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Principal Investigator: Zongdi Feng
Organization: RESEARCH INST NATIONWIDE CHILDREN'S HOSP
Fiscal Year: 2024
Award: $518,025
Funding agency: National Institute of Allergy and Infectious Diseases
Summary
Hepatitis E virus (HEV) infection is a major cause of acute hepatitis worldwide and can often persist in
immunocompromised individuals leading to significant morbidity and mortality. Currently, there are no FDA-
approved vaccines or HEV-specific therapy in the U.S. Our long-term goal is to elucidate the molecular details
of the HEV infectious cycle to aid in the rational design of HEV-specific prevention and treatment. HEV is an
enterically transmitted positive-strand RNA virus with a unique life cycle: the virus is shed as naked particles into
the feces but circulates as quasi-enveloped (eHEV) particles in the bloodstream. The dogma in the field has
been that eHEV mediates virus spread in the infected host, and its biogenesis requires the viral ORF3 protein.
It is proposed that ORF3 usurps the cellular endosomal sorting complex required for transport (ESCRT)
machinery to acquire an envelope from the multivesicular bodies (MVBs). However, in cell culture, HEV can
spread regardless of ORF3 expression, and clinical isolates bearing ORF3 start codon mutations have also been
described. Thus, the release mechanism for HEV is likely to be more complex than previously thought. In HEV-
infected polarized human hepatocyte cultures and human liver chimeric mice, ORF3 almost exclusively localizes
to the apical/canalicular membrane. Moreover, our recently published work shows that ORF3 is required for
apical but not the basolateral release of HEV from polarized human hepatoma cells. This application seeks to
better define the mechanism for HEV release from polarized hepatocytes and to clarify the role of ORF3 in this
process. Our central hypothesis is that ORF3 dictates apical release of HEV but is not required for basolateral
release from polarized hepatocytes. We propose two specific aims to test this hypothesis using in vitro and in
vivo models. Aim 1 will use a highly polarized human hepatoma cell line to elucidate the mechanism for HEV
release from both the apical and basolateral sides of human hepatocytes. We will determine that ORF3 recruits
HEV capsid and ESCRT components to the apical recycling endosomes (AREs)-derived transport vesicles to
facilitate HEV envelopment and exit, and an ORF3-independent mechanism is responsible for HEV release at
the basolateral surface that mediates virus spread. Aim 2 will investigate the HEV release mechanisms in a
newly developed rat model of HEV infection. The role of ORF3, ESCRT, MVBs and AREs will be investigated
using various viral mutants. The morphology and protein composition of HEV virions in the bloodstream will be
characterized and compared to virions released from the apical and basolateral sides of polarized human
hepatoma cells. The completion of this work will shed new light on the unusual HEV life cycle and clarify the role
of ORF3 in HEV infection. The knowledge gained from this research will also have broad implications for
hepatocyte biology and noncytolytic release mechanisms for both nonenveloped and enveloped viruses.
Terms: <Acute Hepatitis><Apical><Bile><Bile Juice><Bile fluid><Biliary><Biogenesis><Biology><Blood Circulation><Bloodstream><Capsid><Cell Body><Cell Culture Techniques><Cell Line><Cell Membrane Permeability><Cell Polarity><CellLine><Cells><Clinical><Common Rat Strains><Complex><Data><Endosomes><Enteral><Enteric><FDA approved><Feces><Genetic Alteration><Genetic Change><Genetic defect><Goals><Hep G2><HepG2><HepG2 cell line><Hepatic Cells><Hepatic Disorder><Hepatic Parenchymal Cell><Hepatitis B><Hepatitis E virus><Hepatocyte><Human><Immunocompromised><Immunocompromised Host><Immunocompromised Patient><Immunosuppressed Host><In Vitro><Individual><Infection><Initiation Codon><Initiator Codon><Knowledge><Life Cycle><Life Cycle Stages><Liver><Liver Cells><Liver diseases><Macaca><Macaque><Mediating><Membrane><Mice><Mice Mammals><Modeling><Modern Man><Molecular><Morbidity><Morbidity - disease rate><Morphology><Multivesicular Body><Murine><Mus><Mutation><Origin of Life><Outcome><Play><Prevention><Process><Proteins><Publishing><RNA Viruses><Rat><Rats Mammals><Rattus><Receptosomes><Recycling><Research><Risk><Role><Side><Sorting><Start Codon><Strains Cell Lines><Surface><Testing><Transmission><Vaccines><Viral><Viral Diseases><Viral Hepatitis B><Viral Pathogenesis><Viremia><Virion><Virus><Virus Diseases><Virus Particle><Work><cell culture><cell cultures><cellular polarity><cultured cell line><genome mutation><hepatic body system><hepatic disease><hepatic organ system><hepatoma cell><hepatopathy><immunosuppressed patient><in vivo Model><insight><life course><liver disorder><membrane permeability><membrane structure><mortality><mutant><novel><palmitoylation><particle><rational design><recruit><serum hepatitis><social role><stool><trafficking><transmission process><vesicle transport><vesicular transport><viraemia><viral infection><viral sepsis><virus infection><virus pathogenesis><virus-induced disease><virusemia>