Hepatitis C virus nonstructural protein 4B is an integral endoplasmic reticulum membrane protein

The hepatitis C virus (HCV) nonstructural protein 4B (NS4B) is a relatively hydrophobic 27-kDa protein of unknown function. A tetracycline-regulated g ene expression system, a novel monoclonal antibody, and in vitro transcript ion-translation were employed to investigate the subcellular localization a nd to characterize the membrane association of this viral protein. When exp ressed individually or in the context of the entire HCV polyprotein, NS4B w as localized in the endoplasmic reticulum (ER), as shown by subcellular fra ctionation, immunofluorescence analyses, and double-label confocal laser sc anning microscopy. In this compartment NS4B colocalized with the other HCV nonstructural proteins. Association of NS4B with the ER membrane occurred c otranslationally, presumably via engagement of the signal recognition parti cle by an internal signal sequence. In membrane extraction and proteinase p rotection assays NS4B displayed properties of a cytoplasmically oriented in tegral membrane protein. Taken together, our findings suggest that NS4B is a component of a membrane-associated cytoplasmic HCV replication complex. A n efficient replication system will be essential to further define the role of NS4B in the viral life cycle. (C) 2001 Academic Press.