A crucial step of HBV (Hepatitis B Virus) virion morphogenesis is the envelopment of the nucleocapsid by the viral envelope proteins, which is triggered by an interaction between the HBV core protein and the large HBV envelope protein. To document this protein–protein interaction, we co-expressed core and large HBV envelope (LHBs) in Huh-7 cells and subjected the cells to microscopy examination by Fluorescence Resonance Energy Transfer (FRET) and Transmission Electron Microscopy (TEM). Our results show that the sole expression of the core protein leads to assembly of capsids that remain individually isolated within the whole cell, but particularly within the nucleus. In the presence of LHBs, capsids were observed as large clusters in a membrane rich region peripheral to the nucleus. In this context, core-LHBs complex co-localize with markers of the late endosome/multivesicular bodies, this co-localization being driven by LHBs. These results thus show that LHBs binds to the core proteins when preassembled into capsid, at membranes of the late endosome, where the inner capsid and the outer envelope meet to assemble a virion.
Hepatitis B virus (HBV) chronic infections affect more than 250 million individuals worldwide and lead to the death of up to one million individuals per year from liver diseases such as, cirrhosis, and hepatocellular carcinoma. The HBV virion is 42 nm in diameter, consisting of (i) an inner nucleocapsid (NC) that contains partially double-stranded relaxed circular HBV DNA (rcDNA) covalently linked to the viral polymerase (P) and (ii) an outer envelope made of lipids and three types of HBV- encoded envelope proteins. The latter bear the HBV surface antigen (HBs) and are referred to the large (LHBs), middle (MHBs) and small (SHBs) proteins.
SHBs, a 226 aas in length protein can self-assemble to form empty subviral particles (SVPs). MHBs protein is larger than SHBs by 55 aas (the pre-S2 domain); it is included in the envelope of HBV virions and SVPs but is dispensable for viral replication and infection. LHBs contains an additional N-terminal pre-S1 domain that is essential at two steps of the HBV life cycle: (i) assembly and release of enveloped HBV virion and (ii) entry into human hepatocytes upon binding to the sodium taurocholate co-transporting polypeptide (NTCP), the cell surface receptor for HBV.
The formation of HBV NC is initiated by an interaction between pre-genomic RNA (pgRNA), polymerase and core proteins. Within the NC, pgRNA is reverse-transcribed into rcDNA and NC envelopment with surface glycoproteins was reported to use the autophagy and the endosomal sorting complexes required for transport (ESCRT) machineries to traffic from ER to multivesicular bodies (MVBs). It has also been reported that the HBV envelope proteins are ubiquitinated to allow for a E3 ubiquitin-protein ligase (NEDD4)-dependent secretion of particles through MVBs using the γ2-adaptin.
However, the mechanism of HBV virion envelopment and its precise intracellular location remain elusive. It is important to note that in addition to DNA-containing virions, an excess of empty virions, i.e. virions containing a genome-free capsid, are assembled, which are indistinguishable by size and shape from DNA-containing virions.
In this study, we aimed at tracing the intracellular path of core and LHBs proteins to identify the cellular compartment in which they meet to promote HBV virion formation.
