A bicistronic subgenomic mRNA encodes both the ORF2 and ORF3 proteins of hepatitis E virus

Abstract

Hepatitis E virus replicons containing the neomycin resistance gene expressed from open reading frames (ORFs) 2 and 3 were transfected into Huh-7 cells, and stable cell lines containing functional replicons were selected by constant exposure to G418 sulfate. Northern blot analyses detected full-length replicon RNA and a single subgenomic RNA. This subgenomic RNA, which was capped, initiated at nucleotide 5122 downstream of the first two methionine codons in ORF3 and was bicistronic; two closely spaced methionine codons in different reading frames were used for the initiation of ORF3 and ORF2 translation.

FIG. 1.

Northern blot analysis of viral positive-strand RNA present in S10-3 cells stably transfected with the HEV replicon HEV/2Neo. (A) Schematic representation of HEV/2Neo. Hatched boxes mark the positions corresponding to the two RNA probes. The positions of the 7.3-kb and 2.2-kb transcripts used as size markers are shown. (B) The total RNA extracted from a mock-transfected (s0) cell line and from three HEV/2Neo-transfected (s2N1, s2N2, and s2N5) cell lines was probed with RNA specific to nt 6583 to 6767. Lane Tx2 contains the in vitro transcript of HEV fragment 5007 to 7210, and lane Tx1 contains the in vitro transcript of the full-length HEV/2Neo genome. (C) The total RNA of a mock-transfected (s0) cell line and two HEV/2Neo-transfected (s2N8-1 and s2N8-2) cell lines was probed with RNA specific to nt 5049 to 5334.

FIG. 2.

(A) Ethidium bromide agarose gel (1.5%) of PCR products amplified with primers (Er5172 and Er5334) for the detection of subgenomic RNAs. PCR products were amplified by 5′ RLM-RACE of total RNA from S10-3 cells stably transfected with HEV/2Neo replicon transcripts. The cell lines s2N5 (5) and s2N8-1 (8-1) are named above each lane, and the use of TAP is indicated by a +. M, 100-bp DNA ladder (Invitrogen). Primer Er5172 amplifies the overlap region of the three ORFs, and primer Er5334 should amplify all 3′-coterminal RNAs. White brackets indicate the PCR products sequenced. (B) Representative chromatograms of sequence analyses of the 5′ terminus of HEV subgenomic RNA of cell line s2N8-1. The sequences of cloned PCR products amplified with primers Er5172 and Er5334 are aligned to HEV/2Neo for comparison. The 5′-terminal viral nucleotides following the 5′ adapter (a) are indicated in colors matching the chromatogram. The sequence of primer Er5172 is in black and in brackets. The HEV/2Neo sequence shows the starts of ORF3 (nt 5104) and ORF2 (nt 5145). The first nucleotides (nt 5104, nt 5113, and nt 5131) of all three ATGs present in the 5′ terminus of ORF3 are indicated, and the third ATG is framed by solid lines.

FIG. 3.

(A) Indirect immunofluorescence microscopy for the detection of HEV ORF2 and ORF3 proteins. S10-3 cells were stained for (a) HEV ORF2 proteins (green) and (b) HEV ORF3 proteins (red) 4 days after transfection with transcripts of pHEV/T+. The nuclei were counterstained with 4′,6′-diamidino-2-phenylindole (DAPI). (B) Western blot analysis of cell lysates of S10-3 cells transfected with transcripts of pSK-HEV (HEVwt), pHEV/T+ (HEV/T+), and pHEV-ORF3/His (ORF3/His) and of mock-transfected cells. Protein markers are indicated to the left in kDa. Proteins were separated on a 16% Tricine gel and detected by rabbit anti-ORF3-specific antibody. The higher molecular mass band seen in ORF3/His represents nonspecific binding to an unknown cellular protein, since it has been seen in other experiments in mock samples.

FIG. 4.

Chromatogram of sequence analysis of the 5′ terminus of HEV subgenomic RNA of cell line s3N. The consensus sequence of the PCR product (s3N × Er5334) specific to the HEV replicon HEV/3Neo is shown in alignment to nt 5104 to 5146 of HEV/3Neo for comparison. The 5′-terminal nucleotide (nt 5122) following the 5′ adapter (a) is indicated. The 5′-terminal sequence of the subgenome is in color matching the chromatogram. The HEV/3Neo sequence shown for comparison indicates the first nucleotides (nt 5104, nt 5113, and nt 5131) of all three ATGs present in the 5′ terminus of ORF3. The third ATG is framed by solid lines.

FIG. 5.

Indirect immunofluorescence microscopy for the detection of HEV ORF2 and ORF3 proteins. S10-3 cells were stained for (a) HEV ORF2 proteins (green) and (b) HEV ORF3 proteins (red) 4 days after transfection with transcripts of pHEV5131Ala. The nuclei were counterstained with 4′,6′-diamidino-2-phenylindole (DAPI). In contrast to HEV ORF2 proteins (a), HEV ORF3 proteins were not detected (b).

FIG. 6.

Detection of HEV ORF2 and HEV ORF3 proteins by specific antibodies at 2 days posttransfection of S10-3 cells with pCMV5122. (A) Indirect immunofluorescence microscopy of cells stained for (a) ORF2 proteins (green) and (b) ORF3 proteins (red). The nuclei were counterstained with 4′,6′-diamidino-2-phenylindole (DAPI). (B) Western blot analysis of ORF2 proteins separated on a 7% Tris-acetate gel. Molecular masses of protein markers are indicated to the left in kDa. (C) Western blot analysis of ORF3 proteins separated on a 10 to 20% Tricine gel. Molecular masses of protein markers are indicated to the left in kDa.