Dual topology of the processed hepatitis C virus protein NS4B is influenced by the NS5A protein

Abstract

Among the least-known hepatitis C virus proteins is the non-structural protein 4B (NS4B). It localizes to the endoplasmic reticulum (ER) membrane and induces membrane changes, resulting in a membranous web that is reported to be the locale for virus replication. A model was presented previously for the topology of recombinant HCV NS4B of the 1a genotype based on in vitro data. In this model, the N-terminal tail of a considerable fraction of the NS4B molecules was translocated into the ER lumen via a post-translational process, giving the protein a dual transmembrane topology. It is now reported that translocation of the N terminus also occurs for processed NS4B expressed in cells in the context of the polyprotein. In the presence of NS5A, however, a lower degree of translocation was observed, which may indicate that NS5A influences the topology of NS4B. In vitro expression studies of NS4B from all major genotypes demonstrated that translocation of the N terminus to the ER lumen is conserved across genotypes. This clearly suggests an important function for this feature. Furthermore, when disrupting a previously reported amphipathic helix (AH) in the N terminus of NS4B, translocation was inhibited. As a disrupted AH also abolished the ability of NS4B to rearrange membranes, these data indicate for the first time an association between translocation of the N terminus and membrane rearrangement. Finally, the present experiments also confirm the predicted location of the first luminal loop to be around aa 112. INTRODUCTIONHepatitis C virus (HCV) is a positive-stranded RNA virus that is classified in the genus Hepacivirus, which, together with the genera Flavivirus and Pestivirus, makes up the family Flaviviridae. The genome is a positive-strand RNA that encodes a polyprotein of about 3000 aa, which is processed proteolytically by cellular and viral proteases. The structural proteins (core, E1 and E2) are located in the Nterminal part of the polyprotein and the non-structural proteins (NS2, NS3, NS4A, NS4B, NS5A and NS5B) in the latter part (Major et al., 2001). Between the two regions there is also a small protein, p7, but the question about to which region it belongs to is still disputed.Little is known about the details of HCV's replication process, but it is generally believed that most of the NS proteins are involved. This assumption is based on comparisons with other members of the family Flaviviridae and the fact that all NS proteins are localized together with newly synthesized viral RNA on the endoplasmic reticulum (ER) or membranes originating from it (El-Hage & Luo, 2003;Gosert et al., 2003;Mottola et al., 2002). Most NS proteins also interact with several other NS proteins, and some of them interact with all (Dimitrova et al., 2003).The NS2 protein is an autoprotease that, together with NS3, digests the junction between them (Hijikata et al., 1993). NS2 has been shown to interact with all other NS proteins, but is not required for replication in a subgenomic replicon (Blight et al., 2000;Dimitrova et al., ...

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