RESUMEN
UNLABELLED: Vaccine manufacturing costs prevent a significant portion of the world's population from accessing protection from vaccine-preventable diseases. To enhance vaccine production at reduced costs, a genome-wide RNA interference (RNAi) screen was performed to identify gene knockdown events that enhanced poliovirus replication. Primary screen hits were validated in a Vero vaccine manufacturing cell line using attenuated and wild-type poliovirus strains. Multiple single and dual gene silencing events increased poliovirus titers >20-fold and >50-fold, respectively. Host gene knockdown events did not affect virus antigenicity, and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9-mediated knockout of the top candidates dramatically improved viral vaccine strain production. Interestingly, silencing of several genes that enhanced poliovirus replication also enhanced replication of enterovirus 71, a clinically relevant virus to which vaccines are being targeted. The discovery that host gene modulation can markedly increase virus vaccine production dramatically alters mammalian cell-based vaccine manufacturing possibilities and should facilitate polio eradication using the inactivated poliovirus vaccine. IMPORTANCE: Using a genome-wide RNAi screen, a collection of host virus resistance genes was identified that, upon silencing, increased poliovirus and enterovirus 71 production by from 10-fold to >50-fold in a Vero vaccine manufacturing cell line. This report provides novel insights into enterovirus-host interactions and describes an approach to developing the next generation of vaccine manufacturing through engineered vaccine cell lines. The results show that specific gene silencing and knockout events can enhance viral titers of both attenuated (Sabin strain) and wild-type polioviruses, a finding that should greatly facilitate global implementation of inactivated polio vaccine as well as further reduce costs for live-attenuated oral polio vaccines. This work describes a platform-enabling technology applicable to most vaccine-preventable diseases.
Asunto(s)
Poliomielitis/prevención & control , Vacunas contra Poliovirus/aislamiento & purificación , Poliovirus/aislamiento & purificación , Poliovirus/fisiología , Tecnología Farmacéutica/métodos , Replicación Viral , Animales , Vacunas Atenuadas/aislamiento & purificación , Células Vero , Cultivo de Virus/métodosRESUMEN
Influenza A virus (IAV) infection causes seasonal epidemics of contagious respiratory illness that causes substantial morbidity and some mortality. Regular vaccination is the principal strategy for controlling influenza virus, although vaccine efficacy is variable. IAV antiviral drugs are available; however, substantial drug resistance has developed to two of the four currently FDA-approved antiviral drugs. Thus, new therapeutic approaches are being sought to reduce the burden of influenza-related disease. A high-throughput screen using a human kinase inhibitor library was performed targeting an emerging IAV strain (H7N9) in A549 cells. The inhibitor library contained 273 structurally diverse, active cell permeable kinase inhibitors with known bioactivity and safety profiles, many of which are at advanced stages of clinical development. The current study shows that treatment of human A549 cells with kinase inhibitors dinaciclib, flavopiridol, or PIK-75 exhibits potent antiviral activity against H7N9 IAV as well as other IAV strains. Thus, targeting host kinases can provide a broad-spectrum therapeutic approach against IAV. These findings provide a path forward for repurposing existing kinase inhibitors safely as potential antivirals, particularly those that can be tested in vivo and ultimately for clinical use.
Asunto(s)
Antivirales/farmacología , Reposicionamiento de Medicamentos/métodos , Virus de la Influenza A/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Replicación Viral/efectos de los fármacos , Antivirales/efectos adversos , Línea Celular , Relación Dosis-Respuesta a Droga , Farmacorresistencia Viral , Sinergismo Farmacológico , Ensayos Analíticos de Alto Rendimiento , Humanos , Subtipo H7N9 del Virus de la Influenza A/efectos de los fármacos , Inhibidores de Proteínas Quinasas/efectos adversos , Seguridad , Bibliotecas de Moléculas PequeñasRESUMEN
COPII vesicles transport proteins from the endoplasmic reticulum to the Golgi apparatus. Previous COPII-cage cryo-EM structures lacked the resolution necessary to determine the residues of Sec13 and Sec31 that mediate assembly and flexibility of the COPII cage. Here we present a 12-Å structure of the human COPII cage, where the tertiary structure of Sec13 and Sec31 is clearly identifiable. We employ this structure and a homology model of the Sec13-Sec31 complex to create a reliable pseudoatomic model of the COPII cage. We combined this model with hydrogen/deuterium-exchange MS analysis to characterize four distinct contact regions at the vertices of the COPII cage. Furthermore, we found that the two-fold symmetry of the Sec31 dimeric region in Sec13-Sec31 is broken upon cage formation and that the resulting hinge is essential to form the proper edge geometry in COPII cages.
Asunto(s)
Vesículas Cubiertas por Proteínas de Revestimiento/química , Proteínas Portadoras/química , Modelos Moleculares , Proteínas de Transporte Vesicular/química , Secuencia de Bases , Vesículas Cubiertas por Proteínas de Revestimiento/genética , Proteínas Portadoras/genética , Cromatografía Líquida de Alta Presión , Microscopía por Crioelectrón/métodos , Medición de Intercambio de Deuterio , Humanos , Procesamiento de Imagen Asistido por Computador , Espectrometría de Masas/métodos , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , Homología de Secuencia , Especificidad de la Especie , Proteínas de Transporte Vesicular/genética , LevadurasRESUMEN
AAV-DJ, a leading candidate vector for liver gene therapy, was created through random homologous recombination followed by directed evolution, selecting for in vivo liver tropism and resistance to in vitro immune neutralization. Here, the 4.5 Å resolution cryo-EM structure is determined for the engineered AAV vector, revealing structural features that illuminate its phenotype. The heparan sulfate receptor-binding site is little changed from AAV-2, and heparin-binding affinity is similar. A loop that is antigenic in other serotypes has a unique conformation in AAV-DJ that would conflict with the binding of an AAV-2 neutralizing monoclonal antibody. This is consistent with increased resistance to neutralization by human polyclonal sera, raising the possibility that changed tropism may be a secondary effect of altered immune interactions. The reconstruction exemplifies analysis of fine structural changes and the potential of cryo-EM, in favorable cases, to characterize mutant or ligand-bound complexes.
Asunto(s)
Cápside/química , Microscopía por Crioelectrón , Dependovirus/química , Modelos Moleculares , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sitios de Unión , Cromatografía de Afinidad , Cristalografía por Rayos X , Heparina/química , Unión Proteica , Estructura Cuaternaria de Proteína , Homología Estructural de Proteína , Propiedades de SuperficieRESUMEN
The use of adeno-associated virus (AAV) as a gene therapy vector is limited by the host neutralizing immune response. The cryo-electron microscopy (EM) structure at 8.5Å resolution is determined for a complex of AAV-2 with the Fab' fragment of monoclonal antibody (MAb) A20, the most extensively characterized AAV MAb. The binding footprint is determined through fitting the cryo-EM reconstruction with a homology model following sequencing of the variable domain, and provides a structural basis for integrating diverse prior epitope mappings. The footprint extends from the previously implicated plateau to the side of the spike, and into the conserved canyon, covering a larger area than anticipated. Comparison with structures of binding and non-binding serotypes indicates that recognition depends on a combination of subtle serotype-specific features. Separation of the neutralizing epitope from the heparan sulfate cell attachment site encourages attempts to develop immune-resistant vectors that can still bind to target cells.
Asunto(s)
Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/química , Anticuerpos Antivirales/inmunología , Dependovirus/química , Dependovirus/inmunología , Sustancias Macromoleculares/ultraestructura , Secuencia de Aminoácidos , Microscopía por Crioelectrón , Mapeo Epitopo , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/inmunología , Datos de Secuencia Molecular , Estructura Molecular , Estructura Cuaternaria de ProteínaRESUMEN
Structural studies have revealed some of the organizing principles and mechanisms involved in the assembly of the COPII coat including the location of the Sec23/24 adapter layer. Previous studies, however, were unable to unambiguously determine the positions of Sec23 and Sec24 in the coat. Here, we have determined a cryogenic electron microscopic structure of Sec13/31 together with Sec23. Electron tomography revealed that the binding of Sec23 induces Sec13/31 to form a variety of different geometries including a cuboctahedron, as was previously characterized for Sec13/31 alone. Single-particle reconstruction of the Sec13/31-23 cuboctahedra revealed that the binding of Sec23 induces a conformational change in Sec13/31, resulting in a more extended conformation. Docking Sec23 crystal structures into the electron microscopy map suggested that Sec24 projects its cargo binding surface out into the large open faces of the coat. These results have implications for the mechanisms by which COPII transports large cargos, cargos with large intracellular domains, and for tethering complexes that must project out of the coat in order to interact with their binding partners. Furthermore, Sec23 binds Sec13/31 at two unique sites in the coat, which suggests that each site may have unique roles in the mechanisms of COPII vesiculation.
Asunto(s)
Vesículas Cubiertas por Proteínas de Revestimiento/química , Vesículas Cubiertas por Proteínas de Revestimiento/ultraestructura , Proteínas Portadoras/química , Proteínas de Transporte Vesicular/química , Vesículas Cubiertas por Proteínas de Revestimiento/metabolismo , Proteínas Portadoras/metabolismo , Microscopía por Crioelectrón , Humanos , Modelos Moleculares , Estructura Cuaternaria de Proteína , Dispersión de Radiación , Proteínas de Transporte Vesicular/metabolismoRESUMEN
Nearly a third of all eukaryotic proteins are transported from the ER to the Golgi apparatus through the secretory pathway using COPII coated vesicles. Evidence suggests that this transport occurs via 500-900 Å vesicles that bud from the ER membrane. It has been shown that procollagen molecules utilize the COPII proteins for transport, but it is unclear how the COPII coat can accommodate these â¼3000 Å long molecules. We now present a cryogenic electron tomographic reconstruction of a Sec13/31 tubule that is approximately 3300 Å long containing a hollow cylindrical interior that is 300 Å in diameter, dimensions that are consistent with those that are required to encapsulate a procollagen molecule wrapped in a membrane and accessory COPII components. This structure suggests a novel mechanism that the COPII coat may employ to transport elongated cargo.
Asunto(s)
Proteínas de Transporte Vesicular/química , Vesículas Cubiertas por Proteínas de Revestimiento/metabolismo , Microscopía por Crioelectrón , Retículo Endoplásmico , Humanos , Proteínas de Transporte Vesicular/ultraestructuraRESUMEN
Adeno-associated virus serotype 2 (AAV-2) is a leading candidate vector for gene therapy. Cell entry starts with attachment to a primary receptor, Heparan Sulfate Proteoglycan (HSPG) before binding to a co-receptor. Here, cryo-electron microscopy provides direct visualization of the virus-HSPG interactions. Single particle analysis was performed on AAV-2 complexed with a 17 kDa heparin fragment at 8.3 A resolution. Heparin density covers the shoulder of spikes surrounding viral 3-fold symmetry axes. Previously implicated, positively charged residues R(448/585), R(451/588) and R(350/487) from another subunit cluster at the center of the heparin footprint. The footprint is much more extensive than apparent through mutagenesis, including R(347/484), K(395/532) and K(390/527) that are more conserved, but whose roles have been controversial. It also includes much of a region proposed as a co-receptor site, because prior studies had not revealed heparin interactions. Heparin density bridges over the viral 3-fold axes, indicating multi-valent attachment to symmetry-related binding sites.
Asunto(s)
Membrana Celular/virología , Dependovirus/química , Dependovirus/ultraestructura , Proteoglicanos de Heparán Sulfato/metabolismo , Heparina/química , Receptores Virales/metabolismo , Cápside/metabolismo , Membrana Celular/metabolismo , Microscopía por Crioelectrón , Células HeLa , Proteoglicanos de Heparán Sulfato/química , Heparina/genética , Heparina/metabolismo , Humanos , Procesamiento de Imagen Asistido por Computador , Microscopía Electrónica , Modelos Moleculares , Estructura Terciaria de Proteína , Receptores de Superficie Celular/ultraestructura , Receptores Virales/genéticaRESUMEN
Adeno-associated virus (AAV) is a small single-stranded DNA member of the family Parvoviradae with at least eight recognized human serotypes, several of which are being studied as candidate vectors for gene therapy. When multiple serotypes are handled in the same laboratory, it is critical to know the serotype of a sample with certainty. Here, a rapid and reliable PCR-based method is presented for the identification of serotypes 2, 3B or 6 and for screening for cross contamination. The PCR assay is based on the use of differentially annealing, serotype-specific primer pairs targeted to the hypervariable regions of the capsid coding region of the AAV genome. Identity is determined by the presence of a PCR product of size specific to each serotype. Multiplexing the reaction allows all serotypes to be queried in a single reaction tube and greatly diminishes the presence of false positives. The method is capable of detecting as little as 25 fg of a contaminating serotype and is potentially extensible to other AAV serotypes.