Viral Proliferation Research Articles

A broadly neutralizing human monoclonal antibody generated from transgenic mice immunized with HCMV particles limits virus infection and proliferation.

Human cytomegalovirus is a herpesvirus that infects a large proportion of the population and can cause significant disease in diverse patient populations whose immune systems are suppressed or compromised. The development and optimization of safe anti-HCMV therapeutics, especially those that have viral targets and inhibition mechanisms different from current HCMV treatments, are of urgent necessity to better public health. Human monoclonal antibodies (mAbs) that prevent HCMV entry of cells were identified by immunizing transgenic mice and screened for broad and effective neutralization capability. Here, we describe one such mAb, which was found to target gH/gL envelope complexes and effectively limit HCMV infection and dissemination. Further, administration of the antibody in combination with the antiviral drug ganciclovir inhibited HCMV in a synergistic manner, highlighting this approach and the use of anti-HCMV mAbs more broadly, as a potential therapeutic strategy for the treatment of diverse patient populations.

Phytochemicals: Promising Inhibitors of Human Rhinovirus Type 14 3C Protease as a Strategy to Fight the Common Cold.

Human rhinovirus 3C protease (HRV-3Cpro) plays a crucial role in viral proliferation, establishing it as a prime target for antiviral therapy. However, research on identifying HRV-3Cpro inhibitors is still limited. This study had two primary objectives: first, to validate the efficacy of an end-point colorimetric assay, previously developed by our team, for identifying potential inhibitors of HRV-3Cpro; and second, to discover phytochemicals in medicinal plants that inhibit the enzyme's activity. Rupintrivir, a well-known inhibitor of HRV-3Cpro, was used to validate the colorimetric assay. Following this, we conducted a two-step in silico screening of 2532 phytochemicals, which led to the identification of eight active compounds: apigenin, carnosol, chlorogenic acid, kaempferol, luteolin, quercetin, rosmarinic acid, and rutin. We subsequently evaluated these candidates in vitro. To further investigate the inhibitory potential of the most promising candidates, namely, carnosol and rosmarinic acid, molecular docking studies were performed to analyze their binding interactions with HRV-3Cpro. The colorimetric assay we previously developed is effective in identifying compounds that selectively inhibit HRV-3Cpro. Carnosol and rosmarinic acid emerged as potent inhibitors, inhibiting HRV-3Cpro activity in vitro by over 55%. Our analysis indicated that carnosol and rosmarinic acid exert their inhibitory effects through a competitive mechanism. Molecular docking confirmed their competitive binding to the enzyme's active site. Carnosol and rosmarinic acid warrant additional investigation for their potential in the development of common cold treatment. By highlighting these compounds as effective HRV-3Cpro inhibitors, our study presents a promising approach for discovering phytochemical inhibitors against proteases from similar pathogens.

Synergistic effects of oral inoculation with a recombinant Lactobacillus plantarum NC8 strain co-expressing interleukin-2 and interleukin-17B on the efficacy of the infectious bronchitis vaccine in chickens

Mucosal vaccination strategies are easier to implement than others in large-scale poultry farming. However, the adjuvants that are approved for veterinary use, which are predominantly aluminum- and oil-emulsion-based adjuvants, are not suitable for mucosal vaccination and carry a risk of adverse reactions. In this study, we engineered a novel Lactobacillus plantarum NC8 strain that co-expresses chicken interleukin-2 (IL-2) and IL-17B, which we designated NC8-ChIL2-17B, and evaluated its potential as an oral immunoadjuvant. The immunomodulatory properties of NC8-ChIL2-17B were evidenced by its ability to activate macrophages and inhibit the proliferation of infectious bronchitis virus (IBV) in vitro. We then confirmed its immunoadjuvant activity in vivo by orally administering NC8-ChIL2-17B along with a commercial IBV vaccine to chicks. The results indicated that NC8-ChIL2-17B enhanced the immune response elicited by the IBV vaccine and increased the levels of IBV-specific IgG and sIgA antibodies produced in response to IBV infection. Additionally, administration of NC8-ChIL2-17B promoted weight gain and beneficially modulated the gut microbiota, resulting in improved chicken performance. These findings suggest that oral administration of NC8-ChIL2-17B is a promising strategy to enhance the immune efficacy of the IBV vaccine in chickens, offering an efficacious alternative adjuvant.

Discovery of Pyrido[1,2-α] Pyrimidinone Mesoionic Compounds as Potential Control Agents Against Potato Virus Y.

Potato virus Y (PVY) relies on aphids and tubers to spread in the field and causes serious economic losses in the potato industry. Here, we found that pyrido[1,2-α] pyrimidinone mesoionic compounds with insecticidal activity against aphids possessed a good inhibitory effect on PVY. Among them, compound 35 had the best inhibitory activity against PVY (EC50 = 104 μg/mL), even superior to that of ningnanmycin (125 μg/mL). The fluorescence and qPCR results confirmed that compound 35 could inhibit the proliferation of PVY in Nicotiana benthamiana. Preliminary experiments on the mechanism of action indicated that compound 35 had good binding affinity with the coat protein (CP), which plays an essential role in aphid-PVY interactions. Molecular docking revealed that compound 35 could bind to the pocket of CP formed by Ser52, Glu204, and Arg208. Compound 35 had substantially lower binding affinity (Kd) values with CPS52A (219 μM), CPE204A (231 μM), and CPR208A (189 μM) than those with CPWT (5.80 μM). A luciferase assay confirmed that mutating Ser52, Glu204, and Arg208 significantly affected the expression level of CP and further reduced virus proliferation. Therefore, the broad-spectrum activity of compound 35 provides a unique strategy for the prevention and treatment of PVY.

Complete Genome Sequence and Construction of an Infectious Bacterial Artificial Chromosome Clone of a Virulent Duck Enteritis Virus Strain XJ

In 2021, a highly virulent strain of duck enteritis virus (DEV), designated as DEV XJ, was isolated from Zhejiang, China, and its complete genome, spanning 162,234 bp with 78 predicted open reading frames (ORFs), was sequenced. While showing relative homology to the DEV CV strain, DEV XJ exhibited distinctions in 38 ORFs, including various immunogenic and virulence-related genes. Amino acid variation analysis, focusing on UL6 and LORF3, indicated a high degree of homology between DEV XJ and the 2085 strain from Europe, as well as the DEV DP-AS-Km-19 strain from India. Subsequently, a full-length infectious bacterial artificial chromosome clone (BAC) of DEV XJ was successfully constructed to delve into the pathogenic mechanisms of this virulent strain. XJ BAC demonstrated substantial similarity to the parental DEV XJ in both in vitro growth properties and the induction of typical pathogenic symptoms in sheldrakes. Furthermore, the US3, LORF3, UL21, and UL36 genes were individually deleted using a two-step RED recombination approach based on the infectious BAC clone. Our findings revealed that the UL21 and UL36 genes play crucial roles in viral proliferation. Although the US3 and LORF3 genes were dispensable for viral replication and cell-to-cell transmission in vitro, they attenuated the replication and transmission efficiency of DEV compared to the WT. In summary, this study accomplished the whole-genome sequencing of a clinically virulent DEV strain and the successful construction of an infectious DEV XJ clone. Moreover, the functional roles of the above-mentioned mutant genes were preliminarily explored through the analysis of their in vitro biological characteristics.

The 6-kilodalton peptide 1 in plant viruses of the family Potyviridae is a viroporin

Potyviridae, the largest family of plant RNA viruses, includes many important pathogens that significantly reduce the yields of many crops worldwide. In this study, we report that the 6-kilodalton peptide 1 (6K1), one of the least characterized potyviral proteins, is an endoplasmic reticulum-localized protein. AI-assisted structure modeling and biochemical assays suggest that 6K1 forms pentamers with a central hydrophobic tunnel, can increase the cell membrane permeability of Escherichia coli and Nicotiana benthamiana, and can conduct potassium in Saccharomyces cerevisiae. An infectivity assay showed that viral proliferation is inhibited by mutations that affect 6K1 multimerization. Moreover, the 6K1 or its homologous 7K proteins from other viruses of the Potyviridae family also have the ability to increase cell membrane permeability and transmembrane potassium conductance. Taken together, these data reveal that 6K1 and its homologous 7K proteins function as viroporins in viral infected cells.

Zebrafish mylipb attenuates antiviral innate immunity through two synergistic mechanisms targeting transcription factor irf3.

IFN regulatory factor 3 (IRF3) is the transcription factor crucial for the production of type I IFN in viral defence and inflammatory responses. The activity of IRF3 is strictly modulated by post-translational modifications (PTMs) to effectively protect the host from infection while avoiding excessive immunopathology. Here, we report that zebrafish myosin-regulated light chain interacting protein b (mylipb) inhibits virus-induced type I IFN production via two synergistic mechanisms: induction of autophagic degradation of irf3 and reduction of irf3 phosphorylation. In vivo, mylipb-null zebrafish exhibit reduced lethality and viral mRNA levels compared to controls. At the cellular level, overexpression of mylipb significantly reduces cellular antiviral capacity, and promotes viral proliferation. Mechanistically, mylipb associates with irf3 and targets Lys 352 to increase K6-linked polyubiquitination, dependent on its E3 ubiquitin ligase activity, leading to autophagic degradation of irf3. Meanwhile, mylipb acts as a decoy substrate for the phosphokinase tbk1 to attenuate irf3 phosphorylation and cellular antiviral responses independent of its enzymatic activity. These findings support a critical role for zebrafish mylipb in the limitation of antiviral innate immunity through two synergistic mechanisms targeting irf3.

Antiviral activity of Vigna radiata extract against feline coronavirus in vitro

Feline infectious peritonitis (FIP) is a fatal illness caused by a mutated feline coronavirus (FCoV). This disease is characterized by its complexity, resulting from systemic infection, antibody-dependent enhancement (ADE), and challenges in accessing effective therapeutics. Extract derived from Vigna radiata (L.) R. Wilczek (VRE) exhibits various pharmacological effects, including antiviral activity. This study aimed to investigate the antiviral potential of VRE against FCoV, addressing the urgent need to advance the treatment of FIP. We explored the anti-FCoV activity, antiviral mechanism, and combinational application of VRE by means of in vitro antiviral assays. Our findings reveal that VRE effectively inhibited the cytopathic effect induced by FCoV, reduced viral proliferation, and downregulated spike protein expression. Moreover, VRE blocked FCoV in the early and late infection stages and was effective under in vitro ADE infection. Notably, when combined with VRE, the polymerase inhibitor GS-441524 or protease inhibitor GC376 suppressed FCoV more effectively than monotherapy. In conclusion, this study characterizes the antiviral property of VRE against FCoV in vitro, and VRE possesses therapeutic potential for FCoV treatment.

Continuous year-round isolation of giant viruses from brackish shoreline soils.

Giant viruses, categorized under Nucleocytoviricota, are believed to exist ubiquitously in natural environments. However, comprehensive reports on isolated giant viruses remain scarce, with limited information available on unrecoverable strains, viral proliferation sites, and natural hosts. Previously, the author highlighted Pandoravirus hades, Pandoravirus persephone, and Mimivirus sp. styx, isolated from brackish water soil, as potential hotspots for giant virus multiplication. This study presents findings from nearly a year of monthly sampling within the same brackish water region after isolating the three aforementioned strains. This report details the recurrent isolation of a wide range of giant viruses. Each month, four soil samples were randomly collected from an approximately 5 × 10 m plot, comprising three soil samples and one water sample containing sediment from the riverbed. Acanthamoeba castellanii was used as a host for virus isolation. These efforts consistently yielded at least one viral species per month, culminating in a total of 55 giant virus isolates. The most frequently isolated species was Mimiviridae (24 isolates), followed by Marseilleviridae (23 isolates), Pandoravirus (6 isolates), and singular isolates of Pithovirus and Cedratvirus. Notably, viruses were not consistently isolated from any of the four samples every month, with certain sites yielding no viruses. Cluster analysis based on isolate numbers revealed that soil samples from May and water and sediment samples from January produced the highest number of viral strains. These findings underscore brackish coastal soil as a significant site for isolating numerous giant viruses, highlighting the non-uniform distribution along coastlines.

Serum/glucocorticoid-regulated kinase 1 contributes to the proliferation of varicella-zoster virus and induction of cyclin B1 expression.

In this study, we investigated the role of serum/glucocorticoid-regulated kinase 1 (SGK1) in varicella-zoster virus (VZV) replication. VZV DNA replication and plaque formation were inhibited by SGK1 knockout and treatment with an SGK1 inhibitor. Furthermore, SGK1 inhibition suppressed the increase in cyclin B1 expression induced by VZV infection. These results suggest that VZV infection induces SGK1 activation, which is required for efficient viral proliferation through the expression of cyclin B1. This is the first study to report that SGK1 is involved in the VZV life cycle.

Design and computational evaluation of a novel multi-epitope hybrid vaccine against monkeypox virus: Potential targets and immunogenicity assessment for pandemic preparedness

Monkeypox is a type of DNA-enveloped virus that belongs to the orthopoxvirus family, closely related to the smallpox virus. It can cause an infectious disease in humans known as monkeypox disease. Although there are multiple drugs and vaccines designed to combat orthopoxvirus infections, with a primary focus on smallpox, the recent spread of the monkeypox virus to over 50 countries have ignited a mounting global concern. This unchecked viral proliferation has raised apprehensions about the potential for a pandemic corresponding to the catastrophic impact of COVID-19. This investigation explored the structural proteins of monkeypox virus as potential candidates for designing a novel hybrid multi-epitope vaccine. The epitopes obtained from the selected proteins were screened to ensure their non-allergenicity, non-toxicity, and antigenicity to trigger T and B-cell responses. The interaction of the vaccine with toll-like receptor-3 (TLR-3) and major histocompatibility complexes (MHCs) was assessed using Cluspro 2.0. To establish the reliability of the docked complexes, a comprehensive evaluation was conducted using Immune and MD Simulations and Normal Mode Analysis. However, to validate the computational results of this study, additional in-vitro and in-vivo research is essential.

Viral entry and translation in brain endothelia provoke influenza-associated encephalopathy

Influenza-associated encephalopathy (IAE) is extremely acute in onset, with high lethality and morbidity within a few days, while the direct pathogenesis by influenza virus in this acute phase in the brain is largely unknown. Here we show that influenza virus enters into the cerebral endothelium and thereby induces IAE. Three-weeks-old young mice were inoculated with influenza A virus (IAV). Physical and neurological scores were recorded and temporal-spatial analyses of histopathology and viral studies were performed up to 72 h post inoculation. Histopathological examinations were also performed using IAE human autopsy brains. Viral infection, proliferation and pathogenesis were analyzed in cell lines of endothelium and astrocyte. The effects of anti-influenza viral drugs were tested in the cell lines and animal models. Upon intravenous inoculation of IAV in mice, the mice developed encephalopathy with brain edema and pathological lesions represented by micro bleeding and injured astrocytic process (clasmatodendrosis) within 72 h. Histologically, massive deposits of viral nucleoprotein were observed as early as 24 h post infection in the brain endothelial cells of mouse models and the IAE patients. IAV inoculated endothelial cell lines showed deposition of viral proteins and provoked cell death, while IAV scarcely amplified. Inhibition of viral transcription and translation suppressed the endothelial cell death and the lethality of mouse models. These data suggest that the onset of encephalopathy should be induced by cerebral endothelial infection with IAV. Thus, IAV entry into the endothelium, and transcription and/or translation of viral RNA, but not viral proliferation, should be the key pathogenesis of IAE.

Detection of Active SARS-CoV-2 3CL Protease in Infected Cells Using Activity-Based Probes with a 2,6-Dichlorobenzoyloxymethyl Ketone Reactive Warhead.

The 3CL protease (3CLpro) is a viral cysteine protease of SARS-CoV-2 and is responsible for the main processing of the viral polyproteins involved in viral replication and proliferation. Despite the importance of 3CLpro as a drug target, the intracellular dynamics of active 3CLpro, including its expression and subcellular localization in SARS-CoV-2-infected cells, are poorly understood. Herein, we report an activity-based probe (ABP) with a clickable alkyne and an irreversible warhead for the SARS-CoV-2 3CL protease. We designed and synthesized two ABPs that contain a chloromethyl ketone (probe 2) or 2,6-dichlorobenzoyloxymethyl ketone (probe 3) reactive group at the P1' site. Labeling of recombinant 3CLpro by the ABPs in the purified and proteome systems revealed that probe 3 displayed ligand-directed and selective labeling against 3CLpro. Labeling of transiently expressed active 3CLpro in COS-7 cells also validated the good target selectivity of probe 3 for 3CLpro. We finally demonstrated that endogenously expressed 3CLpro in SARS-CoV-2-infected cells can be detected by fluorescence microscopy imaging using probe 3, suggesting that active 3CLpro at 5 h postinfection is localized in the juxtanuclear region. To the best of our knowledge, this is the first report investigating the subcellular localization of active 3CLpro by using ABPs. We believe that probe 3 will be a useful chemical tool for acquiring important biological knowledge of active 3CLpro in SARS-CoV-2-infected cells.

E-M349E and NS2A/2B–P1(T) are compensatory mutations of rDTMUV-NS2AB-P1P1’(AA), which regain virus proliferation by enhancing the virus package and restoring NS2A/2B cleavage

Duck Tembusu virus (DTMUV) belongs to the Flaviviridae family and mainly infects ducks. The genome of DTMUV is translated into a polyprotein, which is further cleaved into several protein by viral NS2B3 protease and host proteases. Crucially, the cleavage of the NS2A/2B precursor during this process is essential for the formation of replication complexes and viral packaging. Previous research has demonstrated that alanine mutations in NS2A/2B (P1P1’ (AA)) result in an attenuated strain (rDTMUV-NS2A/2B–P1P1’ (AA)) by disrupting NS2A/2B cleavage. In this study, we investigate the effects of the P1P1’ (AA) mutation on the viral life cycle and explore compensatory mutations in rDTMUV-NS2A/2B–P1P1’ (AA). Infected ducklings exhibit similar body weight gain and viral tissue loads to DTMUV-WT. Compensatory mutations E-M349E and P1(T) emerge, restoring proliferation levels to those of rDTMUV-WT. Specifically, E-M349E enhances viral packaging, while P1(T) reinstates NS2A/2B proteolysis in vitro. Thus, our findings reveal novel compensatory sites capable of restoring the attenuated DTMUV during polyprotein cleavage and packaging.

Novel mutation N588 residue in the NS1 protein of feline parvovirus greatly augments viral replication.

Feline parvovirus (FPV) infection is highly fatal in felines. NS1, which is a key nonstructural protein of FPV, can inhibit host innate immunity and promote viral replication, which is the main reason for the severe pathogenicity of FPV. However, the mechanism by which the NS1 protein disrupts host immunity and regulates viral replication is still unclear. Here, we identified an FPV M1 strain that is regulated by the NS1 protein and has more pronounced suppression of innate immunity, resulting in robust replication. We found that the neutralization titer of the FPV M1 strain was significantly lower than that of the other strains. Moreover, FPV M1 had powerful replication ability, and the FPV M1-NS1 protein had heightened efficacy in repressing interferon-stimulated genes (ISGs) expression. Subsequently, we constructed an FPV reverse genetic system, which confirmed that the N588 residue of FPV M1-NS1 protein is a key amino acid that bolsters viral proliferation. Recombinant virus containing N588 also had stronger ability to inhibit ISGs, and lower ISGs levels promoted viral replication and reduced the neutralization titer of the positive control serum. Finally, we confirmed that the difference in viral replication was abolished in type I IFN receptor knockout cell lines. In conclusion, our results demonstrate that the N588 residue of the NS1 protein is a critical amino acid that promotes viral proliferation by increasing the inhibition of ISGs expression. These insights provide a reference for studying the relationship between parvovirus-mediated inhibition of host innate immunity and viral replication while facilitating improved FPV vaccine production.IMPORTANCEFPV infection is a viral infectious disease with the highest mortality rate in felines. A universal feature of parvovirus is its ability to inhibit host innate immunity, and its ability to suppress innate immunity is mainly accomplished by the NS1 protein. In the present study, FPV was used as a viral model to explore the mechanism by which the NS1 protein inhibits innate immunity and regulates viral replication. Studies have shown that the FPV-NS1 protein containing the N588 residue strongly inhibits the expression of host ISGs, thereby increasing the viral proliferation titer. In addition, the presence of the N588 residue can increase the proliferation titer of the strain 5- to 10-fold without affecting its virulence and immunogenicity. In conclusion, our findings provide new insights and guidance for studying the mechanisms by which parvoviruses suppress innate immunity and for developing high-yielding FPV vaccines.

The topological model of NS4B and its TMD3 in duck TMUV proliferation

Duck Tembusu virus (DTMUV) belongs to the Flaviviridae family and mainly infects ducks. Duck Tembusu virus genome encodes one polyprotein that undergoes cleavage to produce 10 proteins. Among these, NS4B, the largest transmembrane protein, plays a crucial role in the viral life cycle. In this study, we investigated the localization of NS4B and found that it is located in the endoplasmic reticulum, where it co-localizes with DTMUV dsRNA. Subsequently, we confirmed 5 different transmembrane domains of NS4B and discovered that only its transmembrane domain 3 (TMD3) can traverse ER membrane. Then mutations were introduced in the conserved amino acids of NS4B TMD3 of DTMUV replicon and infectious clone. The results showed that V111G, V117G, and I118G mutations enhanced viral RNA replication, while Q104A, T106A, A113L, M116A, H120A, Y121A, and A122G mutations reduced viral replication. Recombinant viruses with these mutations were rescued and studied in BHK21 cells. The findings demonstrated that A113L and H120A mutations led to higher viral titers than the wild-type strain, while Q104A, T106A, V111G, V117G, and Y121A mutations attenuated viral proliferation. Additionally, H120A, M116A, and A122G mutations enhanced viral proliferation. Furthermore, Q104A, T106A, V111G, M116A, V117G, Y121A, and A122G mutants showed reduced viral virulence to 10-d duck embryos. Animal experiments further indicated that all mutation viruses resulted in lower genome copy numbers in the spleen compared to the WT group 5 days postinfection. Our data provide insights into the topological model of DTMUV NS4B, highlighting the essential role of NS4B TMD3 in viral replication and proliferation.

Development of a novel screening questionnaire for brain fog

IntroductionAmidst the widespread proliferation of the COVID-19 virus, brain fog has become one of the most critical issues of public health. Brain fog may lead to sub-health conditions, such as forgetfulness, difficulty thinking, and other related symptoms. Although they are not immediately life-threatening, these sub-health conditions could gradually erode the quality of life. Currently, there is no relevant screening tool for brain fog.ObjectivesThe aim of this study was to develop a reliable screening tool.Methods A web-based brain fog screening questionnaire was developed in the study. It was based on previous studies, which summarized five parts of the most common clinical symptoms after COVID-19: forgetfulness, difficulty thinking, difficulty concentrating, feeling confused, and difficulty finding words or phrases to speak. Unfortunately, these items were used only in a way of yes or no answers in previous studies. Each of these items was expanded to five anchors to evaluate their severity in the study. Cronbach’s alpha coefficient was used to assess internal consistency. K-means clustering was used as a second method to validate the cutoff points. Furthermore, the receiver operating characteristic (ROC) curve was applied to validate the appropriateness of the cutoff point.ResultsThere were 534 participants who completely finished the questionnaire. It includes 183 males and 351 females, and all of them aged between 19 and 81 years. The Cronbach’s Alpha value was 0.821. The cutoff point was at a total score of 6 in terms of K-means. Based on the result, the ROC curve revealed that an area under the curve (AUC) was 0.816 with a confidence interval of 0.784 to 0.849.ConclusionsThe study demonstrated the feasibility and reliability of the web-based screening test for brain fog.Disclosure of InterestNone Declared

Structure elucidation and antiviral activity of a cold water-extracted mannogalactofucan Ts1-1A from Trametes sanguinea against human cytomegalovirus in vitro

In this study, we purified a partially acetylated heteropolysaccharide (Ts1-1A) from the fruit bodies of Trametes sanguinea Lloyd through cold water extraction and serial chromatographic separation. The purified polysaccharide Ts1-1A (12.8 kDa) was characterized as a branched mannogalactofucan with a backbone of alternately connected 1,3-linked α-Fucp and 1,6-linked α-Galp, which was partially substituted by non-reducing end units of β-Manp at O-2 and O-3 positions of 1,6-linked α-Galp. Ts1-1A showed pronounced anti-human cytomegalovirus activity at the concentration of 200 and 500 μg/mL in systematical assessments including morphological changes, western blotting, qPCR, indirect immunofluorescence and tissue culture infective dose assays. Moreover, Ts1-1A exerted its antiviral activity at two distinct stages of viral proliferation manifesting as significantly inhibiting viral protein (IE1/2 and p52) expression and reducing viral gene (UL123, UL44 and UL32) replication in the HCMV-infected WI-38 cells. At viral attachment stage, Ts1-1A interacted with HCMV and prevented HCMV from attaching to its host cells. While at early phase of viral replication stage, Ts1-1A suppressed HCMV replication by downregulating NQO1 and HO-1 proteins related to oxidative stress as an antioxidant. To sum up, Ts1-1A is a promising anti-HCMV agent which could be developed for HCMV infection prevention and therapy.

BmSPP is a virus resistance gene in Bombyx mori.

Signal peptide peptidase (SPP) is an intramembrane protease involved in a variety of biological processes, it participates in the processing of signal peptides after the release of the nascent protein to regulate the endoplasmic reticulum associated degradation (ERAD) pathway, binds misfolded membrane proteins, and aids in their clearance process. Additionally, it regulates normal immune surveillance and assists in the processing of viral proteins. Although SPP is essential for many viral infections, its role in silkworms remains unclear. Studying its role in the silkworm, Bombyx mori , may be helpful in breeding virus-resistant silkworms. First, we performed RT-qPCR to analyze the expression pattern of BmSPP. Subsequently, we inhibited BmSPP using the SPP inhibitor 1,3-di-(N-carboxybenzoyl-L-leucyl-L-leucylaminopropanone ((Z-LL)2-ketone) and downregulated the expression of BmSPP using CRISPR/Cas9 gene editing. Furthermore, we assessed the impact of these interventions on the proliferation of Bombyx mori nucleopolyhedrovirus (BmNPV). We observed a decreased in the expression of BmSPP during viral proliferation. It was found that higher concentration of the inhibitor resulted in greater inhibition of BmNPV proliferation. The down-regulation of BmSPP in both in vivo and in vitro was found to affect the proliferation of BmNPV. In comparison to wild type silkworm, BmSPPKO silkworms exhibited a 12.4% reduction in mortality rate. Collectively, this work demonstrates that BmSPP plays a negative regulatory role in silkworm resistance to BmNPV infection and is involved in virus proliferation and replication processes. This finding suggests that BmSPP servers as a target gene for BmNPV virus resistance in silkworms and can be utilized in resistance breeding programs.

Abstract 5778: Determining the structure and function relationship of the oxysterol-binding protein (OSBP) to guide drug development

Abstract The human Oxysterol Binding Protein Structure and function relationship remains elusive, yet the anti-cancer and antiviral molecules library targeting OSBP and OSBP-Related proteins (ORPs) continues to grow. OSBP is a ubiquitously expressed protein in most eukaryotes, functioning as a master lipid sensor and cholesterol regulator in cells. OSBP and its closely related protein ORP4 are also reported to be essential in several human diseases, including viral infection and cancer cell proliferation. Our lab has shown that the natural product OSW-1 has potential anti-viral and anti-cancer activity through targeting OSBP and ORP4, as confirmed with protein-ligand interactions that showed binding with high affinity. Moreover, several anti-cancer molecules targeting the ORPs provoke different protein conformation changes and, therefore, lead to varying OSBP functions. However, the structure of full-length OSBP is still unknown, which leaves a gap in knowledge regarding the compounds’ mechanism of action or how OSBP could be established as a molecular anti-cancer target. Our goal is to determine the structure and function relationship of OSBP with emphasis on the effect of small molecule ligands on cellular OSBP levels and function. In pursuit of this research goal, we aim to determine the in vitro activity of OSW-1 analogs (OA) isolated in our lab, as well as determine their interaction with OSBP and ORP4 proteins in multiple cancer cell lines. We mainly seek to determine the structure and ligand interaction with the OSBP-ligand binding domain (LBD) and full-length OSBP to shed light on the conformation changes that occur upon ligand binding. Preliminary data indicate that the tested OA compounds are active in multiple cancer cell lines with reduced OSBP levels effect, suggesting a similar mechanism of action to OSW-1. A transient expression of full-length OSBP and LBD showed that the truncated OSBP (LBD) was mostly found in the pellet, which could imply low solubility, while the full-length OSBP showed sufficient solubility. Our efforts are currently focused on purifying full-length OSBP. We are also working towards purifying full-length OSBP with a cleavage site that allows cutting out the LBD, thus solving the insolubility issue. Once obtained, the full-length OSBP and the ligand binding domain will be used for both binding and structure determination studies. Citation Format: Ruth Fiona Bayimenye, Jorge L. Berrios-Rivera, Susan Nimmo, Christina R. Bourne, Anthony W. Burgett. Determining the structure and function relationship of the oxysterol-binding protein (OSBP) to guide drug development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5778.