title: PTB binds to the 3' untranslated region of the human astrovirus type 8: a possible role in viral replication.
abstract: 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.
authors.affiliation: Programa Institucional de Biomedicina Molecular, Sección de Estudios de Posgrado e Investigación, ENMH, Instituto Politécnico Nacional, Col. Fracc. La Escalera-Ticomán, México D.F., México.
authors.forename: Wendy
authors.initials: W
authors.lastname: Espinosa-Hernández
authors.affiliation: Programa Institucional de Biomedicina Molecular, Sección de Estudios de Posgrado e Investigación, ENMH, Instituto Politécnico Nacional, Col. Fracc. La Escalera-Ticomán, México D.F., México.
authors.forename: Dora
authors.initials: D
authors.lastname: Velez-Uriza
authors.affiliation: Departamento de Bioquímica, Centro de Investigación y de Estudios Avanzados del IPN, Col. San Pedro Zacatenco, México D.F., México.
authors.forename: Jesús
authors.initials: J
authors.lastname: Valdés
authors.affiliation: Departamento de Biología Celular, Centro de Investigación y de Estudios Avanzados del IPN, Col. San Pedro Zacatenco, México D.F., México.
authors.forename: Cristina
authors.initials: C
authors.lastname: Vélez-Del Valle
authors.affiliation: Programa Institucional de Biomedicina Molecular, Sección de Estudios de Posgrado e Investigación, ENMH, Instituto Politécnico Nacional, Col. Fracc. La Escalera-Ticomán, México D.F., México.
authors.forename: Juan
authors.initials: J
authors.lastname: Salas-Benito
authors.affiliation: Centro de Investigaciones en Ciencias Microbiológicas, Edificio 103, Instituto de Ciencias, Benemérita Universidad Autónoma de Puebla (BUAP), Col. San Manuel, Puebla, México.
authors.forename: Rebeca
authors.initials: R
authors.lastname: Martínez-Contreras
authors.affiliation: Programa Institucional de Biomedicina Molecular, Sección de Estudios de Posgrado e Investigación, ENMH, Instituto Politécnico Nacional, Col. Fracc. La Escalera-Ticomán, México D.F., México.
authors.forename: Matilde
authors.initials: M
authors.lastname: García-Espítia
authors.affiliation: Programa Institucional de Biomedicina Molecular, Sección de Estudios de Posgrado e Investigación, ENMH, Instituto Politécnico Nacional, Col. Fracc. La Escalera-Ticomán, México D.F., México.
authors.forename: Mariana
authors.initials: M
authors.lastname: Salas-Benito
authors.affiliation: Facultad de Medicina, Departamento de Microbiología y Parasitología, Universidad Nacional Autónoma de México, Circuito interior, Ciudad Universitaria, México D.F., México.
authors.forename: Tania
authors.initials: T
authors.lastname: Vega-Almeida
authors.affiliation: Programa Institucional de Biomedicina Molecular, Sección de Estudios de Posgrado e Investigación, ENMH, Instituto Politécnico Nacional, Col. Fracc. La Escalera-Ticomán, México D.F., México.
authors.forename: Mónica
authors.initials: M
authors.lastname: De Nova-Ocampo
mesh_terms: D020413:3' Untranslated Regions / Q000235:genetics*; D015153:Blotting, Western; D018938:Caco-2 Cells; D017931:DNA Primers / Q000235:genetics; D024202:Electrophoretic Mobility Shift Assay; D005455:Fluorescent Antibody Technique; D006801:Humans; D046911:Macromolecular Substances / Q000378:metabolism*; D001257:Mamastrovirus / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D038941:Polypyrimidine Tract-Binding Protein / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D014779:Virus Replication / Q000502:physiology*
chemical_list: D020413:3' Untranslated Regions; D017931:DNA Primers; D046911:Macromolecular Substances; D034741:RNA, Small Interfering; D038941:Polypyrimidine Tract-Binding Protein