RESUMO
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.
Assuntos
Dependovirus , Vetores Genéticos , Dependovirus/genética , Dependovirus/isolamento & purificação , Vetores Genéticos/genética , Humanos , Capsídeo/química , Capsídeo/metabolismo , Vírion/isolamento & purificação , Vírion/genética , Células HEK293 , Cromatografia de Afinidade/métodos , Ultracentrifugação/métodos , Proteínas do Capsídeo/isolamento & purificação , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismoRESUMO
DEV is an obligatory lytic Pseudomonas phage of the N4-like genus, recently reclassified as Schitoviridae. The DEV genome encodes 91 ORFs, including a 3398 amino acid virion-associated RNA polymerase (vRNAP). Here, we describe the complete architecture of DEV, determined using a combination of cryo-electron microscopy localized reconstruction, biochemical methods, and genetic knockouts. We built de novo structures of all capsid factors and tail components involved in host attachment. We demonstrate that DEV long tail fibers are essential for infection of Pseudomonas aeruginosa but dispensable for infecting mutants with a truncated lipopolysaccharide devoid of the O-antigen. We determine that DEV vRNAP is part of a three-gene operon conserved in 191 Schitoviridae genomes. We propose these three proteins are ejected into the host to form a genome ejection motor spanning the cell envelope. We posit that the design principles of the DEV ejection apparatus are conserved in all Schitoviridae.
Assuntos
Microscopia Crioeletrônica , Genoma Viral , Fagos de Pseudomonas , Pseudomonas aeruginosa , Fagos de Pseudomonas/genética , Fagos de Pseudomonas/ultraestrutura , Genoma Viral/genética , Pseudomonas aeruginosa/virologia , Pseudomonas aeruginosa/genética , RNA Polimerases Dirigidas por DNA/metabolismo , RNA Polimerases Dirigidas por DNA/genética , Vírion/ultraestrutura , Vírion/genética , Fases de Leitura Aberta/genética , Proteínas Virais/genética , Proteínas Virais/metabolismo , Proteínas Virais/química , Óperon/genética , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Proteínas do Capsídeo/química , Capsídeo/metabolismo , Capsídeo/ultraestruturaRESUMO
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.
Assuntos
Dependovirus , Terapia Genética , Músculo Esquelético , Dependovirus/genética , Animais , Humanos , Músculo Esquelético/metabolismo , Camundongos , Terapia Genética/métodos , Masculino , Vetores Genéticos/genética , Integrinas/metabolismo , Integrinas/genética , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Doenças Musculares/terapia , Doenças Musculares/genética , Transdução Genética , Fígado/metabolismo , Capsídeo/metabolismo , Receptores de Vitronectina/metabolismo , Receptores de Vitronectina/genética , Modelos Animais de Doenças , Células HEK293 , Transgenes , Camundongos Endogâmicos C57BL , Antígenos de NeoplasiasRESUMO
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.
Assuntos
Proteínas do Capsídeo , Capsídeo , HIV-1 , Mutação , Montagem de Vírus , Replicação Viral , HIV-1/genética , HIV-1/fisiologia , Montagem de Vírus/genética , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Proteínas do Capsídeo/química , Capsídeo/metabolismo , Humanos , Replicação Viral/genética , Vírion/metabolismo , Vírion/genética , Células HEK293 , Infecções por HIV/virologia , Infecções por HIV/genéticaRESUMO
HIV-1 infection requires passage of the viral core through the nuclear pore of the cell, a process that depends on functions of the viral capsid. Recent studies have shown that HIV-1 cores enter the nucleus prior to capsid disassembly. Interactions of the viral capsid with the nuclear pore complex are necessary but not sufficient for nuclear entry, and the mechanism by which the viral core traverses the comparably sized nuclear pore is unknown. Here we show that the HIV-1 core is highly elastic and that this property is linked to nuclear entry and infectivity. Using atomic force microscopy-based approaches, we found that purified wild type cores rapidly returned to their normal conical morphology following a severe compression. Results from independently performed molecular dynamic simulations of the mature HIV-1 capsid also revealed its elastic property. Analysis of four HIV-1 capsid mutants that exhibit impaired nuclear entry revealed that the mutant viral cores are brittle. Adaptation of two of the mutant viruses in cell culture resulted in additional substitutions that restored elasticity and rescued infectivity and nuclear entry. We also show that capsid-targeting compound PF74 and the antiviral drug Lenacapavir reduce core elasticity and block HIV-1 nuclear entry at concentrations that preserve interactions between the viral core and the nuclear envelope. Our results indicate that elasticity is a fundamental property of the HIV-1 core that enables nuclear entry, thereby facilitating infection. These results provide new insights into the role of the capsid in HIV-1 nuclear entry and the antiviral mechanisms of HIV-1 capsid inhibitors.
Assuntos
Elasticidade , Infecções por HIV , HIV-1 , HIV-1/fisiologia , Humanos , Infecções por HIV/virologia , Infecções por HIV/metabolismo , Internalização do Vírus , Capsídeo/metabolismo , Núcleo Celular/metabolismo , Núcleo Celular/virologia , Simulação de Dinâmica Molecular , Microscopia de Força Atômica , Poro Nuclear/metabolismo , Indóis , Fenilalanina/análogos & derivadosRESUMO
Hepatitis B virus (HBV) exploits the endosomal sorting complexes required for transport (ESCRT)/multivesicular body (MVB) pathway for virion budding. In addition to enveloped virions, HBV-replicating cells nonlytically release non-enveloped (naked) capsids independent of the integral ESCRT machinery, but the exact secretory mechanism remains elusive. Here, we provide more detailed information about the existence and characteristics of naked capsid, as well as the viral and host regulations of naked capsid egress. HBV capsid/core protein has two highly conserved Lysine residues (K7/K96) that potentially undergo various types of posttranslational modifications for subsequent biological events. Mutagenesis study revealed that the K96 residue is critical for naked capsid egress, and the intracellular egress-competent capsids are associated with ubiquitinated host proteins. Consistent with a previous report, the ESCRT-III-binding protein Alix and its Bro1 domain are required for naked capsid secretion through binding to intracellular capsid, and we further found that the ubiquitinated Alix binds to wild type capsid but not K96R mutant. Moreover, screening of NEDD4 E3 ubiquitin ligase family members revealed that AIP4 stimulates the release of naked capsid, which relies on AIP4 protein integrity and E3 ligase activity. We further demonstrated that AIP4 interacts with Alix and promotes its ubiquitination, and AIP4 is essential for Alix-mediated naked capsid secretion. However, the Bro1 domain of Alix is non-ubiquitinated, indicating that Alix ubiquitination is not absolutely required for AIP4-induced naked capsid secretion. Taken together, our study sheds new light on the mechanism of HBV naked capsid egress in viral life cycle.
Assuntos
Capsídeo , Vírus da Hepatite B , Ubiquitina-Proteína Ligases Nedd4 , Ubiquitina-Proteína Ligases , Liberação de Vírus , Humanos , Proteínas de Ligação ao Cálcio , Capsídeo/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Hepatite B/metabolismo , Hepatite B/virologia , Vírus da Hepatite B/metabolismo , Vírus da Hepatite B/fisiologia , Vírus da Hepatite B/genética , Ubiquitina-Proteína Ligases Nedd4/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Liberação de Vírus/fisiologiaRESUMO
Molecular details of genome packaging are little understood for the majority of viruses. In enteroviruses (EVs), cleavage of the structural protein VP0 into VP4 and VP2 is initiated by the incorporation of RNA into the assembling virion and is essential for infectivity. We have applied a combination of bioinformatic, molecular and structural approaches to generate the first high-resolution structure of an intermediate in the assembly pathway, termed a provirion, which contains RNA and intact VP0. We have demonstrated an essential role of VP0 E096 in VP0 cleavage independent of RNA encapsidation and generated a new model of capsid maturation, supported by bioinformatic analysis. This provides a molecular basis for RNA-dependence, where RNA induces conformational changes required for VP0 maturation, but that RNA packaging itself is not sufficient to induce maturation. These data have implications for understanding production of infectious virions and potential relevance for future vaccine and antiviral drug design.
Assuntos
Proteínas do Capsídeo , Montagem de Vírus , Montagem de Vírus/fisiologia , Proteínas do Capsídeo/metabolismo , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Humanos , RNA Viral/genética , RNA Viral/metabolismo , Vírion/metabolismo , Enterovirus/fisiologia , Capsídeo/metabolismo , Infecções por Enterovirus/virologia , Infecções por Enterovirus/metabolismoRESUMO
In response to the escalating antibiotic resistance in multidrug-resistant pathogens, we propose an innovative phagemid-based capsid system to generate CRISPR-Cas13a-loaded antibacterial capsids (AB-capsids) for targeted therapy against multidrug-resistant Staphylococcus aureus. Our optimized phagemid system maximizes AB-capsid yield and purity, showing a positive correlation with phagemid copy number. Notably, an 8.65-fold increase in copy number results in a 2.54-fold rise in AB-capsid generation. Phagemids carrying terL-terS-rinA-rinB (prophage-encoded packaging site genes) consistently exhibit high packaging efficiency, and the generation of AB-capsids using lysogenized hosts with terL-terS deletion resulted in comparatively lower level of wild-type phage contamination, with minimal compromise on AB-capsid yield. These generated AB-capsids selectively eliminate S. aureus strains carrying the target gene while sparing non-target strains. In conclusion, our phagemid-based capsid system stands as a promising avenue for developing sequence-specific bactericidal agents, offering a streamlined approach to combat antibiotic-resistant pathogens within the constraints of efficient production and targeted efficacy.
Assuntos
Antibacterianos , Sistemas CRISPR-Cas , Capsídeo , Staphylococcus aureus Resistente à Meticilina , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/genética , Capsídeo/metabolismo , Capsídeo/efeitos dos fármacos , Antibacterianos/farmacologia , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/tratamento farmacológicoRESUMO
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).
Assuntos
Antivirais , Infecções por Enterovirus , Simulação de Acoplamento Molecular , Replicação Viral , Replicação Viral/efeitos dos fármacos , Humanos , Animais , Antivirais/farmacologia , Antivirais/química , Camundongos , Infecções por Enterovirus/tratamento farmacológico , Infecções por Enterovirus/virologia , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Proteínas do Capsídeo/antagonistas & inibidores , RNA Viral/genética , RNA Viral/metabolismo , Capsídeo/metabolismo , Capsídeo/efeitos dos fármacos , Modelos Animais de Doenças , Enterovirus Humano A/efeitos dos fármacos , Enterovirus Humano A/genética , Enterovirus Humano A/fisiologia , Enterovirus/efeitos dos fármacos , Enterovirus/genética , Linhagem Celular , Replicação do RNARESUMO
Inhibition of Hepatitis B Virus (HBV) replication by small molecules that modulate capsid assembly and the encapsidation of pgRNA and viral polymerase by HBV core protein is a clinically validated approach toward the development of new antivirals. Through definition of a minimal pharmacophore, a series of isoquinolinone-based capsid assembly modulators (CAMs) was identified. Structural biology analysis revealed that lead molecules possess a unique binding mode, exploiting electrostatic interactions with accessible phenylalanine and tyrosine residues. Key analogs demonstrated excellent primary potency, absorption, distribution, metabolism, and excretion (ADME) and pharmacokinetic properties, and efficacy in a mouse model of HBV. The optimized lead also displayed potent inhibition of capsid uncoating in HBV-infected HepG2 cells expressing the sodium-taurocholate cotransporting polypeptide (NTCP) receptor, affecting the generation of HBsAg and cccDNA establishment. Based on these results, isoquinolinone derivative AB-836 was advanced into clinical development. In Phase 1b trials, AB-836 demonstrated >3 log10 reduction in serum HBV DNA, however, further development was discontinued due to the observation of incidental alanine aminotransferase (ALT) elevations.
Assuntos
Antivirais , Desenho de Fármacos , Vírus da Hepatite B , Humanos , Relação Estrutura-Atividade , Vírus da Hepatite B/efeitos dos fármacos , Animais , Antivirais/farmacologia , Antivirais/síntese química , Antivirais/química , Antivirais/farmacocinética , Camundongos , Células Hep G2 , Capsídeo/efeitos dos fármacos , Capsídeo/metabolismo , Proteínas do Capsídeo/metabolismo , Proteínas do Capsídeo/antagonistas & inibidores , Isoquinolinas/farmacologia , Isoquinolinas/química , Isoquinolinas/síntese química , Quinolonas/farmacologia , Quinolonas/síntese química , Quinolonas/química , Montagem de Vírus/efeitos dos fármacosRESUMO
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.
Assuntos
Barreira Hematoencefálica , Dependovirus , Interleucina-3 , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Transcitose , Animais , Humanos , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Capsídeo/metabolismo , Proteínas do Capsídeo/metabolismo , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Dependovirus/genética , Dependovirus/metabolismo , Vetores Genéticos/genética , Vetores Genéticos/administração & dosagem , Células HEK293 , Interleucina-3/metabolismo , Ligação Proteica , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismoRESUMO
The viral capsid performs critical functions during HIV-1 infection and is a validated target for antiviral therapy. Previous studies have established that the proper structure and stability of the capsid are required for efficient HIV-1 reverse transcription in target cells. Moreover, it has recently been demonstrated that permeabilized virions and purified HIV-1 cores undergo efficient reverse transcription in vitro when the capsid is stabilized by addition of the host cell metabolite inositol hexakisphosphate (IP6). However, the molecular mechanism by which the capsid promotes reverse transcription is undefined. Here we show that wild type HIV-1 virions can undergo efficient reverse transcription in vitro in the absence of a membrane-permeabilizing agent. This activity, originally termed "natural endogenous reverse transcription" (NERT), depends on expression of the viral envelope glycoprotein during virus assembly and its incorporation into virions. Truncation of the gp41 cytoplasmic tail markedly reduced NERT activity, suggesting that gp41 licenses the entry of nucleotides into virions. By contrast to reverse transcription in permeabilized virions, NERT required neither the addition of IP6 nor a mature capsid, indicating that an intact viral membrane can substitute for the function of the viral capsid during reverse transcription in vitro. Collectively, these results demonstrate that the viral capsid functions as a nanoscale container for reverse transcription during HIV-1 infection.
Assuntos
Capsídeo , HIV-1 , Transcrição Reversa , HIV-1/fisiologia , HIV-1/metabolismo , Capsídeo/metabolismo , Humanos , Vírion/metabolismo , Proteína gp41 do Envelope de HIV/metabolismo , Proteína gp41 do Envelope de HIV/genética , Montagem de Vírus/fisiologia , Infecções por HIV/virologia , Infecções por HIV/metabolismo , Ácido Fítico/metabolismoRESUMO
The recombinant adeno-associated virus (rAAV) vector is among the most promising viral vectors in gene therapy. However, the limited manufacturing capacity in human embryonic kidney (HEK) cells is a barrier to rAAV commercialization. We investigated the impact of endoplasmic reticulum (ER) protein processing and apoptotic genes on transient rAAV production in HEK293 cells. We selected four candidate genes based on prior transcriptomic studies: XBP1, GADD34 / PPP1R15A, HSPA6, and BCL2. These genes were stably integrated into HEK293 host cells. Traditional triple-plasmid transient transfection was used to assess the vector production capability and the quality of both the overexpressed stable pools and the parental cells. We show that the overexpression of XBP1, HSPA6, and GADD34 increases rAAV productivity by up to 100% and increases specific rAAV productivity by up to 78% in HEK293T cells. Additionally, more prominent improvement associated with ER protein processing gene overexpression was observed when parental cell productivity was high, but no substantial variation was detected under low-producing conditions. We also confirmed genome titer improvement across different serotypes (AAV2 and AAV8) and different cell lines (HEK293T and HEK293); however, the extent of improvement may vary. This study unveiled the importance of ER protein processing pathways in viral particle synthesis, capsid assembly, and vector production. KEY POINTS: ⢠Upregulation of endoplasmic reticulum (ER) protein processing (XBP1, HSPA6, and GADD34) leads to a maximum 100% increase in rAAV productivity and a maximum 78% boost in specific rAAV productivity in HEK293T cells ⢠The enhancement in productivity can be validated across different HEK293 cell lines and can be used for the production of various AAV serotypes, although the extent of the enhancement might vary slightly ⢠The more pronounced improvements linked to overexpressing ER protein processing genes were observed when parental cell productivity was high, with minimal variation noted under low-producing conditions.
Assuntos
Dependovirus , Retículo Endoplasmático , Vetores Genéticos , Proteína 1 de Ligação a X-Box , Humanos , Células HEK293 , Dependovirus/genética , Proteína 1 de Ligação a X-Box/genética , Proteína 1 de Ligação a X-Box/metabolismo , Retículo Endoplasmático/metabolismo , Vetores Genéticos/genética , Expressão Gênica , Proteína Fosfatase 1/genética , Proteína Fosfatase 1/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Capsídeo/metabolismoRESUMO
Symmetry in nature often emerges from self-assembly processes and serves a wide range of functions. Cell surface layers (S-layers) form symmetrical lattices on many bacterial and archaeal cells, playing essential roles such as facilitating cell adhesion, evading the immune system, and protecting against environmental stress. However, the experimental structural characterization of these S-layers is challenging due to their self-assembly properties and high sequence variability. In this study, we introduce the SymProFold pipeline, which utilizes the high accuracy of AlphaFold-Multimer predictions to derive symmetrical assemblies from protein sequences, specifically focusing on two-dimensional S-layer arrays and spherical viral capsids. The pipeline tests all known symmetry operations observed in these systems (p1, p2, p3, p4, and p6) and identifies the most likely symmetry for the assembly. The predicted models were validated using available experimental data at the cellular level, and additional crystal structures were obtained to confirm the symmetry and interfaces of several SymProFold assemblies. Overall, the SymProFold pipeline enables the determination of symmetric protein assemblies linked to critical functions, thereby opening possibilities for exploring functionalities and designing targeted applications in diverse fields such as nanotechnology, biotechnology, medicine, and materials and environmental sciences.
Assuntos
Modelos Moleculares , Capsídeo/química , Capsídeo/ultraestrutura , Capsídeo/metabolismo , Cristalografia por Raios X , Conformação Proteica , Software , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismoRESUMO
Medusavirus is a giant virus classified into an independent family of Mamonoviridae. Amoebae infected with medusavirus release immature particles in addition to virions. These particles were suggested to exhibit the maturation process of this virus, but the structure of these capsids during maturation remains unknown. Here, we apply a block-based reconstruction method in cryo-electron microscopy (cryo-EM) single particle analysis to these viral capsids, extending the resolution to 7-10 Å. The maps reveal a novel network composed of minor capsid proteins (mCPs) supporting major capsid proteins (MCPs). A predicted molecular model of the MCP fitted into the cryo-EM maps clarified the boundaries between the MCP and the underlining mCPs, as well as between the MCP and the outer spikes, and identified molecular interactions between the MCP and these components. Several structural changes of the mCPs under the fivefold vertices of the immature particles were observed, depending on the presence or absence of the underlying internal membrane. In addition, the lower part of the penton proteins on the fivefold vertices was also missing in mature virions. These dynamic conformational changes of mCPs indicate an important function in the maturation process of medusavirus.IMPORTANCEThe structural changes of giant virus capsids during maturation have not thus far been well clarified. Medusavirus is a unique giant virus in which infected amoebae release immature particles in addition to mature virus particles. In this study, we used cryo-electron microscopy to investigate immature and mature medusavirus particles and elucidate the structural changes of the viral capsid during the maturation process. In DNA-empty particles, the conformation of the minor capsid proteins changed dynamically depending on the presence or absence of the underlying internal membranes. In DNA-full particles, the lower part of the penton proteins was lost. This is the first report of structural changes of the viral capsid during the maturation process of giant viruses.
Assuntos
Proteínas do Capsídeo , Capsídeo , Microscopia Crioeletrônica , Modelos Moleculares , Vírion , Microscopia Crioeletrônica/métodos , Proteínas do Capsídeo/metabolismo , Proteínas do Capsídeo/ultraestrutura , Proteínas do Capsídeo/química , Capsídeo/ultraestrutura , Capsídeo/metabolismo , Vírion/ultraestrutura , Vírus Gigantes/ultraestrutura , Vírus Gigantes/genética , Vírus Gigantes/metabolismo , Montagem de Vírus , Conformação ProteicaRESUMO
Virus-like particles (VLPs) have untapped potential for packaging and delivery of macromolecular cargo. To be a broadly useful platform, there needs to be a strategy for attaching macromolecules to the inside or the outside of the VLP with minimal modification of the platform or cargo. Here, we repurpose antiviral compounds that bind to hepatitis B virus (HBV) capsids to create a chemical tag to noncovalently attach cargo to the VLP. Our tag consists of a capsid assembly modulator, HAP13, connected to a linker terminating in maleimide. Our cargo is a green fluorescent protein (GFP) with a single addressable cysteine, a feature that can be engineered in many proteins. The HAP-GFP construct maintained HAP's intrinsic ability to bind HBV capsids and accelerate assembly. We investigated the capacity of HAP-GFP to coassemble with HBV capsid protein and bind to preassembled capsids. HAP-GFP binding was concentration-dependent, sensitive to capsid stability, and dependent on linker length. Long linkers had the greatest activity to bind capsids, while short linkers impeded assembly and damaged intact capsids. In coassembly reactions, >20 HAP-GFP molecules were presented on the outside and inside of the capsid, concentrating the cargo by more than 100-fold compared to bulk solution. We also tested an HAP-GFP with a cleavable linker so that external GFP molecules could be removed, resulting in exclusive internal packaging. These results demonstrate a generalizable strategy for attaching cargo to a VLP, supporting development of HBV as a modular VLP platform.
Assuntos
Capsídeo , Proteínas de Fluorescência Verde , Vírus da Hepatite B , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Fluorescência Verde/química , Capsídeo/química , Capsídeo/metabolismo , Montagem de Vírus , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Vírion/metabolismo , Vírion/química , Propriedades de SuperfícieRESUMO
The enterovirus is a genus of single-stranded, highly diverse positive-sense RNA viruses, including Human Enterovirus A-D and Human Rhinovirus A-C species. They are responsible for numerous diseases and some infections can progress to life-threatening complications, particularly in children or immunocompromised patients. To date, there is no treatment against enteroviruses on the market, except for polioviruses (vaccine) and EV-A71 (vaccine in China). Following a decrease in enterovirus infections during and shortly after the (SARS-Cov2) lockdown, enterovirus outbreaks were once again detected, notably in young children. This reemergence highlights on the need to develop broad-spectrum treatment against enteroviruses. Over the last year, our research team has identified a new class of small-molecule inhibitors showing anti-EV activity. Targeting the well-known hydrophobic pocket in the viral capsid, these compounds show micromolar activity against EV-A71 and a high selectivity index (SI) (5h: EC50, MRC-5 = 0.57 µM, CC50, MRC-5 >20 µM, SI > 35; EC50, RD = 4.38 µM, CC50, RD > 40 µM, SI > 9; 6c: EC50, MRC-5 = 0.29 µM, CC50, MRC-5 >20 µM, SI > 69; EC50, RD = 1.66 µM, CC50, RD > 40 µM, SI > 24; Reference: Vapendavir EC50, MRC-5 = 0.36 µM, CC50, MRC-5 > 20 µM, EC50, RD = 0.53 µM, CC50, RD > 40 µM, SI > 63). The binding mode of these compounds in complex with enterovirus capsids was analyzed and showed a series of conserved interactions. Consequently, 6c and its derivatives are promising candidates for the treatment of enterovirus infections.
Assuntos
Antivirais , Capsídeo , Enterovirus Humano A , Antivirais/farmacologia , Antivirais/química , Antivirais/síntese química , Humanos , Enterovirus Humano A/efeitos dos fármacos , Capsídeo/efeitos dos fármacos , Capsídeo/metabolismo , Relação Estrutura-Atividade , Proteínas do Capsídeo/antagonistas & inibidores , Proteínas do Capsídeo/metabolismo , Proteínas do Capsídeo/química , Estrutura Molecular , Testes de Sensibilidade Microbiana , Relação Dose-Resposta a DrogaRESUMO
Recombinant adeno-associated virus (rAAV) vectors are currently the only proven vehicles for treating ophthalmological diseases through gene therapy. A wide range of gene therapy programs that target ocular diseases are currently being pursued. Nearly 20 years of research have gone into enhancing the efficacy of targeting retinal tissues and improving transgene delivery to specific cell types. The engineered AAV capsid, AAV2.7m8 is currently among the best capsids for transducing the retina following intravitreal (IVT) injection. However, adverse effects, including intraocular inflammation, have been reported following retinal administration of AAV2.7m8 vectors in clinical trials. Furthermore, we have consistently observed that AAV2.7m8 exhibits low packaging titers irrespective of the vector construct design. In this report, we found that AAV2.7m8 packages vector genomes with a higher degree of heterogeneity than AAV2. We also found that genome-loaded AAV2.7m8 stimulated the infiltration of microglia in mouse retinas following IVT administration, while the response to genome-loaded AAV2 and empty AAV2.7m8 capsids produced much milder responses. This finding suggests that IVT administration of AAV2.7m8 vectors may stimulate retinal immune responses in part because of its penchant to package and deliver non-unit length genomes.
Assuntos
Capsídeo , Dependovirus , Terapia Genética , Vetores Genéticos , Retina , Dependovirus/genética , Animais , Vetores Genéticos/genética , Vetores Genéticos/administração & dosagem , Camundongos , Retina/metabolismo , Capsídeo/metabolismo , Terapia Genética/métodos , Genoma Viral , Humanos , Camundongos Endogâmicos C57BL , Transdução Genética/métodos , Microglia/metabolismoRESUMO
Capsid assembly modulators (CAMs) have the potential to cure chronic hepatitis B, as demonstrated in clinical trials. Lead compounds NVR3-778 and 5a were found to exist in normal and flipped conformations through induced fit docking. Therefore, we designed and synthesized series I and II compounds by interchanging the amide and sulfonamide bonds of 5a to modify both the tolerance region and solvent-opening region. Among them, compound 4a (EC50 = 0.24 ± 0.10 µM, CC50 > 100 µM) exhibited potent anti-HBV activity with low toxicity, surpassing the lead compounds NVR3-778 (EC50 = 0.29 ± 0.03 µM, CC50 = 20.78 ± 2.29 µM) and 5a (EC50 = 0.50 ± 0.07 µM, CC50 = 48.16 ± 9.15 µM) in HepAD38 cells. Additionally, compared with the lead compound, 4a displayed a stronger inhibitory effect on HBV capsid protein assembly. Molecular dynamics (MD) simulations confirmed that the normal conformation of 4a had relatively stable conformation at different frames of binding modes. Furthermore, 4a showed better metabolic stability in human plasma than positive control drugs. Therefore, compound 4a could be further structurally modified as a potent lead compound.
Assuntos
Antivirais , Desenho de Fármacos , Vírus da Hepatite B , Vírus da Hepatite B/efeitos dos fármacos , Antivirais/farmacologia , Antivirais/síntese química , Antivirais/química , Humanos , Relação Estrutura-Atividade , Proteínas do Capsídeo/metabolismo , Proteínas do Capsídeo/antagonistas & inibidores , Simulação de Dinâmica Molecular , Estrutura Molecular , Capsídeo/efeitos dos fármacos , Capsídeo/metabolismo , Relação Dose-Resposta a Droga , Testes de Sensibilidade MicrobianaRESUMO
Serine proteases are important environmental contributors of enterovirus biocontrol. However, the structural features of molecular interaction accounting for the susceptibility of enteroviruses to proteases remains unexplained. Here, we describe the molecular mechanisms involved in the recruitment of serine proteases to viral capsids. Among the virus types used, coxsackievirus A9 (CVA9), but not CVB5 and echovirus 11 (E11), was inactivated by Subtilisin A in a host-independent manner, while Bovine Pancreatic Trypsin (BPT) only reduced CVA9 infectivity in a host-dependent manner. Predictive interaction models of each protease with capsid protomers indicate the main targets as internal disordered protein (IDP) segments exposed either on the 5-fold vertex (DE loop VP1) or at the 5/2-fold intersection (C-terminal end VP1) of viral capsids. We further show that a functional binding protease/capsid depends on both the strength and the evolution over time of protease-VP1 complexes, and lastly on the local adaptation of proteases on surrounding viral regions. Finally, we predicted three residues on CVA9 capsid that trigger cleavage by Subtilisin A, one of which may act as a sensor residue contributing to enzyme recognition on the DE loop. Overall, this study describes an important biological mechanism involved in enteroviruses biocontrol.