Identification of the Intragenomic Promoter Controlling Hepatitis E Virus Subgenomic RNA Transcription

Abstract

Approximately 20 million hepatitis E virus (HEV) infections occur annually in both developing and industrialized countries. Most infections are self-limiting, but they can lead to chronic infections and cirrhosis in immunocompromised patients, and death in pregnant women. The mechanisms of HEV replication remain incompletely understood due to scarcity of adequate experimental platforms. HEV undergoes asymmetric genome replication, but it produces an additional subgenomic (SG) RNA encoding the viral capsid and a viroporin in partially overlapping open reading frames. Using a novel transcomplementation system, we mapped the intragenomic subgenomic promoter regulating SG RNA synthesis. This cis-acting element is highly conserved across all eight HEV genotypes, and when the element is mutated, it abrogates particle assembly and release. Our work defines previously unappreciated viral regulatory elements and provides the first in-depth view of the intracellular genome dynamics of this emerging human pathogen.IMPORTANCE HEV is an emerging pathogen causing severe liver disease. The genetic information of HEV is encoded in RNA. The genomic RNA is initially copied into a complementary, antigenomic RNA that is a template for synthesis of more genomic RNA and for so-called subgenomic RNA. In this study, we identified the precise region within the HEV genome at which the synthesis of the subgenomic RNA is initiated. The nucleotides within this region are conserved across genetically distinct variants of HEV, highlighting the general importance of this segment for the virus. To identify this regulatory element, we developed a new experimental system that is a powerful tool with broad utility to mechanistically dissect many other poorly understood functional elements of HEV.

Keywords: hepatitis E; hepatitis E virus; viral hepatitis; viral replication.

FIG 1

HEV ORF1 is able to function in trans to replicate HEV RNA. (a) Schematic representation of the ORF1 transcomplementation system. (b) Representative flow cytometry plots demonstrating efficient ORF1 expression. HepG2C3A cells were transduced with pLVX-ORF1-IRES-zsGreen (wild type [wt] or GAD mutant) or not transduced. Flow cytometric analysis was performed 3 days following transduction to quantify the frequencies of ORF1-expressing cells. FSC, forward scatter. (c) Replication kinetics of HEV RNA in ORF1 transcomplemented HepG2C3A cells. Cell culture supernatants from naive HepG2C3A cells, or HepG2C3A cells transduced with HEV ORF1 or its GAD mutant, were collected at the indicated time points posttransfection with rHEVΔORF2/3[Gluc] Pol- RNA or RNA from its mutants, and Gaussia luciferase (Gluc) activity was quantified. Values are means plus standard deviations (SD) (error bars) (n = 3). Values that are significantly different (P < 0.001 by two-tailed Student’s t test) are indicated by the bar and three asterisks.

FIG 2

Mapping the putative promoter region required for subgenomic RNA synthesis. A series of truncated rHEVΔORF2/3[Gluc] Pol- mutant constructs were generated, and the in vitro-transcribed RNA was transfected into HepG2C3A cells expressing ORF1. Two days posttransfection, supernatants were collected, and Gaussia luciferase activity was quantified. The data are presented as the percentage of Gaussia luciferase activity relative to that of the full-length rHEVΔORF2/3[Gluc] Pol-. The numbering denotes the positions of the Kernow C1/p6 viral genome. Values are means plus SD (n = 3). Values that are significantly different by one-way ANOVA are indicated by asterisks as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001. Values that are not significantly different (n.s.) by one-way ANOVA are also indicated.

FIG 3

Identification of the minimal putative promoter region upstream of the TSS critical for subgenomic RNA synthesis. The truncated mutant viral RNA was transfected into HepG2C3A cells expressing ORF1. Two days posttransfection, supernatants were collected, and Gaussia luciferase activity was quantified. The data are presented as the percentage of Gaussia luciferase activity relative to that of the full-length rHEVΔORF2/3[Gluc] Pol-. The numbering denotes the positions of the Kernow C1/p6 viral genome. Values are means plus SD (n = 3). *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s., not significantly different by one-way ANOVA.

FIG 4

Synthesis of antigenomic HEV RNA is not affected by deletions in the putative promoter region. (a) HEV ORF1 transduced HepG2C3A cells were transfected with the indicated truncated rHEVΔORF2/3[Gluc] Pol- RNA genome. After 2 days, cells were washed, and intracellular total RNA was extracted and subjected to HEV negative-strand RNA-specific RT-qPCR assay to measure the abundance of antigenome. The numbering denotes the positions of the Kernow C1/p6 viral genome. Values are means plus SD (n = 3). *, P < 0.05; **, P < 0.01; ***, P < 0.001. n.s., not significantly different by one-way ANOVA. (b) Consensus sequences of the putative promoter across genotypes 1 to 8. M73218 (genotype 1a, Burma strain) was used as the reference strain for numbering. The consensus sequences (identity of >60%) are shaded. The red or blue colors denote the transversion or transition from the reference sequence.

FIG 5

Mutations in the intragenomic promoter significantly disrupt HEV subgenomic RNA transcription. (a) Schematic diagrams of the putative promoter (WT) and the SgP mutants (SgP^mut1, SgP^mut2, and SgP^mut3). As for the promoter impaired mutant, synonymous mutations are introduced in the ORF1 coding region. M73218 (genotype 1a, Burma strain) was used as the reference strain for numbering. (b to e) rHEVΔORF2/3[Gluc] WT, SgP mutant(s), or GAD mutant RNA were delivered into HepG2C3A cells. Cell culture medium was collected at the indicated time points, and Gaussia luciferase activity of Kernow C1/p6-Gluc (b), SAR55-Gluc (c), pSHEV3-Gluc (d), and TW6196E-Gluc (e) was determined. Black arrows indicate the time point when cells were washed at day 2 posttransfection, and the Gaussia luciferase before and after wash were plotted. Values are means plus SD (n = 3). ***, P < 0.001 by two-tailed Student’s t test.

FIG 6

Full-length genome mutated in the intragenomic promoter is significantly impaired in its ability to produce the infectious virus. (a) Schematic diagrams of the putative promoter (WT) and the SgP mutant (SgPmut). As for the promoter impaired mutant, synonymous mutations are introduced in the ORF1 coding region. (b and c) Transfection of in vitro-transcribed WT, SgPmut or GAD (Pol-) RNA of Kernow-C1/p6 (gt 3), TW6196E (gt 4) into Huh7.5 cells. Cell lysate supernatant was collected after 5 days transfection to infect naive Huh7.5 cells. Quantification of HEV RNA Kernow-C1/p6 (b) and TW6196E (c) 3 days following infection by quantitative RT-PCR. Values are means plus SD (n = 4). *, P < 0.05; **, P < 0.01; ***, P < 0.001 by one-way ANOVA. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (d) Representative flow cytometric plots of Huh7.5 cells infected with Kernow-C1/p6 (top) or TW6196E (bottom) 5 days following infection.

FIG 7

A genetic element downstream of the transcription start site (TSS) is essential for subgenomic RNA transcription. (a) Consensus sequences of the putative promoter (5080 nt to 5132 nt) across genotypes 1 to 8. (b) The truncated mutant viral RNA was transfected into HepG2C3A cells expressing ORF1. Two days posttransfection, supernatants were collected, and Gaussia luciferase activity was quantified. The data are presented as the percentage of Gaussia luciferase activity relative to that of the full-length rHEVΔORF2/3[Gluc] Pol- (1#). The numbering denotes positions within the viral genome. Values are means plus SD (n = 3). *, P < 0.05; **, P < 0.01; ***, P < 0.001 by one-way ANOVA. The numbering of the sequences was referred to M73218 (genotype 1a, Burma strain). The ORF1 coding sequences (blue), the transcriptional start site of HEV subgenomic RNA (green), and the ORF3 start codon (red) are indicated.

FIG 8

Mechanism of HEV RNA transcription. (a) The HEV genome is a positive-sense (+) single-stranded RNA that is capped at the 5′ end and polyadenylated at the 3′ end. It is organized into three open reading frames (ORFs), where ORF1 contains the RdRp as well as other nonstructural proteins, ORF2 contains the capsid protein, and ORF3 is a viroporin critical for viral release. ORF2 and ORF3 are partially overlapping and are separated from ORF1 by a junctional region (JR) containing a stem-loop structure. (b) Once the viral particle enters the cell and uncoats, ORF1 is translated by host ribosomes into the ORF1 polyprotein containing the RdRp. It is undetermined whether posttranslational processing of the ORF1 polyprotein occurs. (c) The RdRp transcribes a full-length negative-sense (−) intermediate strand. (d) The negative-sense strand is used as a template to produce more full-length (+) RNA to be packaged into progeny virions, as well as a subgenomic (SG) RNA that encodes ORF2 and ORF3. This SG RNA is transcribed from the (−) intermediate strand using a SgP. (e) Sequence for the SgP region based on the Kernow C1/p6 strain of genotype 3 HEV. (f) ORF2 capsid protein and ORF3 viroporin are translated from the SG RNA. These proteins are needed for packaging, assembly, and release of progeny virions.