Combining RNA Interference and RIG-I Activation to Inhibit Hepatitis E Virus Replication

Abstract

Hepatitis E virus (HEV) poses a significant global health threat, with an estimated 20 million infections occurring annually. Despite being a self-limiting illness, in most cases, HEV infection can lead to severe outcomes, particularly in pregnant women and individuals with pre-existing liver disease. In the absence of specific antiviral treatments, the exploration of RNAi interference (RNAi) as a targeted strategy provides valuable insights for urgently needed therapeutic interventions against Hepatitis E. We designed small interfering RNAs (siRNAs) against HEV, which target the helicase domain and the open reading frame 3 (ORF3). These target regions will reduce the risk of viral escape through mutations, as they belong to the most conserved regions in the HEV genome. The siRNAs targeting the ORF3 efficiently inhibited viral replication in A549 cells after HEV infection. Importantly, the siRNA was also highly effective at inhibiting HEV in the persistently infected A549 cell line, which provides a suitable model for chronic infection in patients. Furthermore, we showed that a 5' triphosphate modification on the siRNA sense strand activates the RIG-I receptor, a cytoplasmic pattern recognition receptor that recognizes viral RNA. Upon activation, RIG-I triggers a signaling cascade, effectively suppressing HEV replication. This dual-action strategy, combining the activation of the adaptive immune response and the inherent RNAi pathway, inhibits HEV replication successfully and may lead to the development of new therapies.

Keywords: HEV; RIG-I; RNA 5′triphosphate; RNAi therapy; siRNA.

Figure 1

Organization of the HEV genome and siRNA target selection. (A) Overview of the HEV genome. The (+)ssRNA genome is 7.2 kb long. It has a 7-methylguanosine cap at the 5′ end and polyadenylation at the 3′-end. There are three conserved ORFs. ORF1 encodes the non-structural polyproteins and has several functional domains: methyltransferase (Met), Y domain, papain-like cysteine protease (PCP), hypervariable region (HVR), X domain, helicase (Hel), and RNA-dependent RNA polymerase (RdRP). ORF2 encodes the capsid structural protein. ORF3 encodes a multi-functional phosphoprotein. Expression is mediated by a 7.2 kb subgenomic bicistronic RNA. In addition to these three ORFs, HEV-1 has an ORF4 that overlaps the X and Hel domains. (B) Alignment of all HEV-3 subgenotypes to identify conserved regions within the genotype. Non-matching sequences are shown in black—conserved regions in gray. (C) Representation of suitable siRNA target sequences in the helicase and ORF3 regions. Identification was performed using the online programs Eurofins Genomics (blue), Horizon Discovery (red), OligoWalk Web Server (green), and siDirect2.0 (purple). Selected siRNA target sequences are shown in yellow.

Figure 2

Relative change in luciferase activity by the designed siRNAs. A dual luciferase reporter assay was used to evaluate the silencing activity of the designed siRNAs. (A) Graphic representation of the psiCheck2 vectors where GOI is the HEV-3c ORF3 or HEV-3c helicase DNA sequence. (B) Co-transfection of dual-luciferase vectors (500 ng) and siRNA (50 nM) was performed in HeLa cells. The relative Renilla/Firefly (Ren/Luc) activity was determined 48 h after transfection. The relative activity was normalized against a non-regulatory control (siCon) and set to 100%, ns = not significant, **** p ≤ 0.0001.

Figure 3

Changes in viral copy number by siRNA treatment of A549/pers-HEV cells. The viral RNA copy number was determined by qPCR after siRNA transfection of A549/pers-HEV cells. Transfection of 50 nM siRNA was performed 24 h after cell seeding. (A) At 48 h post-transfection, viral RNA was isolated, and the viral copies/mL were determined by qPCR. (B) A second transfection (TF) was then conducted 48 h after seeding. Viral RNA was isolated 96 h after the initial transfection, and the viral RNA was determined by RT-qPCR. The viral RNA was normalized against a non-regulatory control (siCon) and set to 100%. Mean ± SD of three independent experiments (n = 3) are shown. ns = not significant, *** p ≤ 0.001, **** p ≤ 0.0001.

Figure 4

Inhibition of HEV replication by siRNA in infected A549/D3 cells. Cells were transfected and subsequently infected with viral isolates. At 96 h post-infection, viral RNA was isolated, and viral copies/mL were determined by qPCR. The viral copy number was normalized against a non-regulatory control (siCon) and set to 100%. (A) Transfection with 50 nM siRNA was performed 24 h after seeding. Infection was performed 24 h after transfection with viral isolates at an MOI of 1.0. (B) The A549/D3 cells were transfected with different concentrations (0.01 to 50 nM) of siORF3.1, and 24 h after transfection, the cells were infected with virus isolates at a MOI of 1.0 for 16 h. Mean ± SD of three independent experiments (n = 3) are shown. ** p ≤ 0.01, **** p ≤ 0.0001.

Figure 5

Design, synthesis, and functional testing of 5′ triphosphate siRNA. (A) Sequence and structure of 3p-siORF3.1 and 3p-siCon. The siRNA forms a 19 bp double strand with a UU overhang at the 3′-end and a triphosphate at the 5′-end of the sense strand. (B) Successful hybridization and purity of 3p-siRNAs (3p-siORF3.1 and 3p-siCon) were verified by a 15% urea polyacrylamide gel (1 = hybridization, 2 = RNase A digestion, 3 = final purification). The chemically synthesized siRNAs (siORF3.1 and siCon) and the antisense single strands ssORF3.1 and ssCon were used as controls. (C) Efficiency of RIG-I expression by Western blot analysis. A549/D3 cells were stimulated for 48 h with different concentrations of 3p-siCon, 3p-siORF3.1 (10 and 50 nM), poly I:C (0.5 and 0.25 ng/mL) or siORF3.1 (50 nM). Protein extracts from non-transfected A549/D3 cells served as negative control (NC). Actin was used as an internal control. (D) Dual-luciferase reporter assays were used to evaluate the silencing activity of the designed siRNAs. Co-transfection of dual-luciferase vectors (500 ng) and siRNA (50 nM) was performed in HeLa cells. The relative Renilla/Firefly (Ren/Luc) activity was determined 48 h after transfection. The relative activity was normalized against a non-regulatory control (siCon) and set to 100%. (E) Inhibition of HEV replication by 3p-siRNA: A549/D3 cells were transfected with the indicated siRNAs (siCon, siORF3.1, 3p-siCon, 3p-siORF3.1) at a concentration of 50 nM. The cells were infected with HEV at an MOI of 1.0, 24 h after transfection, and incubated for 16 h. At 96 h after infection, viral RNA was isolated, and viral load was determined by qPCR. The viral copy number was normalized against a non-regulatory control (siCon) and set to 100%. Mean ± SD of three independent experiments (n = 3) are shown. ns = not significant, **** p ≤ 0.0001.