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

PLoS One. 2014 Nov 18;9(11):e113113. doi: 10.1371/journal.pone.0113113. eCollection 2014.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 3' Untranslated Regions / genetics*
  • Blotting, Western
  • Caco-2 Cells
  • DNA Primers / genetics
  • Electrophoretic Mobility Shift Assay
  • Fluorescent Antibody Technique
  • Humans
  • Macromolecular Substances / metabolism*
  • Mamastrovirus / genetics
  • Mamastrovirus / metabolism*
  • Polymerase Chain Reaction
  • Polypyrimidine Tract-Binding Protein / genetics
  • Polypyrimidine Tract-Binding Protein / metabolism*
  • RNA, Small Interfering / genetics
  • Real-Time Polymerase Chain Reaction
  • Virus Replication / physiology*

Substances

  • 3' Untranslated Regions
  • DNA Primers
  • Macromolecular Substances
  • RNA, Small Interfering
  • Polypyrimidine Tract-Binding Protein

Grants and funding

This work was partially supported by Secretaria de Investigación y Posgrado-Instituto Politécnico Nacional grants 20080174, 20090206, 20100054, 20110182, 20120179, and 20130689; Consejo Nacional de Ciencia y Tecnología grants 99682 (MDO), 49355M, and 127557M; and Instituto de Ciencia Tecnología grant 71/2012 (JV). WEH and DVU were Programa Institucional de Formación de Investigadores-Instituto Politécnico Nacional scholars. DVU was supported by a scholarship from Consejo Nacional de Ciencia y Tecnología. MDO and JSB received Comisión de Operación y Fomento de Actividades Académicas-Instituto Politécnico Nacional/Estimulo al Desempeño de los Investigadores fellowships. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.