Síntesis de nuevas formulaciones para la vehiculización de la curcumina como estrategia antiviral para el tratamiento de las infecciones causadas por el virus zika

El poli(D,L-láctido-co-glicólido) (PLGA) es un copolímero de gran interés para aplicaciones medicinales, debido a que es bioreabsorbible, biocompatible, no tóxico, y su cinética de degradación puede modificarse por la relación de copolimerización de los monómeros. En este estudio, se sintetizó PLGA por apertura de los anillos de los dímeros cíclicos de los monómeros D,L-láctido y glicólido en presencia de octoato estannoso como iniciador y alcohol laurílico como co-iniciador. El PLGA fue caracterizado por técnicas espectroscópicas y térmicas, resultando una relación equimolecular para ambos monómeros, con una temperatura de transición vítrea de aproximadamente 35ºC, características que lo hacen apropiado para liberación controlada de medicamentos. Por otro lado, la curcumina es una sustancia de origen natural de gran interés biológico, que inhibe la actividad de la inosina monofosfato deshidrogenasa (IMPDH) enzima blanco para el descubrimiento de drogas antivirales, en especial las causadas por el flavivirus Zika (ZIKV). Teniendo en cuenta la difícil en la administración de la curcumina como fármaco, se sintetizaron micropartículas de PLGA que encapsulen dicha sustancia, las cuales fueron caracterizadas y se evaluó su efectividad en el tratamiento del ZIKV mediante diferentes ensayos biológicosFil: Pacho, María Natalia. Universidad de Buenos AiresFil: D’Accorso, Norma B. Universidad de Buenos AiresFil: Damonte, Elsa. Universidad de Buenos AiresFil: García, Cybele. Universidad de Buenos Aire

Differential requirements in endocytic trafficking for penetration of dengue virus.

The entry of DENV into the host cell appears to be a very complex process which has been started to be studied in detail. In this report, the route of functional intracellular trafficking after endocytic uptake of dengue virus serotype 1 (DENV-1) strain HW, DENV-2 strain NGC and DENV-2 strain 16681 into Vero cells was studied by using a susceptibility to ammonium chloride assay, dominant negative mutants of several members of the family of cellular Rab GTPases that participate in regulation of transport through endosome vesicles and immunofluorescence colocalization. Together, the results presented demonstrate that in spite of the different internalization route among viral serotypes in Vero cells and regardless of the viral strain, DENV particles are first transported to early endosomes in a Rab5-dependent manner. Then a Rab7-dependent pathway guides DENV-2 16681 to late endosomes, whereas a yet unknown sorting event controls the transport of DENV-2 NGC, and most probably DENV-1 HW, to the perinuclear recycling compartments where fusion membrane would take place releasing nucleocapsid into the cytoplasm. Besides the demonstration of a different intracellular trafficking for two DENV-2 strains that shared the initial clathrin-independent internalization route, these studies proved for the first time the involvement of the slow recycling pathway for DENV-2 productive infection

Blockade of dengue virus entry into myeloid cells by endocytic inhibitors in the presence or absence of antibodies.

BACKGROUND:Dengue is the most prevalent arthropod-borne viral human disease in tropical and subtropical regions, caused by four dengue virus (DENV) serotypes. In spite of the increasing global incidence, no specific antiviral therapy is available. Cells of the mononuclear phagocyte lineage are the main targets either for direct antibody (Ab)-independent or Ab-mediated human DENV infection, usually associated to the severe forms of disease. Since the virus entry may be a convenient therapeutic alternative, this study aimed to investigate the mode of DENV internalization into myeloid cells in the absence and presence of DENV Ab and evaluate the inhibitory activity of diverse biochemical inhibitors of endocytosis. METHODOLOGY/PRINCIPAL FINDINGS:By infectivity assays and quantitative RT-PCR determinations, it was demonstrated that DENV-2 entry into U937 and K562 cells in the absence of Ab was highly inhibited by the early treatment with ammonium chloride, chlorpromazine and dynasore, but it was not affected by methyl-β-cyclodextrin, indicating that DENV-2 utilizes a low pH-dependent, clathrin- and dynamin-mediated endocytic infectious pathway for the direct entry into both human myeloid cells. To study the Ab-mediated entry of DENV, the experimental conditions for enhancement of infection were established by inoculating immune complexes formed with DENV-2 and the Ab 2H2 or 3H5. The internalization of DENV-2-2H2 or DENV-2-3H5 complexes in both myeloid cells was also dependent on acid pH and dynamin but a differential requirement of the clathrin-mediated endocytic route was observed depending on the FcγR involved in the complex uptake: the infection through FcγRII was dependent on clathrin-coated vesicles whereas the internalization pathway mediated by FcγRI was independent of clathrin. This property was not serotype-specific. CONCLUSIONS/SIGNIFICANCE:DENV entry into myeloid cells in the absence or presence of Ab can be blocked by diverse biochemical inhibitors affecting the cellular factors involved in endocytosis. The identification of the virus-host interactions involved in virus penetration may allow the finding of host-targeted antivirals widely active against diverse pathogenic flaviviruses with similar requirements for virus entry

Dengue-3 Virus Entry into Vero Cells: Role of Clathrin-Mediated Endocytosis in the Outcome of Infection

<div><p>The endocytic uptake and intracellular trafficking for penetration of DENV-3 strain H-87 into Vero cells was analyzed by using several biochemical inhibitors and dominant negative mutants of cellular proteins. The results presented show that the infective entry of DENV-3 into Vero cells occurs through a non-classical endocytosis pathway dependent on low pH and dynamin, but non-mediated by clathrin. After uptake, DENV-3 transits through early endosomes to reach Rab 7-regulated late endosomes, and according with the half-time for ammonium chloride resistance viral nucleocapsid is released into the cytosol approximately at 12 min post-infection. Furthermore, the influence of the clathrin pathway in DENV-3 infective entry in other mammalian cell lines of human origin, such as A549, HepG2 and U937 cells, was evaluated demonstrating that variable entry pathways are employed depending on the host cell. Results show for the first time the simultaneous coexistence of infective and non -infective routes for DENV entry into the host cell, depending on the usage of clathrin-mediated endocytosis.</p></div

Transport of DENV particles to late endosomes: Rab7 dependence.

<p>A. Cells transiently transfected with the GFP-tagged versions of Rab7 wt and DN T22N and the plasmid pGFP-C1 were infected with DENV-1 HW, DENV-2 NGC or 16681. After 24 h of infection cultures were fixed and processed to visualize GFP transgene expression and viral antigen by immunofluorescence staining using mouse anti-E glycoprotein antibody and TRITC-labelled anti-mouse IgG. B. For quantification of samples shown in A, 250 transfected cells with similar levels of GFP expression were screened and cells positive for viral antigen were scored. C. Cells transiently transfected as in A were infected with JUNV. At 24 h p.i. cells were fixed and infection was assessed by immunofluorescence using mouse anti-JUNV NP antibody and TRITC-labelled anti-mouse IgG.</p

Transport of DENV particles to recycling endosomes.

<p>Cells transiently transfected with the GFP-tagged versions of Rab22 wt and Q64L (A), Rab11 wt and S25N (B) and the plasmid pGFP-C1 were infected with DENV-1 HW, DENV-2 NGC or 16681. After 24 h of infection cultures were fixed and processed to visualize GFP transgene expression and viral antigen by immunofluorescence staining using mouse anti-E glycoprotein antibody and TRITC-labelled anti-mouse IgG. C. For quantification of samples shown in A and B, 250 transfected cells with similar levels of GFP expression were screened and cells positive for viral antigen were scored. D. Cells expressing GFP-Rab7 wt or Green Lantern-Rab11 wt were infected at 24 h post-transfection with DENV-2 NGC or 16681 during 60 min at 4°C and then shifted at 37°C. After 15 min cells were fixed and processed to visualize GFP transgene expression and viral antigen by immunofluorescence staining using mouse anti-C glycoprotein antibody and TRITC-labelled anti-mouse IgG. Cells were visualized with a confocal microscope.</p

Effect of blockade of clathrin-mediated endocytosis on DENV-1 and DENV-2 strains.

<p>A. Cells were treated with 50 µM chlorpromazine and then infected with reference strains and clinical isolates of DENV-1 or DENV-2. Virus yields were quantified by PFU at 48 h p.i. and results are expressed as % of inhibition of virus multiplication with respect to a control of infected cells without drug treatment. Each value is the mean±SD of three independent experiments. B. Cells were treated with 50 µM chlorpromazine or left untreated and incubated with TRITC-labelled transferrin. C. Cells transiently transfected with the constructs GFP-DIIIΔ2 or GFP-EH29 were infected with DENV-2 strain 16681. After 24 h cells were fixed and viral antigen expression was visualized by immunofluorescence staining using mouse anti-E glycoprotein antibody and TRITC-labelled anti-mouse IgG.</p