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 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: Antigenomic RNA; Genomic RNA; Hepatitis D virus; Nuclear export; Nuclear export element.

Figure 1

Intracellular localization of hepatitis delta virus gRNA (A) and agRNA (B). HuH-7 cells were transfected with plasmids pDL542 and pDL481, respectively, and virus RNA was detected by in situ hybridization with a dig-11-dUTP labeled probe. Both gRNA and agRNA can be observed in the nucleus and cytoplasm (green).

Figure 2

Nucleo-cytoplasmic distribution of hepatitis delta virus gRNA and agRNA. HuH-7 cells were trasnfected with plasmids pDL542 and pDL481, respectively. A: The relative quantification of HDV RNA was performed by real time-polymerase chain reaction using the 2^-∆∆Ct method. Results are presented as the cytoplasmic /nuclear ratio (C/N) and correspond to the mean of three independent experiments. Bars indicate the standard deviation; B: Western blotting analysis of nuclear and cytoplasmic HuH-7 cell protein fractions. Equivalent amounts of nuclear (lanes 1 and 3) and cytoplasmic (lanes 2 and 4) protein fractions used for quantification of gRNA (lanes 1 and 2) and agRNA (lanes 3 and 4) were separated in 12% SDS-PAGE gels. The possible contamination of nuclear fractions was monitored by using an anti-GAPDH antibody.

Figure 3

Analysis of chloranphenicol acetyl-transferase expression in HuH-7 cells transfected with plasmids pDM138, pDM138 (PRE+), pDM138 (PRE-), pDM138 A1S-pDM138 A10S (A), and pDM138 A1AS-pDM138 A10AS (B). In order to normalize for transfection efficiency, cells were co-transfected with plasmid pSV-β-Gal (Promega). Chloranphenicol acetyl-transferase (CAT) and β-Gal expression levels were determined by ELISA. Normalization of CAT expression levels was calculated by dividing the values obtained for the CAT protein by the values obtained for the β-Gal protein. The results correspond to the mean of three independent experiments. Bars represent the standard deviation.

Figure 4

Northern blotting analysis of reporter chloranphenicol acetyl-transferase mRNA in total (A) and cytoplasmic (B) fractions of HuH-7 cells transfected with plasmids pDM138 (PRE+), pDM138 A7AS, and pDM138 A9AS (lanes 1, 2 and 3 respectively). Hybridization was performed using a dig-11-dUTP labeled probe. A peroxidase conjugated anti-digoxigenin antibody was used to detect the hybridized probe.

Figure 5

Analysis of chloranphenicol acetyl-transferase expression in HuH-7 cells transfected with plasmids pDM138, pDM138 (PRE+), pDM138 (PRE-), pDM138 A7AS (214-417), pDM138-314-417, pDM138-214-322, pDM138-269-379, pDM138-244-379, pDM138-269-417, pDM138-214-403, pDM138-244-403, and pDM138-244-417. Chloranphenicol acetyl-transferase (CAT) and β-Gal expression levels were determined by enzyme-linked immunosorbent assay. The CAT expression values were normalized for transfection efficiency by transfecting HuH-7 cells with plasmid pSV-β-Gal (Promega) followed by determination of β-Gal expression. CAT expression values were divided by the corresponding β-Gal expression values, and the displayed results correspond to the mean of three independent experiments.

Figure 6

Nucleo-cytoplasmic distribution of hepatitis delta virus RNA in HuH-7 cells transfected with plasmids pDL481, pDl542, pDL481ΔNEE, pDL481ΔNEEδ, and pDL481Δδ. The RNA in nuclear and cytoplasmic cell fractions was determined by real time-polymerase chain reaction using the 2^-∆∆Ct method. Results are presented as the cytoplasmic /nuclear ratio (C/N) and correspond to the mean of three independent experiments. Bars indicate the standard deviation.

Figure 7

Analysis of chloranphenicol acetyl-transferase expression in HuH-7 cells transfected with plasmids pDM138 (PRE-), pDM138 (PRE+), and pDM138 A7AS, in the absence (black columns) and presence of 10 nmol/L leptomycin B (grey columns). In order to normalize for transfection efficiency, cells were co-transfected with plasmid pSV-β-Gal (Promega). Chloranphenicol acetyl-transferase (CAT) and β-Gal expression levels were determined by enzyme-linked immunosorbent assay. Normalization of CAT expression levels was calculated by dividing the values obtained for the CAT protein by the values obtained for the β-Gal protein. The results correspond to the mean of three independent experiments. Bars represent the standard deviation.