Summary Project description 2nd funding period

Over 240 million people are chronically infected with the human Hepatitis B virus (HBV), and ~15-25 million of
them are co-infected with Hepatitis D virus (HDV). In the absence of curative treatments, the two viruses are
major causes of fatal liver diseases and liver cancer. They use a common cellular entry pathway via sodium
taurocholate co-transporting polypeptide (NTCP), since HDV utilizes HBV envelope proteins and thereby
hijacks HBV-infected cells. However, the viruses differ in how they establish chronic infections and maintain
their nuclear episomal genomes. Alas, the complex organization of both viral genomes prevents the insertion
of reporters without perturbing replication competence, thus hampering efforts to dissect the viral infection
pathways by direct visualization. Notably, in the current funding phase, we established new labeling strategies
for HBV/HDV envelope and HBV capsids that preserve virus infectivity. Using NTCP-expressing cell lines or
primary hepatocytes, we managed to visualize HBV entry by confocal live-cell microscopy and characterized
heparan sulfate proteoglycans (HSPGs) and NTCP as cellular factors mediating viral uptake.

In the second funding phase, we will expand and refine these results, and investigate early infection events by
tracking fluorescently labeled HBV/HDV via confocal and super-resolution microscopy (with Müller, Kräusslich)
from the site of membrane fusion to the nucleus. This includes (i) the characterization of fusion sites and of
key players of endocytic pathways by using Adeno-associated viral (AAV)-mediated transduction of shRNA or
cDNA into primary hepatocytes. Also, (ii) we will engineer AAV vectors for robust and controlled expression of
the two central components of the amber suppression/click labeling machinery, i.e. tRNA and tRNA synthetase
(with Lemke, Müller). This will yield novel tools for tagging HBV/HDV proteins directly in living cells, including
physiologically relevant primary hepatocytes. Finally, (iii) we will use the new vectors together with our existing
fluorophores in the envelope to produce dually-labeled HBV and HDV (HBV envelope/capsid, HDV
envelope/Delta antigen), and will analyze the contribution of nuclear reimport of HBV nucleocapsid (NC) and
HDV ribonucleoprotein complex (RNP) for maintenance of viral persistence (utilizing CLEM technology from
Funaya/Schwab, and benefitting from novel dyes from Johnsson).

This project will break new scientific ground with the establishment of original labeling and visualization
technologies that will, in turn, permit to address seminal questions on hepatitis virus infection, replication and
spread in liver cells. For the first time, we thus anticipate the ability to resolve the pathways that control HBVNC/
HDV-RNP trafficking in newly versus persistently infected hepatocytes, and to delineate factors that
differentially regulate nuclear import of HBV-NC versus envelopment and exit. Of note, the unique strategies
for pulse-chase in cellulo click labeling of proteins that we will implement for this purpose are not only pivotal
for this project, but will also support several other SFB members (Dao Thi, Müller, Kräusslich, Lemke) who aim
at dissecting temporal, spatial and functional aspects of virus replication and spread in live human cells.