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1.
Nanomedicine ; 44: 102572, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35671983

RESUMO

We previously reported that hydroxylated oxime ether lipids (OELs) efficiently deliver functional Dicer substrate siRNAs (DsiRNAs) in cells. Here, we explored in vivo utility of these OELs, using OEL4 as a prototype and report that surface modification of the OEL4 formulations was essential for their in vivo applications. These surface-modified OEL4 formulations were developed by inclusion of various PEGylated lipids. The vesicle stability and gene knock-down were dependent on the PEG chain length. OEL4 containing DSPE-PEG350 and DSPE-PEG1000 (surprisingly not DSPE2000) promoted gene silencing in cells. In vivo studies demonstrated that OEL4 vesicles formulated using 3 mol% DSPE-PEG350 accumulate in human lung cancer (A549-luc2) xenografts in mice and exhibit a significant increase in tumor to liver ratios. These vesicles also showed a statistically significant reduction of luciferase signal in tumors compared to untreated mice. Taken together, the scalable OEL4:DSPE-PEG350 formulation serves as a novel candidate for delivery of RNAi therapeutics.


Assuntos
Éter , Neoplasias Pulmonares , Animais , Éteres , Xenoenxertos , Humanos , Lipídeos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/terapia , Camundongos , Oximas , Polietilenoglicóis , RNA Interferente Pequeno/genética
2.
J Biol Chem ; 295(1): 1-12, 2020 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-31649031

RESUMO

African swine fever virus (ASFV) is a complex nucleocytoplasmic large DNA virus (NCLDV) that causes a devastating swine disease currently present in many countries of Africa, Europe, and Asia. Despite intense research efforts, relevant gaps in the architecture of the infectious virus particle remain. Here, we used single-particle cryo-EM to analyze the three-dimensional structure of the mature ASFV particle. Our results show that the ASFV virion, with a radial diameter of ∼2,080 Å, encloses a genome-containing nucleoid surrounded by two distinct icosahedral protein capsids and two lipoprotein membranes. The outer capsid forms a hexagonal lattice (triangulation number T = 277) composed of 8,280 copies of the double jelly-roll major capsid protein (MCP) p72, arranged in trimers displaying a pseudo-hexameric morphology, and of 60 copies of a penton protein at the vertices. The inner protein layer, organized as a T = 19 capsid, confines the core shell, and it is composed of the mature products derived from the ASFV polyproteins pp220 and pp62. Also, an icosahedral membrane lies between the two protein layers, whereas a pleomorphic envelope wraps the outer capsid. This high-level organization confers to ASFV a unique architecture among the NCLDVs that likely reflects the complexity of its infection process and may help explain current challenges in controlling it.


Assuntos
Vírus da Febre Suína Africana/ultraestrutura , Proteínas do Capsídeo/ultraestrutura , Capsídeo/ultraestrutura , Proteínas do Envelope Viral/ultraestrutura , Vírus da Febre Suína Africana/metabolismo , Animais , Proteínas do Capsídeo/química , Chlorocebus aethiops , Microscopia Crioeletrônica , Lipídeos/química , Multimerização Proteica , Células Vero , Proteínas do Envelope Viral/química
3.
Viruses ; 10(8)2018 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-30127286

RESUMO

Human respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract disease in young children. With repeat infections throughout life, it can also cause substantial disease in the elderly and in adults with compromised cardiac, pulmonary and immune systems. RSV is a pleomorphic enveloped RNA virus in the Pneumoviridae family. Recently, the three-dimensional (3D) structure of purified RSV particles has been elucidated, revealing three distinct morphological categories: spherical, asymmetric, and filamentous. However, the native 3D structure of RSV particles associated with or released from infected cells has yet to be investigated. In this study, we have established an optimized system for studying RSV structure by imaging RSV-infected cells on transmission electron microscopy (TEM) grids by cryo-electron tomography (cryo-ET). Our results demonstrate that RSV is filamentous across several virus strains and cell lines by cryo-ET, cryo-immuno EM, and thin section TEM techniques. The viral filament length varies from 0.5 to 12 µm and the average filament diameter is approximately 130 nm. Taking advantage of the whole cell tomography technique, we have resolved various stages of RSV assembly. Collectively, our results can facilitate the understanding of viral morphogenesis in RSV and other pleomorphic enveloped viruses.


Assuntos
Vírus Sincicial Respiratório Humano/ultraestrutura , Vírion/ultraestrutura , Montagem de Vírus/fisiologia , Células A549 , Animais , Brônquios/virologia , Linhagem Celular , Chlorocebus aethiops , Microscopia Crioeletrônica/métodos , Tomografia com Microscopia Eletrônica/métodos , Células Epiteliais/ultraestrutura , Células Epiteliais/virologia , Células HeLa , Humanos , Microtomia , Vírus Sincicial Respiratório Humano/fisiologia , Células Vero , Vírion/fisiologia
4.
Nat Commun ; 9(1): 1736, 2018 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-29712906

RESUMO

Measles virus (MeV) remains a major human pathogen, but there are presently no licensed antivirals to treat MeV or other paramyxoviruses. Here, we use cryo-electron tomography (cryo-ET) to elucidate the principles governing paramyxovirus assembly in MeV-infected human cells. The three-dimensional (3D) arrangement of the MeV structural proteins including the surface glycoproteins (F and H), matrix protein (M), and the ribonucleoprotein complex (RNP) are characterized at stages of virus assembly and budding, and in released virus particles. The M protein is observed as an organized two-dimensional (2D) paracrystalline array associated with the membrane. A two-layered F-M lattice is revealed suggesting that interactions between F and M may coordinate processes essential for MeV assembly. The RNP complex remains associated with and in close proximity to the M lattice. In this model, the M lattice facilitates the well-ordered incorporation and concentration of the surface glycoproteins and the RNP at sites of virus assembly.


Assuntos
Hemaglutininas Virais/ultraestrutura , Vírus do Sarampo/ultraestrutura , Ribonucleoproteínas/ultraestrutura , Proteínas Virais de Fusão/ultraestrutura , Proteínas da Matriz Viral/ultraestrutura , Vírion/ultraestrutura , Linhagem Celular , Microscopia Crioeletrônica , Fibroblastos/ultraestrutura , Fibroblastos/virologia , Células HeLa , Hemaglutininas Virais/metabolismo , Humanos , Vírus do Sarampo/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas Virais de Fusão/metabolismo , Proteínas da Matriz Viral/metabolismo , Vírion/metabolismo , Montagem de Vírus , Liberação de Vírus
5.
Nat Commun ; 7: 13916, 2016 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-28000669

RESUMO

Respiratory syncytial virus (RSV) is a leading cause of infant hospitalization and there remains no pediatric vaccine. RSV live-attenuated vaccines (LAVs) have a history of safe testing in infants; however, achieving an effective balance of attenuation and immunogenicity has proven challenging. Here we seek to engineer an RSV LAV with enhanced immunogenicity. Genetic mapping identifies strain line 19 fusion (F) protein residues that correlate with pre-fusion antigen maintenance by ELISA and thermal stability of infectivity in live RSV. We generate a LAV candidate named OE4 which expresses line 19F and is attenuated by codon-deoptimization of non-structural (NS1 and NS2) genes, deletion of the small hydrophobic (SH) gene, codon-deoptimization of the attachment (G) gene and ablation of the secreted form of G. OE4 (RSV-A2-dNS1-dNS2-ΔSH-dGm-Gsnull-line19F) exhibits elevated pre-fusion antigen levels, thermal stability, immunogenicity, and efficacy despite heavy attenuation in the upper and lower airways of cotton rats.


Assuntos
Infecções por Vírus Respiratório Sincicial/imunologia , Vacinas contra Vírus Sincicial Respiratório/imunologia , Vírus Sincicial Respiratório Humano/imunologia , Vacinas Atenuadas/imunologia , Animais , Anticorpos Antivirais/imunologia , Linhagem Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Estabilidade de Medicamentos , Humanos , Camundongos Endogâmicos BALB C , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Infecções por Vírus Respiratório Sincicial/virologia , Vacinas contra Vírus Sincicial Respiratório/genética , Vírus Sincicial Respiratório Humano/genética , Vírus Sincicial Respiratório Humano/fisiologia , Sigmodontinae , Temperatura , Vacinas Atenuadas/genética , Células Vero , Proteínas Virais/genética , Proteínas Virais/imunologia
6.
J Am Chem Soc ; 138(50): 16274-16282, 2016 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-27936625

RESUMO

Sequence-specific peptides have been demonstrated to self-assemble into structurally defined nanoscale objects including nanofibers, nanotubes, and nanosheets. The latter structures display significant promise for the construction of hybrid materials for functional devices due to their extended planar geometry. Realization of this objective necessitates the ability to control the structural features of the resultant assemblies through the peptide sequence. The design of a amphiphilic peptide, 3FD-IL, is described that comprises two repeats of a canonical 18 amino acid sequence associated with straight α-helical structures. Peptide 3FD-IL displays 3-fold screw symmetry in a helical conformation and self-assembles into nanosheets based on hexagonal packing of helices. Biophysical evidence from TEM, cryo-TEM, SAXS, AFM, and STEM measurements on the 3FD-IL nanosheets support a structural model based on a honeycomb lattice, in which the length of the peptide determines the thickness of the nanosheet and the packing of helices defines the presence of nanoscale channels that permeate the sheet. The honeycomb structure can be rationalized on the basis of geometrical packing frustration in which the channels occupy defect sites that define a periodic superlattice. The resultant 2D materials may have potential as materials for nanoscale transport and controlled release applications.


Assuntos
Nanoporos , Peptídeos/química , Modelos Moleculares , Conformação Proteica em alfa-Hélice
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