RESUMEN
The production of Adeno-associated virus (AAV) vectors in the lab setting has typically involved expression in adherent cells followed by purification through ultracentrifugation in density gradients. This production method is, however, not easily scalable, presents high levels of cellular impurities that co-purify with the virus, and results in a mixture of empty and full capsids. Here we describe a detailed AAV production protocol that overcomes these limitations through AAV expression in suspension cells followed by AAV affinity purification and AAV polishing to separate empty and full capsids, resulting in high yields of ultra-pure AAV that is highly enriched in full capsids.
Asunto(s)
Dependovirus , Vectores Genéticos , Dependovirus/genética , Dependovirus/aislamiento & purificación , Vectores Genéticos/genética , Humanos , Cápside/química , Cápside/metabolismo , Virión/aislamiento & purificación , Virión/genética , Células HEK293 , Cromatografía de Afinidad/métodos , Ultracentrifugación/métodos , Proteínas de la Cápside/aislamiento & purificación , Proteínas de la Cápside/genética , Proteínas de la Cápside/química , Proteínas de la Cápside/metabolismoRESUMEN
Current adeno-associated virus (AAV) gene therapy using nature-derived AAVs is limited by non-optimal tissue targeting. In the treatment of muscular diseases (MD), high doses are often required but can lead to severe adverse effects. Here, we rationally design an AAV capsid that specifically targets skeletal muscle to lower treatment doses. We computationally integrate binding motifs of human integrin alphaV beta6, a skeletal muscle receptor, into a liver-detargeting capsid. Designed AAVs show higher productivity and superior muscle transduction compared to their parent. One variant, LICA1, demonstrates comparable muscle transduction to other myotropic AAVs with reduced liver targeting. LICA1's myotropic properties are observed across species, including non-human primate. Consequently, LICA1, but not AAV9, effectively delivers therapeutic transgenes and improved muscle functionality in two mouse MD models (male mice) at a low dose (5E12 vg/kg). These results underline the potential of our design method for AAV engineering and LICA1 variant for MD gene therapy.
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Dependovirus , Terapia Genética , Músculo Esquelético , Dependovirus/genética , Animales , Humanos , Músculo Esquelético/metabolismo , Ratones , Terapia Genética/métodos , Masculino , Vectores Genéticos/genética , Integrinas/metabolismo , Integrinas/genética , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Enfermedades Musculares/terapia , Enfermedades Musculares/genética , Transducción Genética , Hígado/metabolismo , Cápside/metabolismo , Receptores de Vitronectina/metabolismo , Receptores de Vitronectina/genética , Modelos Animales de Enfermedad , Células HEK293 , Transgenes , Ratones Endogámicos C57BL , Antígenos de NeoplasiasRESUMEN
Tumor neoantigen peptide vaccines hold potential for boosting cancer immunotherapy, yet efficiently co-delivering peptides and adjuvants to antigen-presenting cells in vivo remains challenging. Virus-like particle (VLP), which is a kind of multiprotein structure organized as virus, can deliver therapeutic substances into cells and stimulate immune response. However, the weak targeted delivery of VLP in vivo and its susceptibility to neutralization by antibodies hinder their clinical applications. Here, we first designed a novel protein carrier using the mammalian-derived capsid protein PEG10, which can self-assemble into endogenous VLP (eVLP) with high protein loading and transfection efficiency. Then, an engineered tumor vaccine, named ePAC, was developed by packaging genetically encoded neoantigen into eVLP with further modification of CpG-ODN on its surface to serve as an adjuvant and targeting unit to dendritic cells (DCs). Significantly, ePAC can efficiently target and transport neoantigens to DCs, and promote DCs maturation to induce neoantigen-specific T cells. Moreover, in mouse orthotopic liver cancer and humanized mouse tumor models, ePAC combined with anti-TIM-3 exhibited remarkable antitumor efficacy. Overall, these results support that ePAC could be safely utilized as cancer vaccines for antitumor therapy, showing significant potential for clinical translation.
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Antígenos de Neoplasias , Vacunas contra el Cáncer , Células Dendríticas , Animales , Vacunas contra el Cáncer/inmunología , Vacunas contra el Cáncer/genética , Vacunas contra el Cáncer/administración & dosificación , Ratones , Antígenos de Neoplasias/inmunología , Antígenos de Neoplasias/genética , Humanos , Células Dendríticas/inmunología , Vacunas de Partículas Similares a Virus/inmunología , Vacunas de Partículas Similares a Virus/administración & dosificación , Vacunas de Partículas Similares a Virus/genética , Proteínas de la Cápside/inmunología , Proteínas de la Cápside/genética , Péptidos/inmunología , Femenino , Ratones Endogámicos C57BL , Línea Celular Tumoral , VacunaciónRESUMEN
The inner capsid protein of rotavirus, VP6, emerges as a promising candidate for next-generation vaccines against rotaviruses owing to its abundance in virion particles and high conservation. However, the formation of inclusion bodies during prokaryotic VP6 expression poses a significant hurdle to rotavirus research and applications. Here, we employed experimental and computational approaches to investigate inclusion body formation and aggregation-prone regions (APRs). Heterologous recombinant VP6 expression in Escherichia coli BL21(DE3) cells resulted in inclusion body formation, confirmed by transmission electron microscopy revealing amorphous aggregates. Thioflavin T assay demonstrated incubation temperature-dependent aggregation of VP6 inclusion bodies. Computational predictions of APRs in rotavirus A VP6 protein were performed using sequence-based tools (TANGO, AGGRESCAN, Zyggregator, Waltz, FoldAmyloid, ANuPP, Camsol intrinsic) and structure-based tools (SolubiS, CamSol structurally corrected, Aggrescan3D). A total of 24 consensus APRs were identified, with 21 of them being surface-exposed in VP6. All identified APRs display a predominance of hydrophobic amino acids, ranging from 33 to 100%. Computational identification of these APRs corroborates our experimental observation of VP6 inclusion body or aggregate formation. Characterization of VP6's aggregation propensity facilitates understanding of its behaviour during prokaryotic expression and opens avenues for protein engineering of soluble variants, advancing research on rotavirus VP6 in pathology, therapy, and diagnostics.
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Antígenos Virales , Proteínas de la Cápside , Escherichia coli , Cuerpos de Inclusión , Rotavirus , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Antígenos Virales/genética , Antígenos Virales/metabolismo , Cuerpos de Inclusión/metabolismo , Rotavirus/genética , Rotavirus/metabolismo , Agregado de Proteínas , Simulación por Computador , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
BACKGROUND: Global cyclical outbreaks of human enterovirus infections has positioned human enterovirus A71 (EV-A71) as a neurotropic virus of clinical importance. However, there remains a scarcity of internationally approved antivirals and vaccines. METHODS: In pursuit of repurposing drugs for combating human enteroviruses, we employed a comprehensive pharmacophore- and molecular docking-based virtual screen targeting EV-A71 capsid protein VP1-4, 3C protease, and 3D polymerase proteins. Among 15 shortlisted ligand candidates, we dissected the inhibitory mechanism of Tanomastat in cell-based studies and evaluated its in vivo efficacy in an EV-A71-infected murine model. FINDINGS: We demonstrated that Tanomastat exerts dose-dependent inhibition on EV-A71 replication, with comparable efficacy profiles in enterovirus species A, B, C, and D in vitro. Time-course studies suggested that Tanomastat predominantly disrupts early process(es) of the EV-A71 replication cycle. Mechanistically, live virus particle tracking and docking predictions revealed that Tanomastat specifically impedes viral capsid dissociation, potentially via VP1 hydrophobic pocket binding. Bypassing its inhibition on entry stages, we utilized EV-A71 replication-competent, 3Dpol replication-defective, and bicistronic IRES reporter replicons to show that Tanomastat also inhibits viral RNA replication, but not viral IRES translation. We further showed that orally administered Tanomastat achieved 85% protective therapeutic effect and alleviated clinical symptoms in EV-A71-infected neonatal mice. INTERPRETATION: Our study establishes Tanomastat as a broad-spectrum anti-enterovirus candidate with promising pre-clinical efficacy, warranting further testing for potential therapeutic application. FUNDING: MOE Tier 2 grants (MOE-T2EP30221-0005, R571-000-068-592, R571-000-076-515, R571-000-074-733) and A∗STARBiomedical Research Council (BMRC).
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Antivirales , Infecciones por Enterovirus , Simulación del Acoplamiento Molecular , Replicación Viral , Replicación Viral/efectos de los fármacos , Humanos , Animales , Antivirales/farmacología , Antivirales/química , Ratones , Infecciones por Enterovirus/tratamiento farmacológico , Infecciones por Enterovirus/virología , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Proteínas de la Cápside/antagonistas & inhibidores , ARN Viral/genética , ARN Viral/metabolismo , Cápside/metabolismo , Cápside/efectos de los fármacos , Modelos Animales de Enfermedad , Enterovirus Humano A/efectos de los fármacos , Enterovirus Humano A/genética , Enterovirus Humano A/fisiología , Enterovirus/efectos de los fármacos , Enterovirus/genética , Línea Celular , Replicación de ARNRESUMEN
The HIV-1 capsid is composed of capsid (CA) protein hexamers and pentamers (capsomers) that contain a central pore hypothesised to regulate capsid assembly and facilitate nucleotide import early during post-infection. These pore functions are mediated by two positively charged rings created by CA Arg-18 (R18) and Lys-25 (K25). Here we describe the forced evolution of viruses containing mutations in R18 and K25. Whilst R18 mutants fail to replicate, K25A viruses acquire compensating mutations that restore nearly wild-type replication fitness. These compensating mutations, which rescue reverse transcription and infection without reintroducing lost pore charges, map to three adaptation hot-spots located within and between capsomers. The second-site suppressor mutations act by restoring the formation of pentamers lost upon K25 mutation, enabling closed conical capsid assembly both in vitro and inside virions. These results indicate that there is no intrinsic requirement for K25 in either nucleotide import or capsid assembly. We propose that whilst HIV-1 must maintain a precise hexamer:pentamer equilibrium for proper capsid assembly, compensatory mutations can tune this equilibrium to restore fitness lost by mutation of the central pore.
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Proteínas de la Cápside , Cápside , VIH-1 , Mutación , Ensamble de Virus , Replicación Viral , VIH-1/genética , VIH-1/fisiología , Ensamble de Virus/genética , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Proteínas de la Cápside/química , Cápside/metabolismo , Humanos , Replicación Viral/genética , Virión/metabolismo , Virión/genética , Células HEK293 , Infecciones por VIH/virología , Infecciones por VIH/genéticaRESUMEN
Nanoparticles have gained attention as potential antiviral agents, but the effects of graphene oxide nanoparticles (GONPs) on influenza virus remain unclear. In this study, we evaluated the antiviral activity of GONPs against influenza virus strain A/Hunan-Lengshuitan/11197/2013(H9N2). Our results show that GONPs with a diameter of 4 nm exerted an antiviral effect, whereas those with a diameter of 400 nm had no effect. Treatment with 4-nm GONPs reduced viral titers by more than 99% and inhibited viral nucleoprotein expression in a dose-dependent manner. We also confirmed that 4-nm GONPs inhibited the infectivity of H9N2 in MDCK cells. A transmission electron microscopic analysis revealed morphological abnormalities in the GONP-treated virus, including the destruction of the envelope glycoprotein spikes and an irregular shape, suggesting that GONPs cause the destruction of the viral coat proteins. Our results highlight the potential utility of GONPs in the prevention and treatment of viral infections, especially those of emerging and re-emerging viruses.
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Antivirales , Grafito , Subtipo H9N2 del Virus de la Influenza A , Nanopartículas , Grafito/farmacología , Grafito/química , Subtipo H9N2 del Virus de la Influenza A/efectos de los fármacos , Subtipo H9N2 del Virus de la Influenza A/fisiología , Subtipo H9N2 del Virus de la Influenza A/genética , Animales , Perros , Antivirales/farmacología , Células de Riñón Canino Madin Darby , Nanopartículas/química , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
Human noroviruses (HuNoVs) are a leading cause of acute viral gastroenteritis worldwide. Infectious outbreaks due to recombinant NoV genotype called GII.P16-GII.2 have been frequently reported since 2016. In this study, we expressed the major capsid protein VP1 from three GII.2 NoV strains using the recombinant baculovirus expression system. The assembly, histo-blood group antigen (HBGA)-binding patterns, and cross-blocking abilities of VP1 proteins were investigated. All the three NoV VP1 proteins successfully assembled into virus-like particles (VLPs). The HBGA-binding assay demonstrated a temporal binding pattern. The latest isolate bound to saliva samples of all blood types. Sequence alignment suggested that the observed gain in HBGA-binding ability was attributed to a limited number of amino acid mutations. Using chimeric VP1 proteins, we demonstrated that synergistic effects resulted in enhanced binding ability. Bile salts increased GII.2 VLP avidity for HBGAs except GII.2-2011/M1. In vitro blockade assay of salivary HBGA-VLP binding demonstrated the presence of cross-blocking effects among different strains. This study provides insight into the evolutionary binding characteristics and cross-blocking effects of GII.2 NoVs to facilitate the development of measures to control this type of viruses.
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Antígenos de Grupos Sanguíneos , Proteínas de la Cápside , Norovirus , Norovirus/genética , Norovirus/metabolismo , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Proteínas de la Cápside/inmunología , Humanos , Antígenos de Grupos Sanguíneos/metabolismo , Infecciones por Caliciviridae/virología , Infecciones por Caliciviridae/inmunología , Unión Proteica , Genotipo , Saliva/virología , Gastroenteritis/virología , Secuencia de AminoácidosRESUMEN
BACKGROUND: Human adenovirus (HAdV) is an important pathogen causing acute respiratory infection (ARI) in children. Many countries, including China, have experienced sporadic or outbreaks related to HAdV-4, and death cases were reported. However, there is little research on HAdV-4 and the epidemic situation of HAdV-4 in China is little known. This study was designed to comprehend the prevalence and genetic characteristics of HAdV-4 in ARI children in China. METHODS: Respiratory tract samples from ARI children hospitalized in six hospitals of Northern and Southern China from 2017 to 2020 were collected for HAdV detection and typing. Clinical information was collected from HAdV-4 positive patients for clinical characteristics and epidemiological analysis. The main capsid proteins and the whole genome sequences were amplified and sequenced for bioinformatics analysis. RESULTS: There were 2847 ARI children enrolled, and 156 (5.48%) HAdV positive samples were detected. Eleven HAdV-4 positive samples were identified, accounting for 0.39% of the total samples and 7.05% of the HAdV positive samples. The main manifestations were fever and cough. Two children had conjunctivitis. Two children were diagnosed with severe pneumonia and developed respiratory failure. One of them developed hemophagocytic syndrome and checked in pediatric intensive care unit (PICU). This child had ventricular septal defect. All the children recovered. The isolated strains of HAdV-4 obtained in this study and the reference strains from China located in the same phylogenetic branch (HAdV-4a), while the prototype strain and vaccine strains formed another branch (HAdV-4p). Upon comparison with the prototype strain, there were a few amino acid mutations existing in three major capsid proteins. According to recombination analysis, no new recombination was found. CONCLUSIONS: The detection rate of HAdV-4 in children hospitalized with ARI was 0.39% in the total samples and 7.05% of all HAdV positive samples. HAdV-4 isolates obtained in this study and other reference strains from China belonged to the HAdV-4a subtype. Our data provided reference for the monitoring, prevention and control of HAdV-4, as well as the research and development of vaccines and drugs.
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Infecciones por Adenovirus Humanos , Adenovirus Humanos , Filogenia , Infecciones del Sistema Respiratorio , Humanos , China/epidemiología , Adenovirus Humanos/genética , Adenovirus Humanos/aislamiento & purificación , Adenovirus Humanos/clasificación , Infecciones del Sistema Respiratorio/virología , Infecciones del Sistema Respiratorio/epidemiología , Infecciones por Adenovirus Humanos/epidemiología , Infecciones por Adenovirus Humanos/virología , Masculino , Preescolar , Femenino , Estudios Prospectivos , Lactante , Niño , Proteínas de la Cápside/genética , PrevalenciaRESUMEN
Adeno-associated virus (AAV)-based gene therapy is experiencing a rapid growth in the field of medicine and holds great promise in combating a wide range of human diseases. For successful development of AAV-based products, comprehensive thermal stability studies are often required to establish storage conditions and shelf life. However, as a relatively new modality, limited studies have been reported to elucidate the chemical degradation pathways of AAV products under thermal stress conditions. In this study, we first presented an intriguing difference in charge profile shift between thermally stressed AAV8 and AAV1 capsids when analyzed by anion exchange chromatography. Subsequently, a novel and robust peptide mapping protocol was developed and applied to elucidate the underlying chemical degradation pathways of thermally stressed AAV8 and AAV1. Compared to the conventional therapeutic proteins, the unique structure of AAV capsids also led to some key differences in how modifications at specific sites may impact the overall charge properties. Finally, despite the high sequency identity, the analysis revealed that the opposite charge profile shifts between thermally stressed AAV8 and AAV1 could be mainly attributed to a single modification unique to each serotype.
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Dependovirus , Mapeo Peptídico , Dependovirus/genética , Dependovirus/química , Cromatografía por Intercambio Iónico/métodos , Mapeo Peptídico/métodos , Cápside/química , Calor , Humanos , Proteínas de la Cápside/química , Vectores Genéticos/química , Terapia Genética/métodos , Cromatografía Liquida/métodos , Cromatografía Líquida con Espectrometría de MasasRESUMEN
Adeno-associated viruses (AAVs) are foundational gene delivery tools for basic science and clinical therapeutics. However, lack of mechanistic insight, especially for engineered vectors created by directed evolution, can hamper their application. Here, we adapt an unbiased human cell microarray platform to determine the extracellular and cell surface interactomes of natural and engineered AAVs. We identify a naturally-evolved and serotype-specific interaction between the AAV9 capsid and human interleukin 3 (IL3), with possible roles in host immune modulation, as well as lab-evolved low-density lipoprotein receptor-related protein 6 (LRP6) interactions specific to engineered capsids with enhanced blood-brain barrier crossing in non-human primates after intravenous administration. The unbiased cell microarray screening approach also allows us to identify off-target tissue binding interactions of engineered brain-enriched AAV capsids that may inform vectors' peripheral organ tropism and side effects. Our cryo-electron tomography and AlphaFold modeling of capsid-interactor complexes reveal LRP6 and IL3 binding sites. These results allow confident application of engineered AAVs in diverse organisms and unlock future target-informed engineering of improved viral and non-viral vectors for non-invasive therapeutic delivery to the brain.
Asunto(s)
Barrera Hematoencefálica , Dependovirus , Interleucina-3 , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad , Transcitosis , Animales , Humanos , Barrera Hematoencefálica/metabolismo , Encéfalo/metabolismo , Cápside/metabolismo , Proteínas de la Cápside/metabolismo , Proteínas de la Cápside/genética , Proteínas de la Cápside/inmunología , Dependovirus/genética , Dependovirus/metabolismo , Vectores Genéticos/genética , Vectores Genéticos/administración & dosificación , Células HEK293 , Interleucina-3/metabolismo , Unión Proteica , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismoRESUMEN
A novel virus, temporarily named "Arctic wolf parvovirus" (AWPV), was discovered in a pharyngeal metagenomic library derived from an Arctic wolf (Canis lupus arctos) in China. The genome sequence was assigned GenBase accession number C_AA071902.1. AWPV has a genome comprised of 4,920 base pairs with a nucleotide composition of 36.4% A, 23.4% T, 18.2% G, and 22.0% C, with a GC content of 40.2%. Its structure resembles parvoviruses, containing two open reading frames: the nonstructural (NS) region encoding replication enzymes and the structural (VP) region encoding capsid protein. Pairwise sequence comparison and phylogenetic analysis suggest AWPV may represent a novel species within the genus Protoparvovirus. This discovery enhances our understanding of mammalian virus ecology and potential future infectious diseases.
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Genoma Viral , Sistemas de Lectura Abierta , Parvovirus , Filogenia , Lobos , Animales , Lobos/virología , Parvovirus/genética , Parvovirus/clasificación , Parvovirus/aislamiento & purificación , Proteínas de la Cápside/genética , Infecciones por Parvoviridae/virología , Infecciones por Parvoviridae/veterinaria , Composición de Base , China , ADN Viral/genéticaRESUMEN
Papaya ringspot virus (PRSV) is a catastrophic disease that causes huge yield losses in papaya cultivation around the world. Yield losses in severely infected plants can be upto 100%. Because of this disease, papaya cultivation has been shifted to other crops in some areas of the world. Many conventional methods and breeding approaches are used against this disease, which turns out to be less effective. Considering the yield loss caused by PRSV in papaya, it is high time to focus on alternative control methods. To implement effective management strategies, molecular approaches such as Marker Assisted Breeding (MAS) or transgenic methods involving post-transcriptional gene silencing targeting the genome viz., coat protein, replicase gene, or HC Pro can be pursued. However, the public's reluctance to widely accept the transgenic approach due to health and environmental concerns necessitates a consideration of non-transgenic alternatives. Prioritizing safety and ensuring efficient virus control, non-transgenic approaches which encompass cross-protection, genome editing, and topical applications of dsRNA to induce gene silencing within the host, can be adopted. This review aims to provide comprehensive insights of various molecular tools used in managing PRSV which in turn will help in sustainable agriculture.
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Carica , Enfermedades de las Plantas , Potyvirus , Carica/virología , Carica/genética , Enfermedades de las Plantas/virología , Enfermedades de las Plantas/genética , Potyvirus/genética , Potyvirus/patogenicidad , Plantas Modificadas Genéticamente/genética , Fitomejoramiento/métodos , Resistencia a la Enfermedad/genética , Edición Génica/métodos , Proteínas de la Cápside/genética , Silenciador del GenRESUMEN
Despite the high prevalence of BK polyomavirus (BKPyV) and the associated risk for BKPyV-associated nephropathy (BKPyVAN) in kidney transplant (KTX) recipients, many details on viral processes such as replication, maturation, assembly and virion release from host cells have not been fully elucidated. VP1 is a polyomavirus-specific protein that is expressed in the late phase of its replicative cycle with important functions in virion assembly and infectious particle release. This study investigated the localization and time-dependent changes in the distribution of VP1-positive viral particles and their association within the spectrum of differing cell morphologies that are observed in the urine of KTX patients upon active BKPyV infection. We found highly differing recognition patterns of two anti-VP1 antibodies with respect to intracellular and extracellular VP1 localization, pointing towards independent binding sites that were seemingly associated with differing stages of virion maturation. Cells originating from single clones were stably cultured out of the urine sediment of KTX recipients with suspected BKPyVAN. The cell morphology, polyploidy, virus replication and protein production were investigated by confocal microscopy using both a monoclonal (mAb 4942) and a polyclonal rabbit anti-VP1-specific antibody (RantiVP1 Ab). Immunoblotting was performed to investigate changes in the VP1 protein. Both antibodies visualized VP1 and the mAb 4942 recognized VP1 in cytoplasmic vesicles exhibiting idiomorphic sizes when released from the cells. In contrast, the polyclonal antibody detected VP1 within the nucleus and in cytoplasm in colocalization with the endoplasmic reticulum marker CNX. At the nuclear rim, VP1 was recognized by both antibodies. Immunoblotting revealed two smaller versions of VP1 in urinary decoy cell extracts, potentially from different translation start sites as evaluated by in silico analysis. Oxford Nanopore sequencing showed integration of BKPyV DNA in chromosomes 3, 4 and 7 in one of the five tested primary cell lines which produced high viral copies throughout four passages before transcending into senescence. The different staining with two VP1-specific antibodies emphasizes the modification of VP1 during the process of virus maturation and cellular exit. The integration of BKPyV into the human genome leads to high virus production; however, this alone does not transform the cell line into a permanently cycling and indefinitely replicating one.
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Virus BK , Vesículas Extracelulares , Infecciones por Polyomavirus , Esparcimiento de Virus , Virus BK/fisiología , Virus BK/metabolismo , Virus BK/genética , Humanos , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/virología , Infecciones por Polyomavirus/virología , Infecciones por Polyomavirus/metabolismo , Replicación Viral , Trasplante de Riñón , Virión/metabolismo , Proteínas de la Cápside/metabolismo , Proteínas de la Cápside/genética , Núcleo Celular/metabolismo , Ensamble de Virus , Infecciones Tumorales por Virus/virología , Infecciones Tumorales por Virus/metabolismo , Transformación Celular Viral , Masculino , AnimalesRESUMEN
African swine fever (ASF) is a deadly hemorrhagic disease of domestic and wild swine that was first described in the early 20th century after the introduction of European pigs to Kenya. The etiological agent, the African swine fever virus (ASFV), is a large DNA virus within the Asfarviridae family that is broadly categorized epidemiologically into genotypes based on the nucleotide sequence of B646L, the gene encoding the major capsid protein p72. ASF outbreaks in Africa have been linked historically to 25 genotypes by p72 nucleotide analysis and, recently, to 6 genotypes by amino acid comparison, whereas global outbreaks of ASF outside of Africa have only been linked to 2 genotypes: genotype I, which led to an outbreak in Europe during the 1960s that later spread to South America, and genotype II, responsible for the current pandemic that began in Georgia in 2007 and has since spread to Europe, Asia, and Hispaniola. Here, we present an analysis of the genome of ASFV Spencer, an isolate that was collected in 1951 near Johannesburg, South Africa. While nucleotide analysis of Spencer indicates the p72 coding sequence is unique, differentiating from the closest reference by five nucleotides, the predicted amino acid sequence indicates that it is 100% homologous to contemporary genotype 1. Full genome analysis reveals it is more similar to Mkuzi1979 and encodes genes that share similarity with either genotype 1 or genotype 2 outbreak strains.
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Virus de la Fiebre Porcina Africana , Fiebre Porcina Africana , Brotes de Enfermedades , Genoma Viral , Genotipo , Filogenia , Virus de la Fiebre Porcina Africana/genética , Virus de la Fiebre Porcina Africana/aislamiento & purificación , Virus de la Fiebre Porcina Africana/clasificación , Fiebre Porcina Africana/virología , Fiebre Porcina Africana/epidemiología , Animales , Brotes de Enfermedades/veterinaria , Porcinos , Sudáfrica/epidemiología , Proteínas de la Cápside/genética , Análisis de Secuencia de ADN , Historia del Siglo XXRESUMEN
The application of nanoscale scaffolds has become a promising strategy in vaccine design, with protein-based nanoparticles offering desirable avenues for the biocompatible and efficient delivery of antigens. Here, we presented a novel endogenous capsid-forming protein, activated-regulated cytoskeleton-associated protein (ARC), which could be engineered through the plug-and-play strategy (SpyCatcher3/SpyTag3) for multivalent display of antigens. Combined with the self-assembly capacity and flexible modularity of ARC, ARC-based vaccines elicited robust immune responses against Mpox or SARS-CoV-2, comparable to those induced by ferritin-based vaccines. Additionally, ARC-based nanoparticles functioned as immunostimulants, efficiently stimulating dendritic cells and facilitating germinal center responses. Even without adjuvants, ARC-based vaccines generated protective immune responses in a lethal challenge model. Hence, this study showed the feasibility of ARC as a novel protein-based nanocarrier for multivalent surface display of pathogenic antigens and demonstrated the potential of exploiting recombinant mammalian retrovirus-like protein as a delivery vehicle for bioactive molecules.
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Vacunas contra la COVID-19 , COVID-19 , Nanopartículas , SARS-CoV-2 , Animales , Nanopartículas/química , Ratones , SARS-CoV-2/inmunología , COVID-19/prevención & control , Vacunas contra la COVID-19/inmunología , Vacunas contra la COVID-19/química , Humanos , Ratones Endogámicos BALB C , Proteínas de la Cápside/química , Proteínas de la Cápside/inmunología , Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/metabolismo , Femenino , Células Dendríticas/inmunología , Nanovacunas , Proteínas del Tejido NerviosoRESUMEN
Zika virus (ZIKV), a mosquito-borne Flavivirus of international concern, causes congenital microcephaly in newborns and Guillain-Barré syndrome in adults. ZIKV capsid (C) protein, one of three key structural proteins, is essential for viral assembly and encapsidation. In dengue virus, a closely related flavivirus, the homologous C protein interacts with host lipid systems, namely intracellular lipid droplets, for successful viral replication. Here, we investigate ZIKV C interaction with host lipid systems, showing that it binds host lipid droplets but, contrary to expected, in an unspecific manner. Contrasting with other flaviviruses, ZIKV C also does not bind very-low density-lipoproteins. Comparing with other Flavivirus, capsid proteins show that ZIKV C structure is particularly thermostable and seems to be locked into an auto-inhibitory conformation due to a disordered N-terminal, hence blocking specific interactions and supporting the experimental differences observed. Such distinct structural features must be considered when targeting capsid proteins in drug development.
Asunto(s)
Proteínas de la Cápside , Virus Zika , Virus Zika/química , Virus Zika/metabolismo , Proteínas de la Cápside/química , Proteínas de la Cápside/metabolismo , Humanos , Unión Proteica , Modelos MolecularesRESUMEN
Natural products are a valuable resource for the discovery of novel crop protection agents. A series of γ-butyrolactone derivatives, derived from the simplification of podophyllotoxin's structure, were synthesized and assessed for their efficacy against tobacco mosaic virus (TMV). Several derivatives exhibited notable antiviral properties, with compound 3g demonstrating the most potent in vivo anti-TMV activity. At 500 µg/mL, compound 3g achieved an inactivation effect of 87.8%, a protective effect of 71.7%, and a curative effect of 67.7%, surpassing the effectiveness of the commercial plant virucides ningnanmycin and ribavirin. Notably, the syn-diastereomer (syn-3g) exhibited superior antiviral activity compared to the anti-diastereomer (anti-3g). Mechanistic studies revealed that syn-3g could bind to the TMV coat protein and interfere with the self-assembly process of TMV particles. These findings indicate that compound 3g, with its simple chemical structure, could be a potential candidate for the development of novel antiviral agents for crop protection.
Asunto(s)
4-Butirolactona , Antivirales , Podofilotoxina , Virus del Mosaico del Tabaco , Podofilotoxina/química , 4-Butirolactona/análogos & derivados , 4-Butirolactona/farmacología , Antivirales/síntesis química , Antivirales/farmacología , Virus del Mosaico del Tabaco/efectos de los fármacos , Ensamble de Virus/efectos de los fármacos , Proteínas de la Cápside/metabolismo , Protección de Cultivos , Cristalografía por Rayos X , Relación Estructura-Actividad , Nicotiana/efectos de los fármacos , Nicotiana/metabolismo , Nicotiana/virología , Simulación del Acoplamiento MolecularRESUMEN
Cyclization provides a general strategy for improving the proteolytic stability, cell membrane permeability and target binding affinity of peptides. Insertion of a stable, non-reducible linker into a disulphide bond is a commonly used approach for cyclizing phage-displayed peptides. However, among the vast collection of cysteine reactive linkers available, few provide the selectivity required to target specific cysteine residues within the peptide in the phage display system, whilst sparing those on the phage capsid. Here, we report the development of a cyclopropenone-based proximity-driven chemical linker that can efficiently cyclize synthetic peptides and peptides fused to a phage-coat protein, and cyclize phage-displayed peptides in a site-specific manner, with no disruption to phage infectivity. Our cyclization strategy enables the construction of stable, highly diverse phage display libraries. These libraries can be used for the selection of high-affinity cyclic peptide binders, as exemplified through model selections on streptavidin and the therapeutic target αvß3.
Asunto(s)
Biblioteca de Péptidos , Péptidos Cíclicos , Ciclización , Péptidos Cíclicos/química , Péptidos Cíclicos/metabolismo , Estreptavidina/química , Estreptavidina/metabolismo , Humanos , Proteínas de la Cápside/química , Proteínas de la Cápside/metabolismo , Proteínas de la Cápside/genética , Cisteína/química , Cisteína/metabolismo , Ciclopropanos/química , Péptidos/química , Péptidos/metabolismoRESUMEN
Broadening gene therapy applications requires manufacturable vectors that efficiently transduce target cells in humans and preclinical models. Conventional selections of adeno-associated virus (AAV) capsid libraries are inefficient at searching the vast sequence space for the small fraction of vectors possessing multiple traits essential for clinical translation. Here, we present Fit4Function, a generalizable machine learning (ML) approach for systematically engineering multi-trait AAV capsids. By leveraging a capsid library that uniformly samples the manufacturable sequence space, reproducible screening data are generated to train accurate sequence-to-function models. Combining six models, we designed a multi-trait (liver-targeted, manufacturable) capsid library and validated 88% of library variants on all six predetermined criteria. Furthermore, the models, trained only on mouse in vivo and human in vitro Fit4Function data, accurately predicted AAV capsid variant biodistribution in macaque. Top candidates exhibited production yields comparable to AAV9, efficient murine liver transduction, up to 1000-fold greater human hepatocyte transduction, and increased enrichment relative to AAV9 in a screen for liver transduction in macaques. The Fit4Function strategy ultimately makes it possible to predict cross-species traits of peptide-modified AAV capsids and is a critical step toward assembling an ML atlas that predicts AAV capsid performance across dozens of traits.