Hepatitis B virus (HBV) is a unique, tiny, partially double-stranded, reverse-transcribing DNA virus with proteins encoded by multiple overlapping reading frames. The substitution rate is surprisingly high for a DNA virus, but lower than that of other reverse transcribing organisms. More than 260 million people worldwide have chronic HBV infection, which causes 0.8 million deaths a year. Because of the high burden of disease, international health agencies have set the goal of eliminating HBV infection by 2030. Nonetheless, the intriguing HBV genome has not been well characterized. We summarize data on the HBV genome structure and replication cycle, explain and quantify diversity within and among infected individuals, and discuss advances that can be offered by application of next-generation sequencing technology. In-depth HBV genome analyses could increase our understanding of disease pathogenesis and allow us to better predict patient outcomes, optimize treatment, and develop new therapeutics.

CD81 is a tetraspanin protein that is involved in several essential cellular functions, as well as in the hepatitis C virus (HCV) infection. CD81 interacts with a high stoichiometry with its partner proteins EWI-2, EWI-2wint, and EWI-F. These latter proteins modify the functions of CD81 and can thereby potentially inhibit infection or modulate cell migration. Here, we characterized the cleavage of EWI-2 leading to the production of EWI-2wint, which has been shown to inhibit HCV infection. We determined the regions of EWI-2/EWI-2wint and CD81 that are important for their interaction and their functionality. More precisely, we identified a glycine zipper motif in the transmembrane domain of EWI-2/EWI-2wint that is essential for the interaction with CD81. In addition, we found that palmitoylation on two juxtamembranous cysteines in the cytosolic tail of EWI-2/EWI-2wint is required for their interaction with CD81 as well as with CD9, another tetraspanin. Thus, we have shown that palmitoylation of a tetraspanin partner protein can influence the interaction with a tetraspanin. We therefore propose that palmitoylation not only of tetraspanins, but also of their partner proteins is important in regulating the composition of complexes in tetraspanin networks. Finally, we identified the regions in CD81 that are necessary for its functionality in HCV entry and we demonstrated that EWI-2wint needs to interact with CD81 to exert its inhibitory effect on HCV infection.Tetraspanins comprise a family of evolutionary highly conserved proteins that all contain four transmembrane domains for which they are named, as well as a small and a large extracellular loop. They are ubiquitously expressed proteins that are involved in many cellular functions such as adhesion, migration, co-stimulation, signal transduction, and cell differentiation. Tetraspanins also play an important role in infection by several pathogens (reviewed in Ref. 1). However, their specific function in all these processes remains to be elucidated. Tetraspanins have the special feature to interact with each other and other transmembrane proteins, forming extended cholesterolrich complexes on the cell surface, called tetraspanin-enriched microdomains (TEMs) 6 (2, 3). In these domains, tetraspanins form primary complexes with a limited number of proteins called tetraspanin partners. These primary interactions are direct, highly specific, and occur at high stoichiometry. Tetraspanin molecules can form different partnerships in different cell types. The precise mechanisms of interaction within TEMs remain unknown. However, in addition to specific domains, cellular lipids such as cholesterol, glycosphingolipids, and palmitic acid seem to play an important role in the interaction of tetraspanins with each other and therefore in building the tetraspanin network (1).In this study, we focused on tetraspanin CD81, which was initially characterized as important for B cell proliferation (4), but has since been found in many different cell lines, contributing to a variety of dif...