No abstract

The Flaviviridae family comprises a number of human pathogens, which, although sharing structural and functional features, cause diseases with very different outcomes. This can be explained by the plurality of functions exerted by the few proteins coded by viral genomes, with some of these functions shared among members of a same family, but others being unique for each virus species. These non-canonical functions probably have evolved independently and may serve as the base to the development of specific therapies for each of those diseases. Here it is discussed what is currently known about the non-canonical roles of dengue virus (DENV) non-structural proteins (NSPs), which may account for some of the effects specifically observed in DENV infection, but not in other members of the Flaviviridae family. This review explores how DENV NSPs contributes to the physiopathology of dengue, evasion from host immunity, metabolic changes, and redistribution of cellular components during infection.

Understanding protein structure and dynamics, which govern key cellular processes, is crucial for basic and applied research. Intrinsically disordered protein (IDP) regions display multifunctionality via alternative transient conformations, being key players in disease mechanisms. IDP regions are abundant, namely in small viruses, allowing a large number of functions out of a small proteome. The relation between protein function and structure is thus now seen from a different perspective: as IDP regions enable transient structural arrangements, each conformer can play different roles within the cell. However, as IDP regions are hard and time-consuming to study via classical techniques (optimized for globular proteins with unique conformations), new methods are required. Here, employing the dengue virus (DENV) capsid (C) protein and the immunoglobulin-binding domain of streptococcal protein G, we describe a straightforward NMR method to differentiate the solvent accessibility of single amino acid N-H groups in structured and IDP regions. We also gain insights into DENV C flexible fold region biological activity. The method, based on minimal pH changes, uses the well-established 1H-15N HSQC pulse sequence and is easily implementable in current protein NMR routines. The data generated are simple to interpret, with this rapid approach being an useful first-choice IDPs characterization method.

Zika virus (ZIKV) became an important public health concern because infection was correlated to the development of microcephaly and other neurological disorders. Although the structure of the virion has been determined by cryo-electron microscopy, information about the nucleocapsid is lacking. We used nuclear magnetic resonance to determine the solution structure and dynamics of full length ZIKV capsid protein (ZIKVC). Although most of the protein is structured as described for the capsid proteins of Dengue and West Nile viruses and for truncated ZIKVC (residues 23−98), here we show important differences in the α-helix 1 and N-terminal intrinsically disordered region (IDR). We distinguished two dynamical regions in the ZIKVC IDR, a highly flexible N-terminal end and a transitional disordered region, indicating that it contains ordered segments rather than being completely flexible. The unique size and orientation of α-helix 1 partially occlude the protein hydrophobic cleft. Measurements of the dynamics of α-helix 1, surface exposure, and thermal susceptibility of each backbone amide 1 H in protein structure revealed the occlusion of the hydrophobic cleft by α1/α1′ and supported α-helix 1 positional uncertainty. On the basis of the findings described here, we propose that the dynamics of ZIKVC structural elements responds to a structure-driven regulation of interaction of the protein with intracellular hydrophobic interfaces, which would have an impact on the switches that are necessary for nucleocapsid assembly. Subtle differences in the sequence of α-helix 1 have an impact on its size and orientation and on the degree of exposure of the hydrophobic cleft, suggesting that α-helix 1 is a hot spot for evolutionary adaptation of the capsid proteins of flaviviruses.

Dengue virus (DENV) causes a major arthropod-borne viral disease, with 2.5 billion people living in risk areas. DENV consists in a 50 nm-diameter enveloped particle in which the surface proteins are arranged with icosahedral symmetry, while information about nucleocapsid (NC) structural organization is lacking. DENV NC is composed of the viral genome, a positive-sense single-stranded RNA, packaged by the capsid (C) protein. Here, we established the conditions for a reproducible in vitro assembly of DENV nucleocapsid-like particles (NCLPs) using recombinant DENVC. We analyzed NCLP formation in the absence or presence of oligonucleotides in solution using small angle X-ray scattering, Rayleigh light scattering as well as fluorescence anisotropy, and characterized particle structural properties using atomic force and transmission electron microscopy imaging. The experiments in solution comparing 2-, 5- and 25-mer oligonucleotides established that 2-mer is too small and 5-mer is sufficient for the formation of NCLPs. The assembly process was concentration-dependent and showed a saturation profile, with a stoichiometry of 1:1 (DENVC:oligonucleotide) molar ratio, suggesting an equilibrium involving DENVC dimer and an organized structure compatible with NCLPs. Imaging methods proved that the decrease in concentration to sub-nanomolar concentrations of DENVC allows the formation of regular spherical NCLPs after protein deposition on mica or carbon surfaces, in the presence as well as in the absence of oligonucleotides, in this latter case being surface driven. Altogether, the results suggest that in vitro assembly of DENV NCLPs depends on DENVC charge neutralization, which must be a very coordinated process to avoid unspecific aggregation. Our hypothesis is that a specific highly positive spot in DENVC α4-α4’ is the main DENVC-RNA binding site, which is required to be firstly neutralized to allow NC formation.

The latex of Euphorbia tirucalli L. (Aveloz) is popularly used to treat cancers from diverse sources such as: breast, prostate, lung and kidney. Furthermore, high dilutions of this latex (latex-HDs) have been successfully used in the treatment of tumors, although the mechanisms involved in this antitumoral activity are not yet known. The present study aimed to investigate the cytotoxic effects of latex-HDs in 2 human cell lines, including non-tumoral melanocytes (MelanA line) and human breast cancer cells (MCF7 line). Latex-HDs were obtained through the interaction of two procedures: 1:100 dilution in mass and agitation, using 2 solvents, water and 70% ethanol in homeopathic preparations 5, 15 and 30cH, following the Brazilian Homeopathic Pharmacopeia. Cell lines were incubated with 1 % and 10 % of latex-HDs for 24 hours. Controls treated with similar preparations without latex were incubated under the same conditions. Cell viability was analyzed by MTT assay and morphological features were assessed by May-Grunwald-Giemsa method. Cell ultrastructure was analyzed by transmission electron microscopy. Metabolic alterations were detected by spectrophotometric assay for the enzyme 6-phosphofructo-1-kinase (PFK-1) activity. 1% of ethanolic latex-HDs induced no alterations in MelanA cells viability, however Aveloz 15cH induced an increase in MCF7 cells viability. After aqueous treatment, MelanA cells viability decreased in almost all systems, whereas in MCF7 cells, an increase was detected in dynamized water systems and Aveloz 15CH. However, only water 15cH increased cell viability in comparison to control and non-dynamized water. No alterations in MCF7 morphology and ultrastructure were visualized through light microscopy or electronic microscopy, respectively. Interesting results were detected when we studied the glycolytic metabolism of both cells, giving support to evidences showing that HDs interfere in the metabolism of cell lines. Further studies are needed to better understand the mechanisms involved in this in vitro biological response to HDs. Keywords:  Euphorbia tirucalli L.; Aveloz; PFK-1; High Dilutions; Cell metabolism.   Altas diluições de Euphorbia tirucalli L. (aveloz) modificam a viabilidade e o metabolismo glicolítico de linhas celulares Resumo O látex de Ephorbia tirucalli L. (aveloz) é usado popularmente no tratamento de diversos tipos de câncer incluindo mama, próstata, pulmão e rim. Além do mais, altas diluições desse látex (latex-HDs) tém sido utilizadas com sucesso no tratamento de tumores, embora o mecanismo dessa ação ainda seja desconhecido. O presente trabalho objetivou pesquisar os efeitos citotóxicos de látex-HDs em 2 linhas celulares humanas, incluindo melanócitos não tumorais (línha MelanA) e células de câncer de mama humano (linha MCF7). As látex-HDs foram obtidas através de 2 procedimentos: diluição 1:100 em massa e agitação, com 2 solventes, água e etanol 70%, nas diluições homeopáticas 5, 15cH e 30cH de acordo com a Farmacopéia Homeopática Brasileira. As linhas celulares foram incubadas com látex-HDs 1% e 10% por 24 horas. Foram preparados controles similares sem o látex e incubados sob as mesmas condições. A viabilidade celular foi analisada através do método May-grunwald-Giemsa. A ultrastrutura celular foi analisada através de microscopia eletrônica de transmissão. As alterações metabólicas foram detectadas por ensaio de espectrofotometria para a atividade da anzima 6-fosfofructo-1-quinase (PFK-1). Látex-Hds em etanol 1% não induziram qualquer alteração na viabilidade das células MelanA, mas Aveloz 15cH induziu aumento da viabilidade das células MCF7. Após tratamento, a viabilidade das células MelanA diminuiu em quase todos os sistemas, enquanto que nas células MCF7 foi detectado aumento nos sistemas aquosos e Aveloz 15cH, mas só água 15cH aumentou a viabilidade celular por comparação ao controle e agua não dinamizada. Não foram detectadas alterações na morfologia celular por microscopis de luz quando se estudou o metabolismo glicolítico em ambas as células, demonstrando que os padrões de resposta celular dependem do tipo de solvente, metabolismo celular e procedimento de diluição. esa é a primeira vez que se mostra que as altas diluições interferem no metabolismo de linhas celulares. Outros estudos são necessários para se compreender melhor os mecanismos envolvidos nesta resposta in vitro a altas diluições de aveloz. Palavras-chave:  Euphorbia tirucalli L.; Aveloz; PFK-1; Altas diluições; Metabolismo celular.   Altas Diluciones de Euphorbia tirucalli L. (arbusto de goma) modifican la viabilidad y metabolismo glucolítico de líneas celulares Resumen El látex de Euphorbia tirucalli L. (arbusto de goma) es popularmente utilizado en el tratamiento de diversos tipos de cáncer, incluyendo mama, próstata, pulmón y riñón. Además, altas diluciones de este látex (latex-HDs) han sido utilizadas con éxito en el tratamiento de tumores, aunque el mecanismo de esta acción todavía es desconocido. Este trabajo tuvo por objetivo investigar los efectos citotóxicos de látex-HDs en 2 líneas celulares humanas, incluyendo melanocitos no tumorales (línea MelanA) y células de cáncer de mama humano (línea MCF7). Las latex-HDs fueron obtenidas mediante 2 procedimientos; dilución 1:100 en masa y agitación, con 2 solventes, agua y etanol 70%, en las diluciones homeopáticas 5, 15 y 30cH, según la Farmacopea Homeopática Brasileña. Las líneas celulares fueron incubadas con latex-HDs 1% y 10% durante 24 horas. Fueron preparados controles similares sin látex e incubados bajo las mismas condiciones. La viabilidad celular fue analizada por el método May-Grunwald-Giemsa. La ultra-estructura celular fue analizada por microscopía electrónica de transmisión. Alteraciones metabólicas fueron detectadas por ensayo de espectrofotometría para la actividad de la enzima 6-fosfofructo-1-kinasa (PFK-1). Látex-HDs en etanol 1% no indujeran ninguna alteración en la viabilidad de las células MelanA, sin embargo Euphorbia tirucalli 15cH indujo aumento de la viabilidad de las células MCF7. Después del tratamiento, la viabilidad de las células MelanA disminuyó en casi todos los sistemas, mientras que en las células MCF7 fue detectado aumento en los sistemas acuosos y Euphorbia tirucalli 15cH, pero solamente agua 15cH aumentó la viabilidad celular en comparación con el control y agua no dinamizada. No fueron detectadas alteraciones en la morfología celular por microscopía de luz cuando se estudió el metabolismo glucolítico de ambas células demostrando los modelos de respuesta celular dependen del tipo de solvente, metabolismo celular y procedimiento de dilución. Esta es la primera vez que se demostró que altas diluciones interfieren en el metabolismo de líneas celulares. Otros estudios son necesarios para entender mejor los mecanismos implicados en esta respuesta in vitro a las altas diluciones de Euphorbia tirucalli. Palabras-clave:  Euphorbia tirucalli L.; Aveloz; PFK-1; Altas diluciones; metabolismo celular.   Correspondence author:  Carla Holandino Quaresma, cholandino@yahoo.com How to cite this article:  Aquino CL, Barbosa GM, Barbosa GM, Varricchio MCBA, Veiga VF, Kuster R, Zancan P, Sola-Penna M, Quaresma CH. High dilutions of Euphorbia tirucalli L. (AVELOZ) modify the viability and glycolytic metabolism of cell lines. Int J High Dilution Res [online]. 2008 [cited YYYY Mmm DD]; 7(24): 132-139. Available from: http://journal.giri-society.org/index.php/ijhdr/article/view/283/361. ÂÂÂ

No abstract

The SARS-CoV-2 nucleocapsid protein (N) is a multifunctional promiscuous nucleic acid-binding protein, which plays a major role in nucleocapsid assembly and discontinuous RNA transcription, facilitating the template switch of transcriptional regulatory sequences (TRS). Here, we dissect the structural features of the N protein N-terminal domain (N-NTD), either with or without the SR-rich motif (SR), upon binding to single and double-stranded TRS DNA, as well as their activities for dsTRS melting and TRS-induced liquid-liquid phase separation (LLPS). Our study gives insights on specificity for N-NTD/N-NTD-SR interaction with TRS, including an unfavorable energetic contribution to binding along with hydrogen bonds between the triple-thymidine (TTT) motif in the dsTRS and β-sheet II due to the defined position and orientation of the DNA duplex, a well-defined pattern (ΔH > 0 and ΔS > 0 for ssTRS, and ΔH < 0 and ΔS < 0 for dsTRS) for the thermodynamic profile of binding, and a preference for TRS in the formation of liquid condensates when compared to a non-specific sequence. Moreover, our results on DNA binding may serve as a starting point for the design of inhibitors, including aptamers, against N, a possible therapeutic target essential for the virus infectivity.