Viral Proteins Research Articles

Recent advances in solid‐state nuclear magnetic resonance studies on membrane fusion proteins

Membrane fusion is an essential biological process that merges two separate lipid bilayers into a whole one. Membrane fusion proteins facilitate this process by bringing lipid bilayers in close proximity to reduce the repulsive energy between membranes. Along with their interactions with membranes, the structures and dynamics of membrane fusion proteins are key to elucidating the mechanisms of membrane fusion. Solid‐state NMR (SSNMR) spectroscopy has unique advantages in determining the structures and dynamics of membrane fusion proteins in their membrane‐bound states. It has been extensively applied to reveal conformational changes in intermediate states of viral membrane fusion proteins and to characterize the critical lipid–membrane interactions that drive the fusion process. In this review, we summarize recent advancements in SSNMR techniques for studying membrane fusion proteins and their applications in elucidating the mechanisms of membrane fusion.

Exploring the Antiviral Potential of Artemisia annua Through JAK-STAT Pathway Targeting: A Network Pharmacology Approach

Background: Artemisia annua, a plant with antiviral potential, has shown promise against various viral infections, yet its mechanisms of action are not fully understood. This study explores A. annua’s antiviral effects using network pharmacology and molecular docking, focusing on key active compounds and their interactions with viral protein targets, particularly within the JAK-STAT signaling pathway—a critical mediator of immune responses to viral infections. Methods: From the TCMSP database, we identified eight active compounds and 335 drug targets for A. annua, with 19 intersecting targets between A. annua compounds and viral proteins. A protein–protein interaction (PPI) network highlighted 10 key hub genes, analyzed further through Gene Ontology (GO) and KEGG pathways to understand their immune and antiviral roles. ADMET properties of the active compound Patuletin (MOL004112) were assessed, followed by 200 ns molecular dynamics simulations to examine its stability in complex with JAK2. Results: PPI analysis identified JAK2, MAPK3, MAPK1, JAK1, PTPN1, HSPA8, TYK2, RAF1, MAPT, and HMOX1 as key hub genes, with JAK2 emerging as a critical regulator of immune and antiviral pathways. ADMET analysis confirmed Patuletin’s favorable pharmacokinetic properties, and molecular dynamics simulations showed a stable Patuletin-JAK2 complex, with FEL analysis indicating minimal disruption to JAK2’s intrinsic flexibility. Conclusions: These findings highlight JAK2 as a promising target in the antiviral activity of A. annua compounds, particularly Patuletin, supporting its potential as an antiviral agent and providing a foundation for further research on A. annua’s therapeutic applications.

Deep learning pipeline for accelerating virtual screening in drug discovery.

In the race to combat ever-evolving diseases, the drug discovery process often faces the hurdles of high-cost and time-consuming procedures. To tackle these challenges and enhance the efficiency of identifying new therapeutic agents, we introduce VirtuDockDL, which is a streamlined Python-based web platform utilizing deep learning for drug discovery. This pipeline employs a Graph Neural Network to analyze and predict the effectiveness of various compounds as potential drug candidates. During the validation phase, VirtuDockDL was instrumental in identifying non-covalent inhibitors against the VP35 protein of the Marburg virus, a critical target given the virus's high fatality rate and limited treatment options. Further, in benchmarking, VirtuDockDL achieved 99% accuracy, an F1 score of 0.992, and an AUC of 0.99 on the HER2 dataset, surpassing DeepChem (89% accuracy) and AutoDock Vina (82% accuracy). Compared to RosettaVS, MzDOCK, and PyRMD, VirtuDockDL outperformed them by combining both ligand- and structure-based screening with deep learning. While RosettaVS excels in accurate docking but lacks high-throughput screening, and PyRMD focuses on ligand-based methods without AI integration, VirtuDockDL offers superior predictive accuracy and full automation for large-scale datasets, making it ideal for comprehensive drug discovery workflows. These results underscore the tool's capability to identify high-affinity inhibitors accurately across various targets, including the HER2 protein for cancer therapy, TEM-1 beta-lactamase for bacterial infections, and the CYP51 enzyme for fungal infections like Candidiasis. To sum up, VirtuDockDL combines user-friendly interface design with powerful computational capabilities to facilitate rapid, cost-effective drug discovery and development. The integration of AI in drug discovery could potentially transform the landscape of pharmaceutical research, providing faster responses to global health challenges. The VirtuDockDL is available at https://github.com/FatimaNoor74/VirtuDockDL .

Rational design of uncleaved prefusion-closed trimer vaccines for human respiratory syncytial virus and metapneumovirus.

Respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) cause human respiratory diseases and are major targets for vaccine development. In this study, we design uncleaved prefusion-closed (UFC) trimers for the fusion protein (F) of both viruses by examining mutations critical to F metastability. For RSV, we assess four previous prefusion F designs, including the first and second generations of DS-Cav1, SC-TM, and 847A. We then identify key mutations that can maintain prefusion F in a native-like, closed trimeric form (up to 76%) without introducing any interprotomer disulfide bond. For hMPV, we develop a stable UFC trimer with a truncated F2-F1 linkage and an interprotomer disulfide bond. Dozens of UFC constructs are characterized by negative-stain electron microscopy (nsEM), x-ray crystallography (11 RSV-F structures and one hMPV-F structure), and antigenic profiling. Using an optimized RSV-F UFC trimer as bait, we identify three potent RSV neutralizing antibodies (NAbs) from a phage-displayed human antibody library, with a public NAb lineage targeting sites Ø and V and two cross-pneumovirus NAbs recognizing site III. In mouse immunization, rationally designed RSV-F and hMPV-F UFC trimers induce robust antibody responses with high neutralizing titers. Our study provides a foundation for future prefusion F-based RSV and hMPV vaccine development.

An in-silico analysis decoding the differences between the viral proteins of the American and the South-East Asian strains of DENV-4

An <i>in-silico</i> analysis decoding the differences between the viral proteins of the American and the South-East Asian strains of DENV-4

Identification of potential antigenic proteins and epitopes for the development of a monkeypox virus vaccine: an in silico approach.

Virus assembly, budding, or surface proteins play important roles such as viral attachment to cells, fusion, and entry into cells. The present study aimed to identify potential antigenic proteins and epitopes that could be used to develop a vaccine or diagnostic assay against the Monkeypox virus (MPXV) which may cause a potential epidemic. To do this, 39 MPXV proteins (including assembly, budding, and surface proteins) were analyzed using an in silico approach. Of these 39 proteins, the F5L virus protein was found to be the best vaccine candidate due to its signal peptide properties, negative GRAVY value, low transmembrane helix content, moderate aliphatic index, large molecular weight, long-estimated half-life, beta wrap motifs, and being stable, soluble, and containing non-allergic features. Moreover, the F5L protein exhibited alpha-helical secondary structures, making it a potential "structural antigen" recognized by antibodies. The other viral protein candidates were A9 and A43, but A9 lacked beta wrap motifs, while A43 had a positive GRAVY value and was insoluble. These two proteins were not as suitable candidates as the F5L protein. The KRVNISLTCL epitope from the F5L protein demonstrated the highest antigen score (2.4684) for MHC-I, while the GRFGYVPYVGYKCI epitope from the A9 protein exhibited the highest antigenicity (1.754) for MHC-II. Both epitopes met the criteria for high antigenicity, non-toxicity, solubility, non-allergenicity, and the presence of cleavage sites. Molecular docking and dynamics (MD) simulations further validated their potential, revealing stable and energetically favorable interactions with MHC molecules. The immunogenicity assessment showed that GRFGYVPYVGYKCI could strongly induce immune responses through both IFN-γ and IL-4 pathways, suggesting its capacity to provoke a balanced Th1 and Th2 response. In contrast, KRVNISLTCL exhibited limited immunostimulatory potential. Overall, these findings lay the groundwork for future vaccine development, indicating that F5L, particularly the GRFGYVPYVGYKCI epitope, may serve as an effective candidate for peptide-based vaccine design against MPXV.

In Silico Investigation of the Interactions Between Cotton Leaf Curl Multan Virus Proteins and the Transcriptional Gene Silencing Factors of Gossypium hirsutum L.

The highly dynamic nature of the Cotton leaf curl virus (CLCuV) complex (causing Cotton leaf curl disease, a significant global threat to cotton) presents a formidable challenge in unraveling precise molecular mechanisms governing viral-host interactions. To address this challenge, the present study investigated the molecular interactions of 6 viral proteins (Rep, TrAP, C4, C5, V2, and βC1) with 18 cotton Transcriptional Gene Silencing (TGS) proteins. Protein-protein dockings conducted for different viral-host protein pairs using Clustered Protein Docking (ClusPro) and Global RAnge Molecular Matching (GRAMM) (216 docking runs), revealed variable binding energies. The interacting pairs with the highest binding affinities were further scrutinized using bioCOmplexes COntact MAPS (COCOMAPS) server, which revealed robust binding of three viral proteins- TrAP, C4, and C5 with 14 TGS proteins, identifying several novel interactions (not reported yet by earlier studies), such as TrAP targeting DCL3, HDA6, and SUVH6; C4 targeting RAV2, CMT2, and DMT1; and C5 targeting CLSY1, RDR1, RDR2, AGO4, SAMS, and SAHH. Visualizing these interactions in PyMol provided a detailed insight into interacting regions. Further assessment of the impact of 18 variants of the C4 protein on interaction with CMT2 revealed no correlation between sequence variation and docking energies. However, conserved residues in the C4 binding regions emerged as potential targets for disrupting viral integrity. Hence, this study provides valuable insights into the viral-host interplay, advancing our understanding of Cotton leaf curl Multan virus pathogenicity and opening novel avenues for devising various antiviral strategies by targeting the host-viral interacting regions after experimental validation.

NS1-mediated DNMT1 degradation regulates human bocavirus 1 replication and RNA processing.

Methylation of the DNA genome plays an important role in viral gene inactivation. However, the role of DNA methylation in human bocavirus (HBoV) remains unclear. In this study, the HBoV1 genomic DNA was found extensively methylated at the CHG and CHH sites. Inhibiting DNA methylation with 5-aza-2'-deoxycytidine (DAC) altered the methylation status and reduced viral DNA production, while enhanced the RNA splicing at D1 and D3 sites and the polyadenylation at the proximal polyadenylation site, (pA)p. Knockdown of DNA methyltransferase 1 (DNMT1) had the same effect on viral DNA synthesis and RNA processing as the DAC treatment, indicating that DNMT1 is the major host methyltransferase involved in viral DNA methylation. In addition, the nonstructural protein NS1 promoted DNMT1 degradation through the ubiquitin-proteasome pathway to regulate viral replication and RNA processing. Collectively, the results suggest that DNA methylation and DNMT1 facilitate HBoV replication and are essential for appropriate NS1 localization in the nucleus. DNMT1 degradation through NS1 promotes the virus RNA processing, leading to viral protein expression.

VP28 interacts with PmRab7 irrespective of its nucleotide state.

In shrimp aquaculture, white spot syndrome virus (WSSV) infections severely impact production. Previous research highlighted the crucial role of the Penaeus monodon Rab7 (PmRab7) protein in WSSV entry, specifically its interaction with the viral envelope protein VP28. PmRab7 exists in two conformations: GDP-bound (inactive) and GTP-bound (active). This study, using ELISA and isothermal titration calorimetry (ITC), reveals that the PmRab7-VP28 interaction occurs irrespective of the nucleotide binding state of PmRab7. Comparing the binding affinity between VP28 and different PmRab7 conformations, including wild-type (WT, 22.5 nM), a fast nucleotide exchange (L129F, 128 nM), a GDP-bound form (T22N, 334 nM), and a favorably GTP-bound form (Q67L, 1990 nM), PmRab7-WT exhibits the strongest binding affinity, especially at a lower temperature (25°C). The binding of PmRab7-WT and VP28 in the presence of excess nucleotide (WT with excess GDP, 924 nM, and WT with excess GTP, 826 nM) shows a 2-fold higher binding affinity than in the absence (WT, 1920 nM) indicating that the addition of excess nucleotide for PmRab7-WT enhanced the affinity for VP28. Together, these findings support the potential of PmRab7-WT as a promising therapeutic candidate for WSSV control in shrimp. Furthermore, from an industrial point of view, the ITC platform developed to study the VP28-PmRab7 interactions provides a high-throughput method for screening additives for shrimp feed that can inhibit this interaction.

Fangchinoline Inhibits Zika Virus by Disrupting Virus Internalization.

The Zika virus (ZIKV) has garnered significant public attention, particularly following the outbreak in Brazil, due to its potential to cause severe damage to the central nervous system and its ability to cross the placental barrier, resulting in microcephaly in infants. Despite the urgency, there remains a lack of targeted therapies or vaccines for the prevention or treatment of ZIKV infection and its related diseases. Fangchinoline (FAN), an alkaloid derived from traditional Chinese medicinal herbs, has a range of biological activities. In this study, we employed both in vitro and in vivo infection models to demonstrate the efficacy of FAN in inhibiting ZIKV. Our findings indicate that FAN effectively suppresses the replication of ZIKV viral RNA and protein, thereby validating its anti-ZIKV capabilities in living organisms. Further analysis through dosing time assays and infectious inhibition assays revealed that FAN exerts its antiviral effects by impeding the early stages of infection, specifically by inhibiting the internalization of ZIKV. These results underscore the potential of FAN as a candidate for anti-ZIKV drug development and offer novel insights into drug design strategies that target the virus's internalization process.

Proteomic signatures of ambient air pollution and risk of non-alcoholic fatty liver disease: A prospective cohort study in the UK Biobank.

Air pollution has been linked with non-alcoholic fatty liver disease (NAFLD), but the underlying mechanisms characterized by perturbations in the circulating proteome profile are largely unknown. Therefore, we included 51,357 participants from the UK Biobank with 2941 plasma proteins measured in blood samples collected between 2006 and 2010, measurements of annual fine particular matter <2.5 μm in diameter (PM2.5) and nitrogen dioxide (NO2), and follow-up data on NAFLD (743 incident cases occurred over a median follow-up of 13.6 years). Multiple linear regression was used to identify proteins associated with PM2.5 and NO2. Cox proportional hazards models were applied to assess associations of PM2.5 and NO2 and identified proteins with incident NAFLD. Mediation analyses were conducted to explore the mediation role of proteins in the associations between air pollution and incident NAFLD. After adjusting for selected covariates, PM2.5 (hazard ratio [HR] = 2.57, 95%CI:1.27, 5.21, per ln increase) and NO2 (HR = 1.43, 95%CI: 1.10, 1.84, per ln increase) were positively associated with incident NAFLD. We identified 138 proteins associated with PM2.5 (92 positively, 46 inversely, FDR <0.05) and 143 with NO2 (100 positively, 43 inversely). Of the proteins that were significantly associated with both PM2.5 and NO2, 93 (79 positively, 14 inversely) and 79 (69 positively, 10 inversely) were significantly associated with incident NAFLD. Furthermore, 84 PM2.5-associated proteins and 66 NO2-associated proteins significantly mediated the corresponding association between air pollutants and incident NAFLD, with the proportion of mediation effects ranging from 3.2 % to 27.3 % for PM2.5 and 2.6 % to 20.8 % for NO2, respectively. Of note, the majority of significant mediating proteins were enriched in pathways of cytokine-cytokine receptor interaction, viral protein interaction with cytokine and cytokine receptor. Our findings suggested that long-term exposure to PM2.5 and NO2 was associated with an increased risk of NAFLD partially by perturbating circulating proteins involved in pathways of inflammation and immunity responses.

Genetic analysis implicates ERAP1 and HLA as risk factors for severe Puumala virus infection.

Puumala virus (PUUV) infections can cause severe illnesses such as Hemorrhagic Fever with Renal Syndrome in humans. However, human genetic risk factors contributing to disease severity are still poorly understood. Our goal was to elucidate genetic factors contributing to PUUV infections and understand the biological mechanisms underlying individual vulnerability to PUUV infections. Leveraging data from the FinnGen study, we conducted a genome-wide association study on severe Hemorrhagic Fever with Renal Syndrome caused by PUUV with 2227 cases. We identified associations at the Human Leukocyte Antigen (HLA) locus and ERAP1 with severe PUUV infection. HLA molecules are canonical mediators for immune recognition and response. ERAP1 facilitates immune system recognition and activation by cleaving viral proteins into smaller peptides which are presented to the immune system via HLA class I molecules. Notably, we identified that the lead variant (rs26653, OR = 0.84, P = 2.9 × 10-8) in the ERAP1 gene was a missense variant changing amino acid arginine to proline. From the HLA region, we showed independent and significant associations with both HLA class I and II genes. Furthermore, we showed independent associations with four HLA alleles with severe PUUV infection using conditional HLA fine mapping. The strongest association was found with the HLA-C*07:01 allele (OR = 1.54, P = 4.0 × 10-24) followed by signals at HLA-B*13:02, HLA-DRB1*01:01, and HLA-DRB1*11:01 alleles (P < 5 × 10-8). Our findings suggest an association of viral peptide processing with ERAP1 and antigen presentation through HLA alleles that may contribute to the development of severe PUUV disease.

Systematic genetic characterization of the human PKR kinase domain highlights its functional malleability to escape a poxvirus substrate mimic

Evolutionary arms races can arise at the contact surfaces between host and viral proteins, producing dynamic spaces in which genetic variants are continually pursued. However, the sampling of genetic variation must be balanced with the need to maintain protein function. A striking case is given by protein kinase R (PKR), a member of the mammalian innate immune system. PKR detects viral replication within the host cell and halts protein synthesis to prevent viral replication by phosphorylating eIF2α, a component of the translation initiation machinery. PKR is targeted by many viral antagonists, including poxvirus pseudosubstrate antagonists that mimic the natural substrate, eIF2α, and inhibit PKR activity. Remarkably, PKR has several rapidly evolving residues at this interface, suggesting it is engaging in an evolutionary arms race, despite the surface’s critical role in phosphorylating eIF2α. To systematically explore the evolutionary opportunities available at this dynamic interface, we generated and characterized a library of 426 SNP-accessible nonsynonymous variants of human PKR for their ability to escape inhibition by the model pseudosubstrate inhibitor K3, encoded by the vaccinia virus gene K3L. We identified key sites in the PKR kinase domain that harbor K3-resistant variants, as well as critical sites where variation leads to loss of function. We find K3-resistant variants are readily available throughout the interface and are enriched at sites under positive selection. Moreover, variants beneficial against K3 were also beneficial against an enhanced variant of K3, indicating resilience to viral adaptation. Overall, we find that the eIF2α-binding surface of PKR is highly malleable, potentiating its evolutionary ability to combat viral inhibition.

Systematic genetic characterization of the human PKR kinase domain highlights its functional malleability to escape a poxvirus substrate mimic.

Evolutionary arms races can arise at the contact surfaces between host and viral proteins, producing dynamic spaces in which genetic variants are continually pursued. However, the sampling of genetic variation must be balanced with the need to maintain protein function. A striking case is given by protein kinase R (PKR), a member of the mammalian innate immune system. PKR detects viral replication within the host cell and halts protein synthesis to prevent viral replication by phosphorylating eIF2α, a component of the translation initiation machinery. PKR is targeted by many viral antagonists, including poxvirus pseudosubstrate antagonists that mimic the natural substrate, eIF2α, and inhibit PKR activity. Remarkably, PKR has several rapidly evolving residues at this interface, suggesting it is engaging in an evolutionary arms race, despite the surface's critical role in phosphorylating eIF2α. To systematically explore the evolutionary opportunities available at this dynamic interface, we generated and characterized a library of 426 SNP-accessible nonsynonymous variants of human PKR for their ability to escape inhibition by the model pseudosubstrate inhibitor K3, encoded by the vaccinia virus gene K3L. We identified key sites in the PKR kinase domain that harbor K3-resistant variants, as well as critical sites where variation leads to loss of function. We find K3-resistant variants are readily available throughout the interface and are enriched at sites under positive selection. Moreover, variants beneficial against K3 were also beneficial against an enhanced variant of K3, indicating resilience to viral adaptation. Overall, we find that the eIF2α-binding surface of PKR is highly malleable, potentiating its evolutionary ability to combat viral inhibition.

Identification of linear B cell epitopes on the E146L protein of African swine fever virus with monoclonal antibodies.

The outbreak and spread of African swine fever virus (ASFV) have caused considerable economic losses to the pig industry worldwide. Currently, to promote the development of effective ASF vaccines, especially subunit vaccines, more antigenic protein targets are urgently needed. In this work, six transmembrane proteins (I329L, E146L, C257L, EP153R, I177L, and F165R) were expressed in mammalian cell lines and screened with pig anti-ASFV serum. It was found that the E146L protein was an immunodominant protein antigen among the six selected proteins. Moreover, the E146L protein induced antibody responses in all immunized pigs. To gain insight into the antigenic characteristics of the E146L protein, three monoclonal antibodies (mAbs; 12H12, 15G1, and 15H10) were generated by immunizing BALB/c mice with the purified E146L protein. The epitopes of the mAbs were further finely mapped through a peptide fusion protein expression strategy. Finally, the epitopes of the mAbs were identified as 48PDESSIAYMRFRN61 of the mAb 12H12, 138TLTGLQRII146 of the mAb 15G1, and 30GWSPFKYSKGNT41 of the mAb 15H10. Furthermore, the epitope of mAb 15H10 was validated as the immunodominant epitope with ASFV-infected pig sera. The chemically synthesized mAb 15H10 epitope peptide (EP1) exhibited the most extensive immunoreactivity with artificially or naturally ASFV-infected pig sera. The epitope 15H10 is located on the surface of the E146L protein and is highly conserved. These findings provide insight into the structure and function of the E146L protein of ASFV.

The Adeno-Associated Virus Replication Protein Rep78 Contains a Strictly C-Terminal Sequence Motif Conserved Across Dependoparvoviruses

Adeno-Associated Viruses (AAVs, genus Dependoparvovirus) are the leading gene therapy vector. Until recently, efforts to enhance their capacity for gene delivery had focused on their capsids. However, efforts are increasingly shifting towards improving the viral replication protein, Rep78. We discovered that Rep78 and its shorter isoform Rep52 contain a strictly C-terminal sequence motif, DDx3EQ, conserved in most dependoparvoviruses. The motif is highly negatively charged and devoid of prolines. Its wide conservation suggests that it is required for the life cycle of dependoparvoviruses. Despite its short length, the motif’s strictly C-terminal position has the potential to endow it with a high recognition specificity. A candidate target of the DDx3EQ motif might be the DNA-binding interface of the origin-binding domain of Rep78, which is highly positively charged. Published studies suggest that this motif is not required for recombinant AAV production, but that substitutions within it might improve production.

Nef mediates neuroimmune response, myelin impairment, and neuronal injury in EcoHIV-infected mice.

The introduction of antiretroviral therapy has markedly improved the management of HIV-associated neurocognitive disorders (HAND). However, HAND still affects nearly half of HIV-infected individuals, presenting significant challenges to their well-being. This highlights the critical need for a deeper understanding of HAND mechanisms. Among HIV viral proteins, Nef is notable for its multifaceted role in HIV pathogenesis, though its specific involvement in HAND remains unclear. To investigate this, we used a murine model infected with Nef-expressing (EcoHIV) and Nef-deficient (EcoHIVΔNef) murine HIV. Comparative analyses revealed increased neuroinflammation and reduced myelin and neuronal integrity in EcoHIV-infected brains compared with those with EcoHIVΔNef. Both viruses induced astrogliosis, with stronger GFAP activation in Nef-deficient infections. These findings suggest that Nef contributes to neuroinflammation, primarily through microglial targeting and demyelination, although other factors may regulate astrogliosis. Our results indicate that Nef may significantly contribute to neuronal injury in EcoHIV-infected mice, offering insights into Nef-induced neuropathology in HAND and guiding future research.

TRIM28 regulates the coagulation cascade inhibited by p72 of African swine fever virus.

In 2018, African swine fever virus (ASFV) emerged in China, causing extremely serious economic losses to the domestic pig industry. Infection with ASFV can cause disseminated coagulation, leading to the consumption of platelets and coagulation factors and severe bleeding. However, the mechanism of virus-induced coagulation has yet to be established. In our study, ASFV downregulated the coagulation process, as detected by D-dimer (D2D) and Factor X (F10) expression in pigs challenged with ASFV HLJ/18. In vitro, ASFV infection increased Factor IX (F9) and Factor XII (F12) expression while downregulating F10 expression in porcine alveolar macrophages (PAMs). African swine fever virus induced both intrinsic and extrinsic coagulation cascades. In addition, several encoded proteins affect the expression of the crucial coagulation protein F10, and among the encoded proteins, p72 inhibits the activity and expression of F10. Proteomic analysis also revealed that p72 is involved in the coagulation cascade. p72 can interact with F10, and its inhibitory functional domains include amino acids 423-432 and amino acids 443-452. Finally, we found that F10 and p72 interact with tripartite motif-containing protein 28 (TRIM28). TRIM28 knockdown resulted in a decrease in F10 expression. Importantly, TRIM28 contributes to the reduction in F10 protein expression regulated by p72. Our findings revealed an inhibitory effect of the viral protein p72 on the ASFV infection-induced coagulation cascade and revealed a role of TRIM28 in reducing F10 expression, revealing a molecular mechanism of ASFV-associated coagulation.

Abstract 4120480: HIV-Nef extracellular vesicles utilize the novel Btk-NFκB-MerTK signaling axis to impair macrophage efferocytosis and promote atherosclerosis: A multiomics study

Background: People living with HIV (PLWH) on anti-retroviral therapy remain at risk for cardiovascular diseases, including atherosclerosis. We hypothesized that persistent viral protein (HIV-Nef) in extracellular vesicles (EVs) modulate macrophage heterogeneity to impair atheroprotective efferocytosis to accelerate cardiovascular disease. Methods and Results: Macrophage heterogeneity was characterized in human primary macrophages (50,931 cells; 4 donors) stimulated with EVs engineered to contain HIV-Nef by simultaneous scRNAseq and scATACseq. Among 16 clusters (Fig.1A), two inflammatory Nef dominant clusters were characterized using gene set enrichment analysis. Gene regulatory network analysis of scRNAseq (pySCENIC) and transcription factor footprinting analysis of sc-ATACseq (ChromVar) suggest elevated NFκB activation in these clusters. Whole cell and sub-cellular compartmentalized proteomics of nucleus, cytosol, cell surface and secreted EVs indicate changes in immune response related biological pathways. Network analysis of our multi-omics data predicts Bruton Tyrosine Kinase (Btk) signaling as a potential contributor to Nef induced macrophage dysregulation. Pathway enrichment analysis of these multiomics dataset suggest Nef influences atheroprotective efferocytosis through the Btk-NFκB signaling axis. Spectral flow cytometry, high content imaging and multiplexed qPCR showed reduction in key effector of efferocytosis, MerTK. Btk inhibition using siRNA, reversible and irreversible Btk inhibitors restored MerTK expression and rescued efferocytosis. Importantly, CRISPRa based overexpression of MerTK in human primary macrophages rescued Nef impaired efferocytosis. Injection of HIV-Nef EVs into male and female C57BL6/J mice impaired efferocytosis of peritoneal and bone marrow derived macrophages which was rescued with Btk inhibition in vivo . Critically, injection of HIV-Nef EV into male and female Ldlr -/- enhanced atherogenesis with larger aortic wall thickness and necrotic core (Fig.1B). Conclusion: Persistent Nef in EV in PLWH may modulate macrophage heterogeneity to impair efferocytosis. These findings may help develop novel atheroprotective therapies by restoring macrophage efferocytosis.

Molecular characterization of Ebola virus, immune response, and therapeutic challenges: a narrative review

AbstractThe Ebola virus (EBOV) remains a major public health challenge due to its complex structure and the lack of appropriate and effective vaccines and therapies. This review characterizes the Ebola virus, its immune response, and its therapeutic challenges. Structural EBOV proteins include the envelope glycoprotein, nucleoprotein, RNA polymerase L, and viral proteins VP30, VP24, VP35, and VP40. The proteins play a role in the virus’s pathogenesis by evading the host's immune response. The immune system evasion mechanisms of EBOV are critical in its pathogenesis. Some vaccines, such as the recombinant vesicular stomatitis virus-Zaire Ebola virus (RVSV-ZEBOV), have proven to be very effective and have been approved by the Food and Drug Administration (FDA) additionally, four other vaccines have been approved including Gam Evac-Combi (licensed in Russia), ad5-EBOV (approved in China), Zabdeno and Mvabea (approved in Europe). However, some challenges remain in developing effective vaccines, such as the selection of immunogens, cross-protecting immunity, long-term protection, mechanism of protection, and rapid response vaccination. Despite the progress made, there is still a need for an effective vaccine that offers durable and broad protection against multiple strains of the Ebola virus. This will be achieved through the collaboration of various organizations and government and Non-Governmental Organization (NGO) agencies.