Although HEV infectious clones are available (13
), the lack of an efficient cell culture for HEV prevents us from directly testing the replication of mutant viruses in vitro
). It has been shown that a green fluorescence protein (GFP) HEV replicon is a good system to study HEV replication in vitro
). Therefore, in this study, we first constructed a genotype 1 HEV enhanced GFP (EGFP) replicon system (Y.-W. Huang and X. J. Meng, unpublished data) by using the Sar55 infectious clone (a gift from Sue Emerson, NIH, Bethesda, MD). We then constructed eight EGFP replicon-based HEV mutants and tested for their effects on HEV replication. Unfortunately, the sensitivity of the EGFP HEV replicon system was low (data not shown).
To definitively assess the roles of the nucleotides of the junction region in HEV replication, we subsequently constructed a novel HEV replicon system by replacing nt 5148 to 5816 of the infectious clone pSK-HEV-2 with the Renilla
luciferase (Rluc) gene (Fig. ). By utilizing the start codon of HEV ORF2, the Rluc HEV replicon expresses Renilla
luciferase (Rluc) (Fig. ), which was used as a reporter for quantifying HEV replication. By using the Rluc HEV replicon as the backbone, we constructed 11 SL mutants, designated as follows (Fig. ; Table ) (sequences shown as the negative polarity complement of the HEV genome): M1 (A5118→U); M2 (C5122→U, G5123→U); M3 (AGA5116 to -5118→UCU); M4 (AAAGA5116 to -5120→UUUCU); M5 (UGUU5110 to -5113→ACAA), which contains mutations on one leg of the SL stem; M6 (AA5119 to -5120→UU); M7 (C5101→G); M8 (CA5124 to -5125→AU); M9 (AGCA5121 to -5124→UUGU), which contains mutations on another leg of the stem; M59 (UGUU5110 to -5113→ACAA, AGCA5121 to -5124→UUGU), with mutations on both legs of the stem; and M78 (C5101→G, CA5124 to -5125→AU), which is a combination of M7 and M8. In addition, the HEV Rluc replicon mutant with a GDD→GAA mutation on RdRp (MGAA) was constructed and used as a negative control. Capped RNA transcripts from each of the 11 mutant replicons along with the MGAA and wild-type replicon were synthesized in vitro
with an mMessage mMachine T7 kit (Ambion) (17
). The capped RNA transcripts of each mutant and control were transfected into the Huh7-S10-3 liver cell line (a gift from Sue Emerson, NIH, Bethesda, MD) (11
) with 1,2-dimyristyl Rosenthal inhibitor ether (DMRIE-C) reagent (Invitrogen). The luciferase activities were measured with a dual luciferase reporter assay system (Promega) at 5 days posttransfection. Firefly luciferase RNA was cotransfected with HEV Rluc replicon RNAs to normalize the Renilla
FIG. 2. Mutational analyses of the predicted stem-loop (SL) structure in the junction region of the HEV genome. (A) Mutations were introduced into the stems and loop sequences of the SL structure in mutants M1 to M9, M59, and M78 for the HEV Rluc replicon. (B) (more ...)
Primers used in the generation of HEV mutants and LNAs used for inhibition of HEV replicon replication
The results showed that the Rluc signal is lower in cells transfected with RNA of mutant M1, M2, M3, M4, M5, M6, M9, or M59 than that in cells transfected with RNA of the wild-type HEV Rluc replicon (Fig. ), with statistically significant differences for mutants M3, M4, M5, M9, and M59. The mutant M3, which changed only the AGA motif, abolished HEV replication as efficiently as did mutant M4 that contained two additional adenosine nucleotide changes compared to M3. Even a single nucleotide mutation of the AGA motif on the loop (M1) inhibited HEV replication, suggesting that the nucleotides on the loop of SL are important for HEV replication and that the AGA motif is critical for HEV replication. The mutation on either leg of the stem (M5 and M9) also significantly inhibits HEV replication. The mutation that broke one base pair (U-G) on one leg of the stem (M2) also inhibited HEV replication, indicating that the structure of SL is also important for HEV replication. Although the HEV EGFP replicon system is not as sensitive as the Rluc system, the results with the EGFP replicon-based SL mutants are qualitatively similar to those with the Rluc-based mutants (data not shown).
It is noteworthy that the conserved AAUAAC sequence in the sense genome of the junction region, which was identified as an important motif for HEV replication in vivo
), has 3 nt overlapped with one leg of the SL stem. The mutations of AAUAAC to AACAUG that resulted in less-efficient replication (19
) actually broke two base pairs on the SL stem and thus may change the SL structure and inhibit HEV replication. However, we failed to rescue HEV replication by replacing the stem with a mutated complement sequence (M59) on the stem of SL (Fig. ), suggesting that both the sequence and structure of the SL play an important role in HEV replication. Elimination of the predicted JC virus (JCV) repeated sequence (25
) and enhancer core motif (41
) (M7) has no significant effect on HEV replication. Furthermore, mutations in the metal response element (MRE) motif CS2 (8
) (M8) reduced HEV replication, and the combination of M7 and M8 mutations (M78) has a similar effect on HEV replication compared to that of the single mutation (M7 or M8), suggesting that these motifs may regulate but are not important for HEV replication.