Mechanism for hepatitis E virus exit from polarized hepatocytes

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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.