Hepatitis E virus replication involves alternating negative- and positive-sense RNA synthesis.
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.
Department of Pathology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029, India.
Satya Pavan Kumar
SP
Varma
Amit
A
Kumar
Neeraj
N
Kapur
Hemlata
H
Durgapal
Subrat Kumar
SK
Acharya
Subrat Kumar
SK
Panda
D017930:Genes, Reporter; D016679:Genome, Viral; D016752:Hepatitis E virus / Q000502:physiology*; D006801:Humans; D008969:Molecular Sequence Data; D012367:RNA, Viral / Q000096:biosynthesis*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013997:Time Factors; D014162:Transfection; D014779:Virus Replication
D012367:RNA, Viral