Identification of a Novel Determinant for Membrane Association in Hepatitis C Virus Nonstructural Protein 4B

Abstract

Nonstructural protein 4B (NS4B) plays an essential role in the formation of the hepatitis C virus (HCV) replication complex. It is a relatively poorly characterized integral membrane protein predicted to comprise four transmembrane segments in its central portion. Here, we describe a novel determinant for membrane association represented by amino acids (aa) 40 to 69 in the N-terminal portion of NS4B. This segment was sufficient to target and tightly anchor the green fluorescent protein to cellular membranes, as assessed by fluorescence microscopy as well as membrane extraction and flotation analyses. Circular dichroism and nuclear magnetic resonance structural analyses showed that this segment comprises an amphipathic ␣-helix extending from aa 42 to 66. Attenuated total reflection infrared spectroscopy and glycosylation acceptor site tagging revealed that this amphipathic ␣-helix has the potential to traverse the phospholipid bilayer as a transmembrane segment, likely upon oligomerization. Alanine substitution of the fully conserved aromatic residues on the hydrophobic helix side abrogated membrane association of the segment comprising aa 40 to 69 and disrupted the formation of a functional replication complex. These results provide the first atomic resolution structure of an essential membrane-associated determinant of HCV NS4B.With 120 to 180 million chronically infected individuals worldwide, hepatitis C virus (HCV) infection represents a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (38). HCV contains a 9.6-kb positive-strand RNA genome that encodes a polyprotein of about 3,000 amino acids (reviewed in references 36 and 51). The polyprotein precursor is co-and posttranslationally processed by cellular and viral proteases to yield the mature structural and nonstructural proteins. The structural proteins include the core and the envelope glycoproteins E1 and E2. The nonstructural proteins include the p7 ion channel polypeptide, the NS2-3 and NS3-4A proteases, an RNA helicase located in the C-terminal twothirds of NS3, the NS4B and NS5A proteins, and the NS5B RNA-dependent RNA polymerase. HCV replication takes place in a membrane-associated complex, composed of viral proteins, replicating RNA, altered cellular membranes, and other host factors (7,18,31,43). Determinants for membrane association of the HCV nonstructural proteins have been mapped and a likely endoplasmic reticulum (ER)-derived membrane alteration, designated the membranous web, was found to harbor the HCV replication complex (7, 18; reviewed in reference 36).NS4B is a 27-kDa integral ER membrane protein (21). The expression of NS4B alone induces the formation of the membranous web (7). Thus, an essential function of NS4B is the induction of the specific membrane alteration that serves as a scaffold for the HCV replication complex. In addition, a nucleotide-binding motif has been proposed to reside in the middle of NS4B (8), and RNA binding properties have recently been reported for NS4B (9).Both the N and the C t...

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

“…7 Recently, Gouttenoire et al proposed the amphipathic a-helix to be located between amino acid 42 to 66. 17 In this publication no membrane association of amino acid 1 to 29 or amino acid 1 to 40 was detected. 17 Most likely, the HCV NS4B protein is anchored to the ER membrane by the four TMDs, initiating ER membrane alterations and recruiting other host and viral factors of the replication complex, 20,21 while the aminoterminal region contains an amphipathic a-helix and mediates membrane association.…”

“…12 The bZIP motif in HCV NS4B is located in the aminoterminal part of the protein, which was reported to mediate membrane association. 7,17 Interestingly, amino acid substitutions within the predicted bZIP motif (Q26H, L46I) have been described to influence viral replication efficacy in the replicon model. 11,25,30 The aim of the present study was to investigate the capability of NS4B to interact with each other due to specific interactions via this bZIP motif.…”

“…Note that a genotype 1b sequence was used within the present study, while Gouttenoire et al used a HCV genotype 1a sequence. 17 outside the core for putative ionic interactions (salt bridges). We used multiple sequence alignments to determine amino acid positions of functional importance within the putative NS4B bZIP motif.…”

“…[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. 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.…”

“…17 Most likely, the HCV NS4B protein is anchored to the ER membrane by the four TMDs, initiating ER membrane alterations and recruiting other host and viral factors of the replication complex, 20,21 while the aminoterminal region contains an amphipathic a-helix and mediates membrane association. 7,17 Exact mechanisms how NS4B causes ER membrane alterations or interacts with other proteins are still unknown.…”