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.
Highlights
Human astroviruses (HAstVs) are a major cause of acute gastroenteritis in children constituting an important public health problem
Our results suggest that PTB binds to the cognate UCUU site, localized in the linker of the 39 untranslated region (39UTR) of HAstV-8, that helix I is required for such binding, and that it is required and participates in viral replication
Several cellular proteins bind to the 39UTR of HAstV-8 RNA
Summary
Human astroviruses (HAstVs) are a major cause of acute gastroenteritis in children constituting an important public health problem. To explore the role of the HAstV-8 39UTR RNA in the regulation of viral functions, we studied the interaction of human CaCo2 cellular factors with RNA probes of this region with or without the PTB binding site. SiRNA-mediated PTB knockdown in HAstV-8 infected CaCo2 cells showed reduced RNA viral genome synthesis and viral yield.
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