RESUMEN
In the last five years, cellular mRNA export has been extensively studied and revealed the existence of two distinct pathways. The majority of mRNA is exported in a TAP dependent pathway and the others RNAs need a CRM1 dependent pathway to reach the cytoplasm. During evolution, viruses acquired sophisticated mechanisms to hijack cellular pathways to export their genomic, subgenomic and messenger RNAs. Some viral nucleic acids code proteins that interact with the cellular export machinery and with viral RNAs. Others harbour specific secondary structures, which are recognized directly or indirectly by CRM-1 or TAP proteins. This review highlights these mechanisms, underlining the importance of virology in understanding the export of these cellular molecules from the nucleus.
RESUMEN
Messengers RNA (mRNA) are thought to be export from the nucleus as ribonucleoprotein complexes (mRNP), whose exact protein composition remains not completely determined. The Gle2/RAE1 protein, a highly conserved member of the WD40 repeat protein family, was first shown to be involved in mRNA export. More recently, a role in the cell cycle was also suggested. To get new insights into the functions of the metazoan protein, the Drosophila melanogaster rae1 (dmrae1) cDNA was first cloned, then the corresponding protein characterized and its function investigated by RNA interference. This paper shows that dmRae1 mainly localises to the nuclear membrane like its homologue in Saccharomyces cerevisiae. However, unlike its homologue and despite this particular sub-cellular distribution, its depletion does not impair the export of polyA+ RNAs. Interestingly, the presence of dmRae1 is important for normal proliferation and, more importantly, for the progression through the G1 phase of the cell cycle. Given that dmRae1 is closely related to the human form, results suggest that the human homologue, hsRAE1, may also play a similar role during the cell cycle.
Asunto(s)
Drosophila melanogaster/genética , Fase G1/genética , Proteínas Asociadas a Matriz Nuclear/genética , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Saccharomyces cerevisiae , Secuencia de Aminoácidos , Animales , Bromodesoxiuridina/metabolismo , Proteínas de Ciclo Celular/genética , Línea Celular , Proteínas de Drosophila/genética , Drosophila melanogaster/citología , Expresión Génica , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/genética , Microscopía Confocal , Datos de Secuencia Molecular , Proteínas Asociadas a Matriz Nuclear/metabolismo , Proteínas de Complejo Poro Nuclear , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Interferencia de ARN , ARN Bicatenario/genética , ARN Bicatenario/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Factores de TiempoRESUMEN
gC1qR, a mitochondrial matrix protein, was identified as the main cellular partner of the hepatitis B virus P22 protein. We demonstrated by immunofluorescence studies that some P22 molecules were colocalized with the endogenous gC1qR in both the cytoplasm and the nucleus but never in the mitochondria. We also showed that the last 34 amino acids of P22 were involved in the association with gC1qR.
Asunto(s)
Receptores de Hialuranos , Glicoproteínas de Membrana , Receptores de Complemento/metabolismo , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Proteínas Portadoras , Núcleo Celular/metabolismo , Células Cultivadas , Citoplasma/metabolismo , Técnica del Anticuerpo Fluorescente , Humanos , Mitocondrias/metabolismo , Proteínas Mitocondriales , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Proteínas Virales/químicaRESUMEN
Vesicular stomatitis virus matrix protein (VSV M) has been shown to inhibit both transcription and nucleocytoplasmic transport. We have isolated a mutant form of M, termed M(D), lacking both inhibitory activities. HeLa cells expressing M, but not M(D), accumulate polyadenylated RNAs within the nucleus. Concomitantly, a fraction of M, but not of the M(D) mutant, localizes at the nuclear rim. Additionally, the nucleoporin Nup98 specifically interacts with M but not with M(D). In Nup98(-/-) cells, both the levels of M at the nuclear envelope and its inhibitory effects on host cell-directed expression of reporter genes were significantly reduced. Together, our data demonstrate that VSV M inhibits host cell gene expression by targeting a nucleoporin and primarily blocking nuclear export.
Asunto(s)
Núcleo Celular/metabolismo , Regulación de la Expresión Génica , Proteínas de Complejo Poro Nuclear/metabolismo , Proteínas de la Matriz Viral/metabolismo , Transporte Activo de Núcleo Celular , Secuencia de Aminoácidos , Animales , Línea Celular , Núcleo Celular/genética , Células HeLa , Humanos , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Mutación/genética , Poro Nuclear/química , Poro Nuclear/metabolismo , Proteínas de Complejo Poro Nuclear/química , Proteínas de Complejo Poro Nuclear/deficiencia , Proteínas de Complejo Poro Nuclear/genética , Unión Proteica , Estructura Terciaria de Proteína , ARN Mensajero/genética , ARN Mensajero/metabolismo , Secuencias Repetitivas de Aminoácido , Especificidad por Sustrato , Transcripción Genética , Proteínas de la Matriz Viral/química , Proteínas de la Matriz Viral/genéticaRESUMEN
Mammalian hepatitis B viruses encode a unique regulatory protein termed X, which is essential for infection and likely plays a role in the carcinogenic process associated with hepadnaviral infection. Among the numerous properties ascribed to X protein, two have been widely documented: promiscuous transcriptional transactivation and proapoptosis. However, full understanding of the mechanisms underlying these activities requires the identification of the genuine X partners among the multiple X-binding host proteins. Here we show that (i) mutations in X protein, which markedly alter affinity for the host protein UVDDBp127, inactivate both transactivation and proapoptosis; (ii) ectopic fusion of a functional UVDDB-binding domain to a deficient binding X mutant restored its activity; (iii) in contrast to the loss-of-binding mutants, a mutant with a strong gain-of-binding exerted trans-dominant negative effects on wt X activity and localized in the nucleus and (iv) increase in intracellular UVDDB concentration enhanced both wt X-mediated transactivation and apoptosis. Taken together, our data provide strong evidence for a common upstream step in X mode of action, consisting of its productive interaction with UVDDB, via a structurally and functionally autonomous module. In addition, they underscore a nuclear location step of the viral protein that depends on its ability to bind UVDDB.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Transactivadores/metabolismo , Apoptosis/fisiología , Transporte Biológico , Línea Celular , Núcleo Celular/metabolismo , Supervivencia Celular , Virus de la Hepatitis B de la Marmota/química , Humanos , Mutación , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Activación Transcripcional , Proteínas Reguladoras y Accesorias ViralesRESUMEN
A fully effective treatment of chronic human hepatitis B virus (HBV) infection is still missing and HBV remains the first etiological agent of liver cancer. Although the viral regulatory X protein is essential for infection, its mode of action remains obscure, due the lack of an in vitro infection system. In the accompanying study, we showed the functional importance of interaction between X and the host protein UVDDB-p127, in the transactivation and apoptotic properties of the viral protein. Here, we addressed the biological role of X-UVDDB interaction in the infectious process using a genetic approach in the woodchuck virus closely related to HBV. We show that (i) mutations in X, which markedly affect UVDDB-binding, also abolished productive infection in woodchucks, (ii) in the few cases where mutant viruses led to infection, compensatory mutations had occurred in the X gene of the viral progeny, which restored correct UVDDB-binding. We conclude that efficient viral replication in vivo requires proper X-UVDDB interaction. The interaction may thus provide a novel therapeutic target for the treatment of hepatitis
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Virus de la Hepatitis B de la Marmota/genética , Virus de la Hepatitis B de la Marmota/patogenicidad , Transactivadores/metabolismo , Animales , Hepatitis B/veterinaria , Hepatitis B/virología , Virus de la Hepatitis B de la Marmota/metabolismo , Marmota , Mutación , Transactivadores/genética , Proteínas Reguladoras y Accesorias Virales , Replicación Viral/genéticaRESUMEN
The PML/SP100 nuclear bodies (NBs) were first described as discrete subnuclear structures containing the SP100 protein. Subsequently, they were shown to contain the PML protein which is part of the oncogenic PML-RARalpha hybrid produced by the t(15;17) chromosomal translocation characteristic of acute promyelocytic leukemia. Yet, the physiological role of these nuclear bodies remains unknown. Here, we show that SP100 binds to members of the heterochromatin protein 1 (HP1) families of non-histone chromosomal proteins. Further, we demonstrate that a naturally occurring splice variant of SP100, here called SP100-HMG, is a member of the high mobility group-1 (HMG-1) protein family and may thus possess DNA-binding potential. Both HP1 and SP100-HMG concentrate in the PML/SP100 NBs, and overexpression of SP100 leads to enhanced accumulation of endogenous HP1 in these structures. When bound to a promoter, SP100, SP100-HMG and HP1 behave as transcriptional repressors in transfected mammalian cells. These observations present molecular evidence for an association between the PML/SP100 NBs and the chromatin nuclear compartment. They support a model in which the NBs may play a role in certain aspects of chromatin dynamics.
Asunto(s)
Antígenos Nucleares , Autoantígenos/metabolismo , Proteínas Portadoras/metabolismo , Núcleo Celular/metabolismo , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Proteínas del Grupo de Alta Movilidad/metabolismo , Leucemia Promielocítica Aguda/metabolismo , Proteínas Nucleares/metabolismo , Secuencia de Aminoácidos , Biomarcadores de Tumor , Homólogo de la Proteína Chromobox 5 , Cromosomas Humanos Par 15 , Cromosomas Humanos Par 17 , Proteína HMGB1 , Células HeLa , Humanos , Leucemia Promielocítica Aguda/mortalidad , Datos de Secuencia Molecular , Unión Proteica , Proteínas Represoras/metabolismo , TransfecciónRESUMEN
We carried out a comparative analysis of several proposed host protein partners of the human hepatitis B virus X protein (HBx) using both the GAL4- and the LexA-based yeast two-hybrid system. We showed that the interaction of HBx with the UV-damaged DNA-binding protein (UVDDB) is positive in both yeast systems, detectable in cotransfected human cells, conserved by rodent hepadnavirus X proteins (known to transactivate in human cells), and tightly correlated with the transactivation proficiency of X-insertion mutants. Taken together, our results strongly suggest that UVDDB is involved in X-mediated transactivation.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Hepadnaviridae/genética , Transactivadores/fisiología , Activación Transcripcional , Sitios de Unión , Proteínas de Unión al ADN/efectos de la radiación , Humanos , Mutación , Rayos Ultravioleta , Proteínas Reguladoras y Accesorias ViralesRESUMEN
In most cases, Acute Promyelocytic Leukemia (APL) is associated with t(15;17) translocation which juxtaposes sequences from PML and retinoic acid receptor alpha (RAR alpha) genes. The generated PML-RAR alpha fusion interferes with wild type RAR alpha-mediated transcription and disrupts subnuclear compartments, known as PML bodies. Both defects are corrected by all trans retinoic acid (ATRA) therapy which induces differentiation of leukemic cells and clinical remission. In a rare APL syndrome associated with t(11;17), fusion of the RAR alpha gene with the PLZF gene, encoding a Zinc-finger protein produces two reciprocal RAR alpha chimeras. Although PLZF-RAR alpha and PML-RAR alpha are similar in their apparent dominant negative effects, t(11;17)-associated APL is refractory to ATRA therapy. In a yeast two-hybrid genetic screening, we isolated clones encoding the GAL4 transactivation domain fused to various parts of PLZF. Using these autonomously transactivating hybrids, similar in structure to the RAR alpha-PLZF fusion, we mapped the DNA-binding domain of PLZF to the last five Zinc-fingers, a region retained in RAR alpha-PLZF chimera and characterized a specific PLZF target sequence. Our data support the hypothesis that RAR alpha-PLZF chimera is not an inert product of reciprocal translocation and may thus contribute to ATRA unresponsiveness of t(11;17)-associated APL.