hCLE/RTRAF-HSPC117-DDX1-FAM98B: A New Cap-Binding Complex That Activates mRNA Translation.

hCLE/C14orf166/RTRAF, DDX1, and HSPC117 are components of cytoplasmic mRNA-transporting granules kinesin-associated in dendrites. They have also been found in cytoplasmic ribosome-containing RNA granules that transport specific mRNAs halted for translation until specific neuronal signals renders them accessible to the translation machinery. hCLE associates to DDX1, HSPC117, and FAM98B in HEK293T cells and all four proteins bind to cap analog-containing resins. Competition and elution experiments indicate that binding of hCLE complex to cap resins is independent of eIF4E; the cap-binding factor needed for translation. Purified hCLE free of its associated proteins binds cap with low affinity suggesting that its interacting proteins modulate its cap association. hCLE silencing reduces hCLE accumulation and that of its interacting proteins and decreases mRNA translation. hCLE-associated RNAs have been isolated and sequenced; RNAs involved in mRNA translation are specifically associated. The data suggest that RNA granules may co-transport RNAs encoding proteins involved in specific functions together with RNAs that encode proteins needed for the translation of these specific RNAs and indicate an important role for hCLE modulating mRNA translation.

Apoptosis, Toll-like, RIG-I-like and NOD-like Receptors Are Pathways Jointly Induced by Diverse Respiratory Bacterial and Viral Pathogens.

Lower respiratory tract infections are among the top five leading causes of human death. Fighting these infections is therefore a world health priority. Searching for induced alterations in host gene expression shared by several relevant respiratory pathogens represents an alternative to identify new targets for wide-range host-oriented therapeutics. With this aim, alveolar macrophages were independently infected with three unrelated bacterial (Streptococcus pneumoniae, Klebsiella pneumoniae, and Staphylococcus aureus) and two dissimilar viral (respiratory syncytial virus and influenza A virus) respiratory pathogens, all of them highly relevant for human health. Cells were also activated with bacterial lipopolysaccharide (LPS) as a prototypical pathogen-associated molecular pattern. Patterns of differentially expressed cellular genes shared by the indicated pathogens were searched by microarray analysis. Most of the commonly up-regulated host genes were related to the innate immune response and/or apoptosis, with Toll-like, RIG-I-like and NOD-like receptors among the top 10 signaling pathways with over-expressed genes. These results identify new potential broad-spectrum targets to fight the important human infections caused by the bacteria and viruses studied here.

hCLE/C14orf166, a cellular protein required for viral replication, is incorporated into influenza virus particles.

The influenza A virus polymerase associates with a number of cellular transcription-related factors, including the RNA polymerase II (RNAP II). We previously described that the cellular protein hCLE/C14orf166 interacts with and stimulates influenza virus polymerase as well as RNAP II activities. Here we show that, despite the considerable cellular shut-off observed in infected cells, which includes RNAP II degradation, hCLE protein levels increase throughout infection in a virus replication-dependent manner. Human and avian influenza viruses of various subtypes increase hCLE levels, but other RNA or DNA viruses do not. hCLE colocalises and interacts with viral ribonucleoproteins (vRNP) in the nucleus, as well as in the cytoplasm late in infection. Furthermore, biochemical analysis of purified virus particles and immunoelectron microscopy of infected cells show hCLE in virions, in close association with viral vRNP. These findings indicate that hCLE, a cellular protein important for viral replication, is one of the very few examples of transcription factors that are incorporated into particles of an RNA-containing virus.

hCLE/C14orf166 associates with DDX1-HSPC117-FAM98B in a novel transcription-dependent shuttling RNA-transporting complex.

hCLE/C14orf166 is a nuclear and cytoplasmic protein that interacts with the RNAP II, modulates nuclear RNA metabolism and is present in cytoplasmic RNA granules involved in localized translation. Here we have studied whether hCLE shares common interactors in the nucleus and the cytosol, which could shed light on its participation in the sequential phases of RNA metabolism. Nuclear and cytoplasmic purified hCLE-associated factors were identified and proteins involved in mRNA metabolism, motor-related proteins, cytoskeletal and translation-related factors were found. Purified hCLE complexes also contain RNAs and as expected some hCLE-interacting proteins (DDX1, HSPC117, FAM98B) were found both in the nucleus and the cytoplasm. Moreover, endogenous hCLE fractionates in protein complexes together with DDX1, HSPC117 and FAM98B and silencing of hCLE down-regulates their nuclear and cytosolic accumulation levels. Using a photoactivatable hCLE-GFP protein, nuclear import and export of hCLE was observed indicating that hCLE is a shuttling protein. Interestingly, hCLE nuclear import required active transcription, as did the import of DDX1, HSPC117 and FAM98B proteins. The data indicate that hCLE probably as a complex with DDX1, HSPC117 and FAM98B shuttles between the nucleus and the cytoplasm transporting RNAs suggesting that this complex has a prominent role on nuclear and cytoplasmic RNA fate.

[Polio vaccines, eradication and posterradication]. / Vacunas antipoliomieliticas, erradicación y posterradicación.

Vaccination against polio generates herd immunity (both with the attenuated (OPV) and inactivated (IPV) vaccines) and this will allow the eradication of the disease. The OPV vaccine produces 2-4 polio cases per cohort of one million children and therefore IPV is used in countries that can afford its cost (about 15 times more expensive than OPV). In 1988 the World Health Assembly established the polio eradication goal as "interruption of wild poliovirus transmission". If the elimination of wild poliovirus were achieved, the use of OPV will produce annually between 250 and 500 cases of polio in the world. From 1999, it was clear that eradication would require ending of immunization with OPV. On the 25th of January, 2013 it is approved the plan for the eradication and containment of all polioviruses, wild or not, so that no child suffers paralytic poliomyelitis. The most important landmarks include the lack of wild polio cases after 2014, the introduction of at least one dose of IPV in all immunization programs and to cease the type 2 OPV vaccination by the end of 2016 and to stop the use of the oral bivalent vaccine in 2019. To achieve all this, a complex scientific work and economic solidarity will be required.

Vacunas antipoliomieliticas, erradicación y posterradicación / Polio vaccines, eradication and posterradication

La vacunación antipoliomielítica genera inmunidad de grupo (con vacunas atenuadas (VPO) e inactivadas (VPI) y ello permitirá la erradicación de la enfermedad. La VPO produce de 2-4 casos de poliomielitis por cohorte de un millón de niños y por ello los países que pueden hacer frente al coste de la VPI (unas 15 veces más cara) la utilizan. En 1988 la Asamblea de la Organización Mundial de la Salud aprobó el objetivo de la erradicación como “la interrupción de la transmisión de poliovirus salvajes”. Si se conseguía su eliminación, el mantenimiento de la VPO produciría al año entre 250 y 500 casos de poliomielitis en el mundo. Desde 1999 era evidente que la erradicación requeriría la cesación de la vacunación con VPO. El 25 de enero del 2013 se aprobó el plan para la erradicación y la contención de todos los virus de la polio, salvajes o no, para que ningún niño sufra una poliomielitis paralítica. Los hitos más importantes incluyen, la no aparición de casos de polio salvaje tras el año 2014, la introducción de al menos una dosis de VPI en todos los programas de vacunación y que se suspenda la vacunación con VPO tipo 2 al final del 2016 y que en 2019 se pueda cesar de utilizar la vacuna bivalente oral. Para todo ello será preciso un trabajo científico complejo y solidaridad financiera (AU)

Vaccination against polio generates herd immunity (both with the attenuated (OPV) and inactivated (IPV) vaccines) and this will allow the eradication of the disease. The OPV vaccine produces 2-4 polio cases per cohort of one million children and therefore IPV is used in countries that can afford its cost (about 15 times more expensive than OPV). In 1988 the World Health Assembly established the polio eradication goal as “interruption of wild poliovirus transmission”. If the elimination of wild poliovirus were achieved, the use of OPV will produce annually between 250 and 500 cases of polio in the world. From 1999, it was clear that eradication would require ending of immunization with OPV. On the 25th of January, 2013 it is approved the plan for the eradication and containment of all polioviruses, wild or not, so that no child suffers paralytic poliomyelitis. The most important landmarks include the lack of wild polio cases after 2014, the introduction of at least one dose of IPV in all immunization programs and to cease the type 2 OPV vaccination by the end of 2016 and to stop the use of the oral bivalent vaccine in 2019. To achieve all this, a complex scientific work and economic solidarity will be required (AU)

Cellular human CLE/C14orf166 protein interacts with influenza virus polymerase and is required for viral replication.

The influenza A virus polymerase associates with a number of cellular transcription-related factors, including RNA polymerase II. We previously described the interaction of influenza virus polymerase subunit PA with human CLE/C14orf166 protein (hCLE), a positive modulator of this cellular RNA polymerase. Here, we show that hCLE also interacts with the influenza virus polymerase complex and colocalizes with viral ribonucleoproteins. Silencing of hCLE causes reduction of viral polymerase activity, viral RNA transcription and replication, virus titer, and viral particle production. Altogether, these findings indicate that the cellular transcription factor hCLE is an important protein for influenza virus replication.

Attenuated strains of influenza A viruses do not induce degradation of RNA polymerase II.

We have previously shown that infection with laboratory-passaged strains of influenza virus causes both specific degradation of the largest subunit of the RNA polymerase II complex (RNAP II) and inhibition of host cell transcription. When infection with natural human and avian isolates belonging to different antigenic subtypes was examined, we observed that all of these viruses efficiently induce the proteolytic process. To evaluate whether this process is a general feature of nonattenuated viruses, we studied the behavior of the influenza virus strains A/PR8/8/34 (PR8) and the cold-adapted A/Ann Arbor/6/60 (AA), which are currently used as the donor strains for vaccine seeds due to their attenuated phenotype. We have observed that upon infection with these strains, degradation of the RNAP II does not occur. Moreover, by runoff experiments we observe that PR8 has a reduced ability to inhibit cellular mRNA transcription. In addition, a hypervirulent PR8 (hvPR8) variant that multiplies much faster than standard PR8 (lvPR8) in infected cells and is more virulent in mice than the parental PR8 virus, efficiently induces RNAP II degradation. Studies with reassortant viruses containing defined genome segments of both hvPR8 and lvPR8 indicate that PA and PB2 subunits individually contribute to the ability of influenza virus to degrade the RNAP II. In addition, recently it has been reported that the inclusion of PA or PB2 from hvPR8 in lvPR8 recombinant viruses, highly increases their pathogenicity. Together, the data indicate that the capacity of the influenza virus to degrade RNAP II and inhibit the host cell transcription machinery is a feature of influenza A viruses that might contribute to their virulence.

hCLE/CGI-99, a human protein that interacts with the influenza virus polymerase, is a mRNA transcription modulator.

The human protein hCLE was previously identified by its interaction with the PA subunit of influenza virus polymerase. It exhibits a sequence similarity of 38% with the yeast Spt16 component of the FACT complex, which is involved in transcriptional regulation. Therefore, we studied the possible relationship of hCLE with the transcription machinery. Here we show that hCLE and different phosphorylated forms of the RNA polymerase II (RNAP II) largest subunit, co-immunoprecipitate and colocalize by confocal microscopy analysis. Furthermore, hCLE was found in nuclear sites of active mRNA synthesis, as demonstrated by its colocalization with spots of in situ Br-UTP incorporation. Silencing of hCLE expression by RNA interference inhibited the synthesis of RNAP II transcripts around 50%. Accordingly, the expression profiling in hCLE-silenced cells studied by microarray analysis showed that, among the genes that exhibited a differential expression under hCLE silencing, more than 90% were down-regulated. Collectively these results indicate that hCLE works as a positive modulator of the RNA polymerase II activity.

An alternative domain containing a leucine-rich sequence regulates nuclear cytoplasmic localization of protein 4.1R.

In red blood cells, protein 4.1 (4.1R) is an 80-kDa protein that stabilizes the spectrin-actin network and anchors it to the plasma membrane. The picture is more complex in nucleated cells, in which many 4.1R isoforms, varying in size and intracellular location, have been identified. To contribute to the characterization of signals involved in differential intracellular localization of 4.1R, we have analyzed the role the exon 5-encoded sequence plays in 4.1R distribution. We show that exon 5 encodes a leucine-rich sequence that shares key features with nuclear export signals (NESs). This sequence adopts the topology employed for NESs of other proteins and conserves two hydrophobic residues that are shown to be critical for NES function. A 4.1R isoform expressing the leucine-rich sequence binds to the export receptor CRM1 in a RanGTP-dependent fashion, whereas this does not occur in a mutant whose two conserved hydrophobic residues are substituted. These two residues are also essential for 4.1R intracellular distribution, because the 4.1R protein containing the leucine-rich sequence localizes in the cytoplasm, whereas the mutant protein predominantly accumulates in the nucleus. We hypothesize that the leucine-rich sequence in 4.1R controls distribution and concomitantly function of a specific set of 4.1R isoforms.