B a c k g r o u n d: Nonstructural protein 4B (NS4B) plays an
essential role in the formation of the hepatitis C virus (HCV)
replication complex. It is an integral membrane protein that has
only poorly been characterized to date, believed to comprise a
central part harboring 4 transmembrane passages (TM) flanked
by 2 cytosolic parts. However, a precise membrane topology of
HCV NS4B is thus far elusive. This work is aimed at enhancing
our understanding of the structure and function of this protein.
Methods: Full-length NS4B as well as a comprehensive panel
of mutants were fused to the green fluorescent protein and
expressed in cultured cells. The impact of point mutations on
HCV infection and replication was assessed by the use of cell
culture-derived HCV and the replicon system. 3-D structures
were determined by nuclear magnetic resonance. Proteinprotein
interactions were investigated by fluorescence
resonance energy transfer and co-immunoprecipitation.
R e s u l t s : We identified two unexpected determinants for
membrane association in the N- and C-terminal parts of HCV
NS4B. The first determinant is an amphipathic alpha-helix that
can traverse the membrane, likely as an oligomer, thereby
constituting a fifth TM. The second is a peculiar "twisted"
amphipathic alpha-helix at the C-terminus of NS4B. Both
determinants are involved in protein-protein interactions and
play important roles in the formation of replication complex.
Conclusions: These results provide the first atomic resolution
structures of essential membrane-associated segments of
NS4B and highlight their essential roles in the assembly of a
functional HCV replication complex.