Background: Hepatitis C virus (HCV) nonstructural protein 2 (NS2)
plays essential roles in particle assembly and polyprotein processing.
It harbors an N-terminal membrane domain comprising three putative
transmembrane s egments ( amino acids [aa] 1-93) a nd a C-terminal
cysteine protease domain (aa 94-217). Given that the latter has been
predicted to be membrane-associated, we aimed to identify molecular
determinants for membrane association of the NS2 protease domain.
Methods: A comprehensive panel of NS2 deletion constructs was
analyzed by fluorescence microscopy, selective membrane extraction,
and m embrane flotation assays. Candidate aa r esidues involved in
membrane association were substituted by site-directed mutagenesis.
Results: The NS2 protease domain alone was found to associate with
membranes. Two N-terminal α-helices comprising aa 102-114 and aa
123-136 were found to m ediate this a ssociation, w ith c onserved
hydrophobic and positively charged aa residues representing the key
determinants. I nterestingly, m utagenesis analyses r evealed that
electrostatic interactions involving a positively charged aa residue in
α-helix aa 123-136 are required for membrane association. Mono- and
bicistronic (i.e. NS2 c leavage-independent) HCV constructs were
prepared to i nvestigate the effect o f these substitutions on RNA
replication and infectious viral particle formation.
Conclusions: T he NS2 protease d omain itself harbors m olecular
determinants for membrane association within α-helices aa 102-114
and aa 1 23-136 which may contribute to p roper p ositioning of t he
active site. These results provide new insights i nto the membrane
topology and t he p oorly understood f unction of t his essential viral
protease.