Assuntos
DNA/química , Exodesoxirribonucleases/química , Candida/genética , Carbocianinas , DNA/genética , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Fúngico/química , DNA Fúngico/genética , Corantes Fluorescentes , Listeria monocytogenes/genética , Técnicas Microbiológicas , Neisseria meningitidis/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Egress of herpes capsids from the nucleus to the plasma membrane is a complex multistep transport event that is poorly understood. The current model proposes an initial envelopment at the inner nuclear membrane of capsids newly assembled in the nucleus. The capsids are then released in cytosol by fusion with the outer nuclear membrane. They are finally reenveloped at a downstream organelle before traveling to the plasma membrane for their extracellular release. Although the trans-Golgi network (TGN) is often cited as a potential site of reenvelopment, other organelles have also been proposed, including the Golgi, endoplasmic reticulum-Golgi intermediate compartment, aggresomes, tegusomes, and early or late endosomes. To clarify this important issue, we followed herpes simplex virus type 1 egress by immunofluorescence under conditions that slowed intracellular transport and promoted the accumulation of the otherwise transient reenvelopment intermediate. The data show that the capsids transit by the TGN and point to this compartment as the main reenvelopment site, although a contribution by endosomes cannot formally be excluded. Given that viral glycoproteins are expected to accumulate where capsids acquire their envelope, we examined this prediction and found that all tested could indeed be detected at the TGN. Moreover, this accumulation occurred independently of capsid egress. Surprisingly, capsids were often found immediately adjacent to the viral glycoproteins at the TGN.
Assuntos
Capsídeo/metabolismo , Glicoproteínas/metabolismo , Herpesvirus Humano 1/fisiologia , Proteínas Virais/metabolismo , Rede trans-Golgi/virologia , Animais , Chlorocebus aethiops , Humanos , Transporte Proteico , Células Vero , Rede trans-Golgi/metabolismoRESUMO
The interaction between nuclear receptors (NRs) and their coactivators, a key step in transcription regulation, requires a short consensus sequence called the LXXLL motif found in the coactivators' structure. Using the AlphaScreen technology, the authors have taken advantage of this receptor-coactivator interaction to develop a highly sensitive assay to identify and characterize compounds modulating NR activity. Estrogen and retinoic acid receptors were chosen as models to demonstrate the versatility of the AlphaScreen technology: (1) the assay can be designed using different antibodies to capture either full-length receptors or receptor domains that have been tagged, (2) the assay can differentiate between ligands that act as agonists or antagonists because only agonists will allow recruitment of the coactivator sequence-derived peptide, and (3) the assay gives the opportunity to screen for antagonists targeting the ligand-binding site or the dimerization interface between the receptor and the coactivator. Titration of the receptor and biotinylated peptide indicates that AlphaScreen is highly sensitive, requiring nanomolar concentration of reagents. Competition isotherms performed with known receptor antagonists demonstrate that the assay is a useful tool to rank the antagonists according to their order of potency. Overall, the results presented here indicate that the versatility, sensitivity, robustness, and ease of execution of the AlphaScreen NR assay will allow for efficient screening of NR modulators.
