Hepatitis E virus (HEV) is the major cause of acute hepatitis in the world. HEV is principally transmitted through the fecal-oral route: the virus enters via the gastrointestinal tract and infects the liver, where it enters polarized hepatocytes from the bloodstream and exits with the bile to be shed into feces. The molecular principles of HEV particle entry, assembly and release in polarized hepatocytes are largely unknown. By establishing polarized surface domains and restricting paracellular flow via their tight junctions, hepatocytes function like all epithelial cells to mediate the vectorial flow of macromolecules in and out of the organ they encompass.
Yet, hepatocytes distinguish themselves from other nonstriated epithelia by their multipolar organization. We previously developed a novel differentiation protocol that allows columnar polarization of human embryonic or induced pluripotent stem cell (hESC/iPSC)-derived hepatocyte-like cells (HLCs) in transwells. The polarization leads to the development of tight-junctions separating the HLCs’ apical membranes from the basal membranes. Polarized HLCs can be infected with HEV on their basal side, with the majority of infectious virus being released to the apical compartment, recapitulating the directionality of infection occurring in vivo. The aim of this proposal is to combine the polarized HLC system with genetic, biochemical, and imaging approaches to identify and characterize polarized trafficking and secretion routes of HEV.
In Aim 1 we will take advantage of the expertise within the SFB in minimally invasive live-cell virus labeling strategies (Müller, Johnsson, Bartenschlager, and Lemke) with the goal of studying HEV assembly and secretion.
In Aim 2, we will apply this strategy to live-track newly synthesized HEV proteins along their biosynthetic pathway in polarized HLCs. In order to map their secretion pathway to either the basal or the apical membrane we will perform live co-localization studies with cellular proteins along the trafficking machinery (4D-analysis). This Aim 2 will be strongly supported by experts in live-cell imaging (Ruggieri and Boulant) as well as single-particle tracking analysis (Hamprecht/Rohr; Z4). We will also characterize a novel differentiation protocol in which HLCs are grown as sandwich cultures to acquire a more natural, multipolar 3D-polarization.
In Aim 3, we will map the viral determinants that are involved in HEV assembly and vectorial trafficking. These efforts should lead to a better understanding of HEV secretion but also of the fine-tuned spatio-temporal dynamics and regulations of the polarized trafficking machinery in hepatocytes in general. These efforts may help develop new strategies to block HEV assembly and secretion and, thus, virus spread.
Transport und Sekretion von Hepatitis E virus in humanen Stammzellen-abgeleiteten polarisierten Leberzellen
Das Hepatitis E Virus (HEV) gilt als Hauptursache für akute Hepatitis weltweit. Die Übertragung von HEV verläuft fäkal-oral, die mitunter auf der Polarisation von Leberzellen beruht. Wir werden humane embryonische oder induzierte pluripotente Stammzellen zu polarisierten Leberzellen differenzieren und diese mit genetischen, biochemischen und mikroskopischen Anwendungen kombinieren um polarisierte HEV Transport- und Sekretionswege zu charakterisieren. Das hier beschriebene Projekt soll zu einem besseren Verständnis der HEV Sekretion sowie der fein abgestimmten Regulation der polarisierten Transportmaschinerie in Leberzellen führen.
Hepatitis E virus life cycle and transmission.
From Fu RM, Decker CC, Dao Thi VL (2019). Cell Culture Models for Hepatitis E Virus. Viruses 11, 608; doi:10.3390/v11070608