Viral and cellular determinants involved in hepadnaviral entry

Abstract

Hepadnaviridae is a family of hepatotropic DNA viruses that is divided into the genera orthohepadnavirus of mammals and avihepadnavirus of birds. All members of this family can cause acute and chronic hepatic infection, which in the case of human hepatitis B virus (HBV) constitutes a major global health problem. Although our knowledge about the molecular biology of these highly liver-specific viruses has profoundly increased in the last two decades, the mechanisms of attachment and productive entrance into the differentiated host hepatocytes are still enigmatic. The difficulties in studying hepadnaviral entry were primarily caused by the lack of easily accessible in vitro infection systems. Thus, for more than twenty years, differentiated primary hepatocytes from the respective species were the only in vitro models for both orthohepadnaviruses (e.g. HBV) and avihepadnaviruses (e.g. duck hepatitis B virus [DHBV]). Two important discoveries have been made recently regarding HBV: (1) primary hepatocytes from tree-shrews; i.e., Tupaia belangeri, can be substituted for primary human hepatocytes, and (2) a human hepatoma cell line (HepaRG) was established that gains susceptibility for HBV infection upon induction of differentiation in vitro. A number of potential HBV receptor candidates have been described in the past, but none of them have been confirmed to function as a receptor. For DHBV and probably all other avian hepadnaviruses, carboxypeptidase D (CPD) has been shown to be indispensable for infection, although the exact role of this molecule is still under debate. While still restricted to the use of primary duck hepatocytes (PDH), investigations performed with DHBV provided important general concepts on the first steps of hepadnaviral infection. However, with emerging data obtained from the new HBV infection systems, the hope that DHBV utilizes the same mechanism as HBV only partially held true. Nevertheless, both HBV and DHBV in vitro infection systems will help to: (1) functionally dissect the hepadnaviral entry pathways, (2) perform reverse genetics (e.g. test the fitness of escape mutants), (3) titrate and map neutralizing antibodies, (4) improve current vaccines to combat acute and chronic infections of hepatitis B, and (5) develop entry inhibitors for future clinical applications.

Figure 1

Schematic presentation of human (HBV) and duck (DHBV) hepatitis B virus. The viral DNA is drawn as a single or double line. The viral polymerase is depicted with the primer domain (pr) and the reverse transcription domain (RT). The nucleocapsid (core or HBc/DHBc) is shown in black. Reported encapsidated cellular proteins are omitted. For HBV the surface proteins L, M and S are shown with the S-domain, the preS2-domain and the preS1-domain, whereas for DHBV, L- and S-surface proteins with preS and S-domain are depicted. St, truncated form of DHBV S-surface protein. Non-infectious subviral particles of HBV are shown in filamentous and spherical form and in larger spheroids in the case of DHBV.

Figure 2

Surface proteins of HBV and DHBV. (A) Schematic organisation of the three surface proteins of HBV and the two surface proteins of DHBV. For HBV, used N- and O-glycosylation sites (NG and OG) are shown, (parenthesis indicate partial glycosylation). DHBV surface proteins are non-glycosylated, but the L-protein is phosphorylated at position 118 within its preS-domain. The L-protein of HBV and DHBV is myristoylated (Myr) at Gly-2 of the N-terminus of preS1 or preS, respectively. In case of HBV the preS1-domain encodes for 108 or 119 amino acids, depending on the genotype. The preS2-domain of MHBs is N-terminally acetylated (Ac) and is 55 amino acids long, while a preS2-domain and therefore a special M-protein is missing in DHBV. (B) Schematic presentation of preS-domains of HBV and DHBV important for virion formation and viral entry. The host-determining regions are depicted in green. The potential receptor binding site within the preS1-domain of HBV is shown with the essential domain (aa 9-18) and the two accessory domains (aa 28-39 and 39-48, respectively). For DHBV, the carboxypeptidase D (dCPD) binding site is depicted with the essential and stabilizing domains. Alpha helical domains are shown in blue (α). Reported trans-location motives (TLM) are marked in boxes. In DHBV preS, two TLMs are predicted (TLM1 and TLM2). TLM1 overlaps with the host-determining region, while TLM2 is located within the stabilizing sequence of the dCPD receptor binding site. In the case of HBV, the carboxyterminus of the 55 aa long preS2-domain was reported to contain a single TLM. The numbering of HBV preS1 is for genotype D (108 aa). P, phosphorylation site; OG, O-glycosylation; myr, myristoylation.