Original Article
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World J Virol. Aug 12, 2013; 2(3): 123-135
Published online Aug 12, 2013. doi: 10.5501/wjv.v2.i3.123
Searching for nuclear export elements in hepatitis D virus RNA
Natália Freitas, Celso Cunha
Natália Freitas, Department of Microbiology, Molecular Genetics, and Immunology, Kansas University Medical Center, Rainbow Boulevard, Kansa, KS 66160, United States
Celso Cunha, Medical Microbiology Unit, Center for Malaria and Tropical Diseases, Institute of Hygiene and Tropical Medicine, Nova University of Lisbon, 1349-008 Lisbon, Portugal
Author contributions: Freitas N designed and performed experiments, interpreted the results and helped draft the manuscript; Cunha C designed the experiments, interpreted the results and wrote the manuscript.
Supported by A grant from Fundação para a Ciência e Tecnologia, Portugal to Freitas N
Correspondence to: Celso Cunha, PhD, Medical Microbiology Unit, Center for Malaria and Tropical Diseases, Institute of Hygiene and Tropical Medicine, Nova University of Lisbon, Rua da Junqueira, 100, 1349-008 Lisbon, Portugal. ccunha@ihmt.unl.pt
Telephone: +35-121-3652620 Fax: +35-121-3632105
Received: May 7, 2013
Revised: July 26, 2013
Accepted: August 8, 2013
Published online: August 12, 2013
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 real-time 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.

Keywords: 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.