Mechanisms and Signals for the Nuclear Import of Proteins

In eukaryotes, the nuclear membrane provides a physical barrier to the passive diffusion of macromolecules from and into the cytoplasm. Nucleocytoplasmic traffic occurs through highly specialized structures known as nuclear pores, and involves the participation of a special class of transport proteins. Active transport across the nuclear pores is an energy-dependent process that relies on the activity of Ran-GTPases both in the nuclear and cytoplasmic compartments

Characterization of the nuclear localization signal of the hepatitis delta virus antigen

AbstractThe delta antigen (HDAg) is the only protein encoded by the hepatitis delta virus (HDV) RNA genome. The HDAg contains an RNA binding domain, a dimerization domain, and a nuclear localization signal (NLS). The nuclear import of HDV RNPs is thought to be one of the first tasks of the HDAg during the HDV replication cycle. Using c-myc–PK fusions with several regions of the HDAg in transfection assays in Huh7 cells, we found that the HDAg NLS consists of a single stretch of 10 amino acids, EGAPPAKRAR, located in positions 66–75. Deletion and mutation analysis of this region showed that both the acidic glutamic acid residue at position 66 and the basic arginine residue at position 75 are essential for promoting nuclear import

Validação do questionário de mapeamento do conhecimento ético em estudantes de enfermagem

INTRODUÇÃO A construção e validação de instrumentos válidos que permitam obter informações sobre o Conhecimento Ético, é necessária, uma vez que se torna essencial que o enfermeiro pense, reflita e oriente a sua prática de acordo com o seu respetivo código deontológico. OBJETIVO Descrever o processo de validação do Questionário Mapeamento do Conhecimento Ético (QMCE). MÉTODOS Participaram 85 estudantes de enfermagem, com média de idades de 20,96 anos. Foi analisada a consistência interna e a validade de constructo. O Questionário Mapeamento do Conhecimento Ético (Cunha et al., 2013), é constituído por 4 Partes: Parte I: Dados Biográficos e Académicos, Parte II: Modo de Agir, Parte III: Escala Tipologia dos Valores (ETV), Parte IV: Escala Valores/Deveres e Éticos (EVDE). RESULTADOS O alfa de Cronbach final da Escala Tipologia dos Valores (alfa=.839), revelou uma boa consistência interna, assim como os da Escala Valores/Deveres e Éticos (alfa=.870). Os estudantes apresentaram na sua maioria um nível de conhecimento positivo, na Parte III Escala Tipologia dos Valores (ETV) e Parte IV: Escala Valores/Deveres e Éticos (EVDE), respetivamente com 50.6% e 55.3%. CONCLUSÕES As propriedades psicométricas do Questionário Mapeamento do Conhecimento Ético certificam a sua qualidade, enquanto instrumento a utilizar na avaliação do nível de conhecimento ético dos estudantes de enfermagem.info:eu-repo/semantics/publishedVersio

Searching for nuclear export elements in hepatitis D virus RNA.

AIM To search for the presence of cis elements in hepatitis D virus (HDV) genomic and antigenomic RNA capable of promoting nuclear export. METHODS We made use of a well characterized chloramphenicol acetyl-transferase reporter system based on plasmid pDM138. Twenty cDNA fragments corresponding to different HDV genomic and antigenomic RNA sequences were inserted in plasmid pDM138, and used in transfection experiments in Huh7 cells. The relative amounts of HDV RNA in nuclear and cytoplasmic fractions were then determined by real-time polymerase chain reaction and Northern blotting. The secondary structure of the RNA sequences that displayed nuclear export ability was further predicted using a web interface. Finally, the sensitivity to leptomycin B was assessed in order to investigate possible cellular pathways involved in HDV RNA nuclear export. RESULTS Analysis of genomic RNA sequences did not allow identifying an unequivocal nuclear export element. However, two regions were found to promote the export of reporter mRNAs with efficiency higher than the negative controls albeit lower than the positive control. These regions correspond to nucleotides 266-489 and 584-920, respectively. In addition, when analyzing antigenomic RNA sequences a nuclear export element was found in positions 214-417. Export mediated by the nuclear export element of HDV antigenomic RNA is sensitive to leptomycin B suggesting a possible role of CRM1 in this transport pathway. CONCLUSION A cis-acting nuclear export element is present in nucleotides 214-417 of HDV antigenomic RNA.publishersversionpublishe

a fascinating and neglected pathogen

Hepatitis delta virus (HDV) is the etiologic agent of the most severe form of virus hepatitis in humans. Sharing some structural and functional properties with plant viroids, the HDV RNA contains a single open reading frame coding for the only virus protein, the Delta antigen. A number of unique features, including ribozyme activity, RNA editing, rolling-circle RNA replication, and redirection for a RNA template of host DNA-dependent RNA polymerase II, make this small pathogen an excellent model to study virus-cell interactions and RNA biology. Treatment options for chronic hepatitis Delta are scarce and ineffective. The disease burden is perhaps largely underestimated making the search for new, specific drugs, targets, and treatment strategies an important public health challenge. In this review we address the main features of virus structure, replication, and interaction with the host. Virus pathogenicity and current treatment options are discussed in the light of recent developments.publishersversionpublishe

Hepatitis delta virus: a peculiar virus.

The hepatitis delta virus (HDV) is distributed worldwide and related to the most severe form of viral hepatitis. HDV is a satellite RNA virus dependent on hepatitis B surface antigens to assemble its envelope and thus form new virions and propagate infection. HDV has a small 1.7 Kb genome making it the smallest known human virus. This deceivingly simple virus has unique biological features and many aspects of its life cycle remain elusive. The present review endeavors to gather the available information on HDV epidemiology and clinical features as well as HDV biology.publishersversionpublishe

The heterogeneous ribonuclear protein C interacts with the hepatitis delta virus small antigen

Background: Hepatitis delta virus (HDV) is considered to be a satellite virus of the Hepatitis B virus. The genome consists of a 1679 nt ssRNA molecule in which a single ORF was identified. This ORF codes for a unique protein, the Delta antigen (HDAg). During transcription, two forms, small (S-HDAg; p24) and large (L-HDAg; p27) of this antigen are derived as a result of an editing mechanism catalyzed by cellular adenosine deaminase 1. Despite its simplicity, little is still known about the host factors that interact with the virus RNA and antigens being to modulate virus replication. Methods: A yeast two-hybrid screening of a human liver cDNA library, using the hepatitis delta virus (HDV) small antigen (S-HDAg) as bait, was performed. Blot overlay and co-immunoprecipitation assays were used in an attempt to confirm the interaction of hnRNPC and S-HDAg. siRNA knockdown assays of hnRNPC were performed to assess the effect on HDV antigen expression. Results: Thirty known proteins were identified as S-HDAg interactors in the yeast two-hybrid screening. One of the identified proteins, hnRNPC, was found to interact with S-HDAg in vitro and in vivo in human liver cells. The interaction of the two proteins is mediated by the C-terminal half of the S-HDAg which contains a RNA-binding domain (aa 98-195). HDV RNA, S-HDAg, and hnRNPC, were also found to co-localize in the nucleus of human liver cells. Knockdown of hnRNPC mRNA using siRNAs resulted in a marked decreased expression of HDV antigens. Conclusions: S-HDAg was found to interact with human liver proteins previously assigned to different functional categories. Among those involved in nucleic acid metabolism, hnRNPC was found to interact in vitro and in vivo in human liver cells. Similar to other RNA viruses, it seems plausible that hnRNPC may also be involved in HDV replication. However, further investigation is mandatory to clarify this question.publishe

In Vivo Interaction of the Hepatitis Delta Virus Small Antigen with the ELAV-Like Protein HuR

The small and large delta antigens (S-HDAg and L-HDAg, respectively) represent two forms of the only protein encoded by the hepatitis delta virus (HDV) RNA genome. Consequently, HDV relies, at a large extent, on the host cell machinery for replication and transcription. Until now, only a limited number of cellular proteins were identified as S-HDAg or L-HDAg partners being involved in the modulation of the virus life cycle. In an attempt to identify cellular S-HDAg-binding proteins we made use of a yeast two-hybrid approach to screen a human liver cDNA library. We were able to identify HuR, a ubiquitously expressed protein involved in RNA stabilization, as an S-HDAg partner both in vitro and in vivo. HuR was found to be overexpressed and colocalize with HDAg in human hepatoma cells. siRNA knockdown of HuR mRNA resulted in inhibition of S-HDAg and L-HDAg expression