Topology of the Membrane-Associated Hepatitis C Virus Protein NS4B

Abstract

Hepatitis C virus (HCV) belongs to the Hepacivirus genus in the Flaviviridae family. Among the least known viral proteins in this family is the nonstructural protein NS4B, which has been suggested to be a part of the replication complex. Hydrophobicity plots indicate a common profile among the NS4B proteins from different members of the Flaviviridae family, suggesting a common function. In order to gain a deeper understanding of the nature of HCV NS4B, we have determined localization and topology of this protein by using recombinant HCV NS4B constructs. The protein localized to the endoplasmic reticulum (ER), but also induced a pattern of cytoplasmic foci positive for markers of the ER. Computer predictions of the membrane topology of NS4B suggested that it has four transmembrane segments. The N and C termini were anticipated to be localized in the cytoplasm, because they are processed by the cytoplasmic NS3 protein. By introducing glycosylation sites at various positions in HCV NS4B, we show that the C terminus is cytoplasmic and the loop around residue 161 is lumenal as predicted. Surprisingly, the N-terminal tail was translocated into the lumen in a considerable fraction of the NS4B molecules, most likely by a posttranslational process. Interestingly, NS4B proteins of the yellow fever and dengue viruses also have their N termini located in the ER lumen due to an N-terminal signal peptide not found in NS4B of HCV. A shared topology achieved in two different ways supports the notion of a common function for NS4B in Flaviviridae.

From Structure to Function: New Insights into Hepatitis C Virus RNA Replication

“…NS4B: Inducer of a Membrane Scaffold for the Viral RC?-TheNS4B is a highly hydrophobic protein predicted to contain four transmembrane domains, while its N-terminal region may contribute to targeting of NS4B to intracellular membranes (29). Overexpression of NS4B in cell culture is sufficient to induce membrane alterations arguing that the main function of this protein is the formation of membranous structures serving as the scaffold of the HCV RC (30).…”

Dimerization of the hepatitis C virus nonstructural protein 4B depends on the integrity of an aminoterminal basic leucine zipper

“…Furthermore, it has been reported that the aminoterminal part of HCV-NS4B, containing the first 33 amino acids, has the ability to translocate at least partially towards the ER lumen. 13,15 Thus, bZIP-mediated NS4B protein interactions with other host and viral factors may depend on different localization of NS4B during the viral life cycle. Nevertheless, it must be discussed whether dimerization of NS4B is mediated solely by the bZIP motif or if other structural elements are involved.…”

“…Amongst others, bioinformatics was used to study protein structure and function as an alternative approach. 12,13 From several experimental and bioinformatics' analyses, it is accepted that NS4B contains at least four transmembrane domains (TMDs) in its middle part, while the topology of a putative fifth TMD close to the aminoterminal part remains controversial. [12][13][14][15] Furthermore, amphipathic a-helix structure elements were predicted within the aminoterminal part and recently reported to be potential targets of HCV replication inhibitors.…”

“…12,13 From several experimental and bioinformatics' analyses, it is accepted that NS4B contains at least four transmembrane domains (TMDs) in its middle part, while the topology of a putative fifth TMD close to the aminoterminal part remains controversial. [12][13][14][15] Furthermore, amphipathic a-helix structure elements were predicted within the aminoterminal part and recently reported to be potential targets of HCV replication inhibitors. 7,16,17 The HCV NS4B protein integrates into the endoplasmatic reticulum (ER) with subsequent specific membrane alterations, described as membranous web.…”

“…7,16,17 The HCV NS4B protein integrates into the endoplasmatic reticulum (ER) with subsequent specific membrane alterations, described as membranous web. 13,18,19 The subcellular structure is considered to harbour the HCV replication complex, since all nonstructural proteins as well as viral genomic RNA were found to co-localize within this ER alterations. 19,20 The aminoterminal amphipathic a-helix (amino acid 1 to 26) has been described to mediate membrane association and correct localization of the replication complex within the ER.…”