Hepatitis E virus replication involves alternating negative- and positive-sense RNA synthesis
Open Access
- 1 December 2010
- journal article
- Published by Microbiology Society in Journal of General Virology
- Vol. 92 (3), 572-581
- https://doi.org/10.1099/vir.0.027714-0
Abstract
Hepatitis E virus (HEV) is the major cause of epidemic hepatitis and many outbreaks of sporadic hepatitis. The virus responsible has a single-stranded, positive-sense RNA. Its replication and the regulatory process involved therein are poorly understood. Much of the HEV biology studied has been done by using full-length capped RNA transcripts (replicons) and transient transfections in cell cultures. We investigated replicon replication using negative-sense strand-specific molecular beacons in live cell imaging, and quantifying intracellular viral RNA using strand-specific real-time PCR every 2 h until 24 h post-transfection. A graph of the copy numbers of both positive- and negative-sense RNA at the different time points was plotted. This showed a temporal separation and alternating cycles of negative- and positive-sense RNA formation. As a control, a dysfunctional replicase mutant (GDD→GAA) was used, which showed no increase in copy number. The live cell imaging corroborated the quantitative data, in that the maximal amount of negative-sense RNA was observed at 8 h post-transfection. The real-time-PCR copy-number analysis of the subgenome showed the presence of a single subgenomic RNA. Using fluorescent protein genes mCherry and EGFP fused in-frame to ORF2 and ORF3 in separate constructs and immunofluorescence, we showed the formation of both proteins pORF2 and pORF3 from a single subgenomic RNA. Our study demonstrated cyclical bursts of virus replication and the role of subgenomic RNA in the HEV life cycle.Keywords
This publication has 31 references indexed in Scilit:
- Structure of the hepatitis E virus-like particle suggests mechanisms for virus assembly and receptor bindingProceedings of the National Academy of Sciences of the United States of America, 2009
- The Hepatitis E Virus Open Reading Frame 3 Product Interacts with Microtubules and Interferes with Their DynamicsJournal of Virology, 2009
- The Hepatitis E Virus ORF3 Protein Modulates Epidermal Growth Factor Receptor Trafficking, STAT3 Translocation, and the Acute-Phase ResponseJournal of Virology, 2008
- Initiation at the Third In-Frame AUG Codon of Open Reading Frame 3 of the Hepatitis E Virus Is Essential for Viral Infectivity In VivoJournal of Virology, 2007
- A Bicistronic Subgenomic mRNA Encodes both the ORF2 and ORF3 Proteins of Hepatitis E VirusJournal of Virology, 2006
- In Vitro Replication of Hepatitis E Virus (HEV) Genomes and of an HEV Replicon Expressing Green Fluorescent ProteinJournal of Virology, 2004
- The 3′ End of Hepatitis E Virus (HEV) Genome Binds Specifically to the Viral RNA-Dependent RNA Polymerase (RdRp)Virology, 2001
- Cloning, sequencing, and expression of the hepatitis E virus (HEV) nonstructural open reading frame 1 (ORF1)Journal of Medical Virology, 2000
- Sequence and gene structure of the hepatitis E virus isolated from MyanmarVirus Genes, 1993
- Evidence for a Virus in Non-A, Non-B Hepatitis Transmitted via the Fecal-Oral RouteIntervirology, 1982