Abstract
BackgroundThe biology of Hepatitis E Virus (HEV), a common cause of epidemic and sporadic hepatitis, is still being explored. HEV exits liver through bile, a process which is essential for its natural transmission by feco-oral route. Though the process of this polarised HEV egress is not known in detail, HEV pORF3 and hepatocyte actin cytoskeleton have been shown to play a role.MethodsOur transcriptome analysis in Hepatitis E virus (HEV) replicon transfected Huh7 cells at 24 and 72 hrs indicated that at 24hrs, both LncBISPR and BST2, expressed by a bidirectional promoter were highly upregulated whereas at 72 hrs, BST2 expression was comparatively reduced accompanied by normal levels of BISPR. These findings were confirmed by qPCR analysis. Co-localisation of BST2 and HEV pORF2 was confirmed in HEV transfected Huh7 by confocal microscopy. To investigate the role of BISPR/BST2 in HEV life cycle, particularly virus egress, we generated Huh7 cells with ~8kb deletion in BISPR gene using Crispr-Cas9 system. The deletion was confirmed by PCR screening, Sanger sequencing and Real time PCR. Virus egress in ΔBISPR Huh7 and Huh7 cells was compared by measuring HEV positive strand RNA copy numbers in cell lysates and culture supernatants at 24 and 72 hrs post HEV replicon transfection and further validated by western blot for HEV pORF2 capsid protein.ResultsΔBISPR Huh7 cells showed ~8 fold increase in virus egress at 24 hrs compared to Huh7 cells. No significant difference in virus egress was observed at 72hrs. Immunohistochemistry in histologically normal liver and HEV associated acute liver failure revealed BST2 overexpression in HEV infected hepatocytes and a dominant canalicular BST2 distribution in normal liver in addition to the cytoplasmic localisation reported in literature.ConclusionsThese findings lead us to believe that BISPR and BST2 may regulate egress of HEV virions into bile in vivo. This system may also be used to scale up virus production in vitro.
Highlights
Hepatitis E virus (HEV) is a common cause of both epidemic and sporadic viral hepatitis
ΔBISPR Huh7 cells showed ~8 fold increase in virus egress at 24 hrs compared to Huh7 cells
No significant difference in virus egress was observed at 72hrs
Summary
Hepatitis E virus (HEV) is a common cause of both epidemic and sporadic viral hepatitis. The factors involved in this polarized virus egress have been identified as a small viral protein pORF3 [2,3] and hepatocyte cytoskeleton [4]. Virus replication [5], recycling and cytoplasmic retrotranslocation [6,7] of HEV capsid protein (pORF2) have been described to occur on endoplasmic reticulum. Virus egress occurs in HEV infected cell lines, established either by infection with virus isolated from feces or by HEV replicon transfection, its quantity is limited. This has been a major problem for developing an attenuated vaccine, which shall possibly work better than current subunit virus like particles with truncated capsid protein. Editor: Jason Blackard, University of Cincinnati College of Medicine, UNITED STATES
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