Searching for nuclear export elements in hepatitis D virus RNA

Abstract

AIM:To search for the presence of cis elements in hepatitis D virus (HDV) genomic and antigenomic RNA capable of promoting nuclear export. METHODS:We made use of a well characterized chloramphenicol acetyl-transferase reporter system based on plasmid pDM138. Twenty cDNA fragments corresponding to different HDV genomic and antigenomic RNA sequences were inserted in plasmid pDM138, and used in transfection experiments in Huh7 cells. The relative amounts of HDV RNA in nuclear and cytoplasmic fractions were then determined by realtime polymerase chain reaction and Northern blotting. The secondary structure of the RNA sequences that displayed nuclear export ability was further predicted using a web interface. Finally, the sensitivity to leptomycin B was assessed in order to investigate possible cellular pathways involved in HDV RNA nuclear export. RESULTS:Analysis of genomic RNA sequences did not allow identifying an unequivocal nuclear export element. However, two regions were found to promote the export of reporter mRNAs with efficiency higher than the negative controls albeit lower than the positive control. These regions correspond to nucleotides 266-489 and 584-920, respectively. In addition, when analyzing antigenomic RNA sequences a nuclear export element was found in positions 214-417. Export mediated by the nuclear export element of HDV antigenomic RNA is sensitive to leptomycin B suggesting a possible role of CRM1 in this transport pathway. CONCLUSION:A cis-acting nuclear export element is present in nucleotides 214-417 of HDV antigenomic RNA.© 2013 Baishideng. All rights reserved.Key words: Hepatitis D virus; Genomic RNA; Antigenomic RNA; Nuclear export; Nuclear export element Core tip: Hepatitis D virus (HDV) replicates in the nucleus and export of HDV RNPs to the cytoplasm is thought to be mediated by cis-elements present in virus RNA. We used a chloramphenicol acetyl-transferase reporter system in an attempt to identify the RNA sequences that mediate export to the cytoplasm. Several cDNA constructs coding for different HDV RNA (genomic and antigenomic) sequences were tested. Our results show that a cis-acting nuclear export element is present in positions 214-417 of antigenomic RNA. Two regions in genomic RNA were found to promote nuclear export with efficiency higher than the negative control although lower that the positive control.Freitas N, Cunha C. Searching for nuclear export elements in hepatitis D virus RNA. World J Virol 2013; 2(3): 123-135 Available from:

Cellular Factors Involved in the Hepatitis D Virus Life Cycle

“…The recruitment of both NXF1 and Aly as well as other RNA-binding proteins belonging to the transcription export (TREX) complex that could associate either with HDAg or HDV mRNA remains to be identified. The proline-rich NES of L-HDAg interacts with the RNA helicase UAP56, responsible for the recruitment of Aly, which in turn recruits NXF1 as the export receptor, facilitating the export of RNPs [ 33 , 116 , 117 ]. For a detailed review of mRNA export, see [ 118 , 119 ].…”