrupt envelope integrity and cause envelope proteins to lose their activity. Here, we systematically compared discontinuous, continuous, and optimized continuous sucrose density gradient centrifugation protocols for purifying Autographa californica multiple nucleopolyhedrovirus (AcMNPV). Through cryo-EM, we demonstrated that our optimized continuous sucrose gradient protocol significantly increased the proportion of AcMNPV budded virions with intact envelopes from 36% to 81%, while preserving the metastable prefusion conformation of the fusion protein GP64. This advancement should prove useful for structural studies of viral envelope proteins and may enhance applications in gene therapy and vaccine development utilizing enveloped viruses. Full article

The endosomal sorting complexes required for transport (ESCRT) comprise a fundamental cellular machinery with remarkable versatility in membrane remodeling. It is multifunctional in the multivesicular body (MVB) biogenesis, exosome formation and secretion, virus budding, cytokinesis, plasma membrane repair, neuron pruning, and autophagy. ESCRT’s involvement in cellular mechanisms extends beyond basic membrane trafficking. By directly interacting with autophagy-related (ATG) proteins and facilitating autophagosome-lysosome fusion, ESCRT ensures cellular homeostasis. Dysregulation in ESCRT function has been implicated in cancer, neurodegenerative disorders, and infectious diseases, underscoring its critical role in numerous pathologies. Hepatitis B virus (HBV) is an enveloped virus that exploits ESCRT and autophagy pathways for viral replication, assembly, and secretion. This review synthesizes recent mechanistic insights into ESCRT’s multifaceted roles, particularly focusing on its interactions with autophagy formation and the HBV lifecycle. Full article

Groundnut bud necrosis orthotospovirus (GBNV), a tripartite single-stranded RNA virus, poses a significant threat to United States agriculture. GBNV is a quarantine pathogen, and its introduction could lead to severe damage to economically important crops, such as groundnuts, tomatoes, potatoes, peas, and soybeans. For the rapid and accurate detection of GBNV at points of entry, TaqMan reverse transcriptase–quantitative polymerase chain reaction (RT-qPCR) assays were developed and the results validated using conventional reverse transcriptase–polymerase chain reaction (RT-PCR) followed by Sanger sequencing. These assays target highly conserved regions of the nucleocapsid (NP) and movement (MP) proteins within the viral genome. Multiplex GBNV detection assays targeting the NP and MP genes, as well as an internal control plant gene, ACT11, showed efficiency rates between 90% and 100% and R² values of 0.98 to 0.99, indicating high accuracy and precision. Moreover, there was no significant difference in sensitivity between multiplex and singleplex assays, ensuring reliable detection across various plant tissues. This rapid, sensitive, and specific diagnostic assay will provide a valuable tool at ports of entry to prevent the entry of GBNV into the United States. Full article

Our study’s aim was to evaluate the clinicopathological profile of colorectal cancer (CRC) patients from North-East Romania in relation to the Kirsten rat sarcoma viral oncogene homolog (KRAS). We designed a retrospective study on 108 CRC patients using the fully automated real-time PCR-based molecular testing system, Idylla^TMKRAS Mutation Test (Biocartis, Mechelen, Belgium). Of the patients, 64 (59.3%) were men and 62 (57.4%) were older than the group average, with left bowel location in 38 cases (35.2%), adenocarcinoma NOS in 102 cases (94.4%), mixed histological pattern in 65 cases (60.2%), T3 in 60 patients (55.6%), N2 in 46 patients (42.6%), and 7–12 tumour buds registered in 58 tumours (53.7%). A total of 54 tumour samples (50%) showed KRAS mutation. Statistical comparative analyses associated KRAS mutations with the histopathological pattern (p = 0.018), tumour grade (p = 0.030), depth of invasion (pT) (p < 0.001), lymph node involvement (pN) (p < 0.001), venous vascular invasion (p = 0.048), and tumour buds’ number (p = 0.007). Our results demonstrate the relationship between KRAS mutation and clinicopathological features, with possible impact in clinical tumour stratification and therapeutic management. Full article

The USDA-ARS collection of insect viruses at Beltsville, MD, USA, contains samples of an alphabaculovirus from larvae of the tufted apple bud moth, Platynota idaeusalis Walker, as well as a presumptive betabaculovirus from the same host species. The viruses in these samples—Platynota idaeusalis nucleopolyhedrovirus isolate 2680 (PlidNPV-2680) and Platynota idaeusalis granulovirus isolate 2683 (PlidGV-2683)—were characterized by electron microscopy of their occlusion bodies (OBs) and determination and analysis of their genome sequences. Scanning and transmission electron microscopy of the OBs revealed morphologies typical for alphabaculoviruses and betabaculoviruses. Sequencing viral DNA resulted in circular genomes of 121,881 bp and 106,633 bp for PlidNPV-2680 and PlidGV-2683, respectively. Similar numbers of ORFs (128 for PlidNPV-2680, 125 for PlidGV-2683) were annotated, along with ten homologous regions (hrs) in the PlidNPV-2680 genome and five intergenic regions of tandem direct repeats (drs) in the PlidGV genome. Phylogenetic inference from core gene alignments suggested that PlidMNPV-2680 represents a unique lineage within the genus Alphabaculovirus, while PlidGV-2683 was grouped with clade b betabaculoviruses. A comparison of the PlidNPV-2680 and PlidGV-2683 genomes revealed a 1516 bp region in PlidNPV-2680 that exhibited 97.5% sequence identity to a region of the PlidGV-2683 genome, suggesting that recombination had occurred recently between viruses from these lineages. Full article

The global burden of respiratory syncytial virus (RSV) and severe associated disease is prodigious. RSV-specific vaccines have been launched recently but there is no antiviral medicine commercially available. RSV polymerase (L) protein is one of the promising antiviral targets, along with fusion and nucleocapsid proteins. During medicinal chemistry campaigns, two potent L-protein inhibitors (PC786 and PC751) were identified. Both compounds inhibited the RSV A/B-induced cytopathic effect in HEp-2 cells equally, but PC786 was more potent than PC751 in bronchial epithelial cells. Repeated treatment with escalating concentrations on RSV A2-infected HEp-2 cells revealed both inhibitors led to a Y1631H mutation in the L protein, but only PC786 induced a mutation in the M protein (V153A). By L protein fragment and M protein binding analysis, we showed that the M protein interacts with the 1392–1735 amino acid region of the L protein, where PC786 potentially binds. In addition, PC786 treatment or PC786-induced mutant RSV was found to increase M-protein nuclear localisation later in infection, concomitant with delayed fusion protein localisation at the budding viral filaments. As M protein is known to play a key role in virus assembly and budding late in infection, our data suggests that disrupting the interaction between the M and L proteins could provide a novel target for antiviral development. Full article

The respiratory syncytial virus (RSV) matrix protein plays key roles in the virus life cycle and is essential for budding, as it stimulates the optimal membrane curvature necessary for the emergence of viral particles. Resveratrol, a polyphenol (3,4′,5-trihydroxy-trans-stilbene) produced by plants, exhibits pharmacological effects, including anti-inflammatory and antiviral activities. In this study, resveratrol was tested in HEp-2 (Epidermoid carcinoma of the larynx cell) cells for its post-infection effects, and recombinant M protein was produced to characterize the biophysical mechanisms underlying this interaction. The CC50 (Cytotoxic concentration 50%) value for resveratrol was determined to be 297 μM over 48 h, and the results from the HEp-2 cell cultures demonstrated a viral inhibition of 42.7% in the presence of resveratrol, with an EC50 (Half maximal effective concentration) of 44.26 μM. This mechanism may occur through interaction with the M protein responsible for the budding of mature viral particles. Biophysical assays enabled us to characterize the interaction of the M/resveratrol complex as an entropically driven bond, guided by hydrophobic interactions at the dimerization interface of the M protein, which is essential for the stabilization and formation of the oligomers necessary for viral budding. These findings suggest that one of the targets for resveratrol binding is the M protein, indicating a potential site for blocking the progression of the infection. Full article