Abstract
ABSTRACTApproximately 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.
Highlights
20 million hepatitis E virus (HEV) infections occur annually in both developing and industrialized countries
To map the intragenomic subgenomic promoter (SgP) that is critical for SG RNA synthesis off the FL (Ϫ) strand, we devised an experimental system in which HEV RNA transcription is uncoupled from protein translation
Intragenomic Promoter for HEV SG RNA Transcription well in HepG2C3A cells (Fig. 1b). wt or PolϪ ORF1-expressing cells were subsequently transfected with in vitro-transcribed RNA from a recombinant, PolϪ version of an HEV genome derived from the Kernow C1/p6 genome [14] in which ORF2 and ORF3 are replaced by a secreted version of Gaussia luciferase (Gluc) [14], termed rHEVΔORF2/3[Gluc] Pol- (Fig. 1a)
Summary
20 million hepatitis E virus (HEV) infections occur annually in both developing and industrialized countries. 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. Due to the lack of adequate experimental systems, it remains unclear where precisely the cis-acting element regulating subgenomic (SG) RNA synthesis is and what the kinetics and dynamics of the different viral RNAs [FL (ϩ), (Ϫ) strand and SG] are. These are critical questions, and answering them will provide important insights into the mechanism of HEV genome replication
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