PTB binds to the 3' untranslated region of the human astrovirus type 8: a possible role in viral replication

• Published in: PloS one 2014-11, Vol.9 (11), p.e113113-e113113
• Main Authors: Espinosa-Hernández, Wendy, Velez-Uriza, Dora, Valdés, Jesús, Vélez-Del Valle, Cristina, Salas-Benito, Juan, Martínez-Contreras, Rebeca, García-Espítia, Matilde, Salas-Benito, Mariana, Vega-Almeida, Tania, De Nova-Ocampo, Mónica
• Format: Article
• Language: English
• Subjects: 3' Untranslated regions; 3' Untranslated Regions - genetics; Analysis; Binding sites; Biology and Life Sciences; Blotting, Western; Caco-2 Cells; Cellular proteins; Crosslinking; Cytoplasm; Dengue fever; DNA Primers - genetics; DNA probes; Electrophoretic mobility; Electrophoretic Mobility Shift Assay; Fluorescent Antibody Technique; Genomes; Genomics; Homology; Humans; Immunofluorescence; Macromolecular Substances - metabolism; Mamastrovirus - genetics; Mamastrovirus - metabolism; Nuclei; Nuclei (cytology); Polymerase Chain Reaction; Polypyrimidine Tract-Binding Protein - genetics; Polypyrimidine Tract-Binding Protein - metabolism; Protection and preservation; Proteins; Real-Time Polymerase Chain Reaction; Replication; Ribonucleic acid; RNA; RNA probes; RNA, Small Interfering - genetics; RNA-mediated interference; siRNA; Virus replication; Virus Replication - physiology; Viruses
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0113113

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.