Luces y sombras entre familias y escuelas : una relación protagónica en el escenario educativo actual

Fil: Ferreyra, Horacio Ademar. Universidad Católica de Córdoba. Facultad de Educación; ArgentinaFil: Bonetti, Olga Concepción. Universidad Católica de Córdoba. Facultad de Educación; ArgentinaFil: Di Francesco, Adriana Carlota. Universidad Católica de Córdoba. Facultad de Educación; Argentin

Luces y sombras entre familias y escuelas: una relación protagónica en el escenario educativo actual

Fil: Ferreyra, Horacio Ademar. Universidad Católica de Córdoba. Facultad de Educación; ArgentinaFil: Bonetti, Olga Concepción. Universidad Católica de Córdoba. Facultad de Educación; ArgentinaFil: Di Francesco, Adriana Carlota. Universidad Católica de Córdoba. Facultad de Educación; Argentin

EducaFarma 11.0

Memoria ID2022-036. Ayudas de la Universidad de Salamanca para la innovación docente, curso 2022-2023

Educafarma 10.0

Memoria ID-030. Ayudas de la Universidad de Salamanca para la innovación docente, curso 2021-2022

PTB Binds to the 3’ Untranslated Region of the Human Astrovirus Type 8: A Possible Role in Viral Replication

<div><p>The 3′ untranslated region (3′UTR) of human astroviruses (HAstV) consists of two hairpin structures (helix I and II) joined by a linker harboring a conserved PTB/hnRNP1 binding site. The identification and characterization of cellular proteins that interact with the 3′UTR of HAstV-8 virus will help to uncover cellular requirements for viral functions. To this end, mobility shift assays and UV cross-linking were performed with uninfected and HAstV-8-infected cell extracts and HAstV-8 3′UTR probes. Two RNA-protein complexes (CI and CII) were recruited into the 3′UTR. Complex CII formation was compromised with cold homologous RNA, and seven proteins of 35, 40, 45, 50, 52, 57/60 and 75 kDa were cross-linked to the 3′UTR. Supermobility shift assays indicated that PTB/hnRNP1 is part of this complex, and 3′UTR-crosslinked PTB/hnRNP1 was immunoprecipitated from HAstV-8 infected cell-membrane extracts. Also, immunofluorescence analyses revealed that PTB/hnRNP1 is distributed in the nucleus and cytoplasm of uninfected cells, but it is mainly localized perinuclearly in the cytoplasm of HAstV-8 infected cells. Furthermore, the minimal 3′UTR sequences recognized by recombinant PTB are those conforming helix I, and an intact PTB/hnRNP1-binding site. Finally, small interfering RNA-mediated PTB/hnRNP1 silencing reduced synthesis viral genome and virus yield in CaCo2 cells, suggesting that PTB/hnRNP1 is required for HAstV replication. In conclusion, PTB/hnRNP1 binds to the 3′UTR HAstV-8 and is required or participates in viral replication.</p></div

PTB bind to the HAstV8 virus 3′UTR.

<p><b>A)</b> Supermobility shift assay from uninfected or HAstV-8 infected CaCo2 S10 extracts were incubated without (lane 2) or with anti-hnRNP1 (lane 3) or with a non-related antibody (lane 4) after of the 3′UTR labeled RNA (lane 1). Supershifted complexes are indicated as CIII. <b>B)</b> Uninfected (U) and infected CaCo2 cells cytoplasmic and nuclear cell extracts (8 h p.i.) prepared as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113113#pone-0113113-g002" target="_blank">Figure 2D</a> were probed with monoclonal anti-PTB and polyclonal anti-hnRNP1 antibodies by western blotting. <b>C)</b> Colocalization of hnRNP1 (green) in Mock infected (upper panel) or infected CaCo2 cells (lower panel). Cells were fixed at 12 h p.i. and were subjected to indirect immunofluorescence. The HAstV-8 infected positive cells were labeled with anti-HAstV CQFG sera (red). Nuclei are shown by DAPI (blue) staining. The images of cell were acquired with a confocal lasser scanning microscope LSM 710 (Carl Zeiss). <b>D)</b> 3′UTR RNA was cross-linked with 60 µg of HAstV-8-infected CaCo2 cells extracts (lanes 2 to 6 and 8) or mutant mPTB RNA labeled (lane 7) and immunoprecipitated with anti-hnRNP1 antibody (lanes 7 and 8) or with a non-related antibody (lane 5). The lanes 4 and 6 are internal controls. Probes are indicated with asterisks and the arrow indicates the position of the immunoprecipitated labeled PTB/hnRNP1.</p

CaCo2 cellular proteins interact with the HAstV-8 3′UTR.

<p><b>A)</b> Schematic representation of the 3′UTR from HAstV-8 and their short versions used to produce RNAs. The complete 3′UTR and mPTB version consisting of nt 6,674 to 6,759, H1 and HI/mPTB shorts versions consisting of nt 6,674 to 6,706. The versions HI/mPTB and mPTB has a UCUU PTB binding site within nt 6700 to 6704 changed to CGAA. The amplicons were used for RNA probe synthesis. <b>B)</b> Competition gel mobility shift assay. Different amounts of non-radiolabeled competitor RNA were incubated with 5 µg of S10 extracts from HAstV-8-infected CaCo2 cells before addition of the labeled 3′UTR RNA. Free probe (lane 1); (lane 2) no competitor; 20-fold (lanes 3 and 5) or 60-fold molar excess (lanes 4 and 6); (lanes 3 and 4) cold homologous RNA or heterologous RNA (lanes 5 and 6). Complex CI and specific complex CII are indicated. <b>C)</b> Labeled 3′UTR RNA (nt 6674 to 6759) was incubated without extract (lane 1) or with 20 µg of S10 from uninfected (lane 2) or HAstV-8-infected CaCo2 cells (lane 3). After UV crosslinking, the proteins were separated by SDS-10%PAGE and detected by autoradiography. Arrows indicate cross-linked proteins with the molecular masses in kilo daltons. <b>D)</b> Thirty micrograms of uninfected (U) and infected CaCo2 cells cytoplasmic and nuclear cell extracts were analyzed by western blotting with monoclonal anti- Actin and polyclonal anti-Lamin A antibodies.</p

CaCo2 cellular proteins interact with the HAstV-8 3′UTR.

<p><b>A)</b> Diagram of the HAstV-8 genome. The blowout represents the 3′UTR, showing the minimal energy model of its secondary structure. The 3′UTRs of all astrovirus contain one consensus UCUU PTB binding site (light gray) and a second UCUC element PTB (dark gray). Hyphens indicate gaps in the sequence. <b>B)</b> The RNA structures of the viral 3′UTR of all HAstVs were predicted with the MFold software. Comparisons of Helix I-linker structures and of Helix II structures are shown in the upper and lower panels, respectively. Residues within the single stranded regions of the putative PTB binding sites appear underlined.</p

His-PTB binds to the HAstV-8 3′UTR.

<p>UV crosslinking assay carried out with radiolabeled HAstV-8 3′UTR (<b>A</b>), mPTB (<b>B</b>), HI (<b>C</b>) and HI/mPTB (<b>D</b>) were incubated with 0.50 and 1 µg of recombinant His-PTB or 1 µg of BSA (C). Free probes are shown in lanes 1 and F, respectively. The arrow and asterisk indicate the His-PTB protein cross-linked to the viral RNA. The molecular mass markers (MWM) are shown. The secondary structures of the RNA probes are shown below. Consensus PTB site is indicated as continuous line in HI, PTB sequence changes appear undelined in probes HI/mPTB and mPTB.</p

Effect of PTB knockdown on the replication of HAstV8.

<p><b>A)</b> CaCo-2 cells were transfected with the indicated siRNA and infected with HAstV-8 following the schedule. <b>B)</b> Total protein was harvesting to demonstrate PTB knockdown by western blot, Actin was used as loading control. PTB signal intensity was quantified by densitometry. <b>C)</b> The nsP1b protein level was monitored by western-blotting in transfected and infected CaCo2 cells 15 hp.i. with polyclonal 1b-2 and actin antibodies. nsP1b signal intensity was quantified by densitometry as describe above. Signal intensity is expressed as percent of expression reduction with respect to actin signals. <b>D)</b> Relative RNA level of HAstV-8 in transfected and an infected cell were determined by qRT-PCR. The results shown are from triplicate assays; error bars represent standard deviations (<i>P</i><0.0001, two-tailed Student <i>t</i> test). <b>E)</b> The viral yield was determined by focus forming units per ml (ffu/ml) from supernatants recovered 72 h post-transfection of CaCo-2 cells, with the indicated siRNA and HAstV-8 infection (15 hp.i.). The Y axis represents the virus release percentage of 15 hours post infection after 72 hours post-transfection time. Duplicate experiments rendered identical results.</p